Claudin 18.2-targeting antibody-drug conjugate CMG901 in patients with advanced gastric or gastro-oesophageal junction cancer (KYM901): a multicentre, open-label, single-arm, phase 1 trial.
The Lancet. Oncology
BACKGROUND:CMG901 is a novel first-in-class antibody-drug conjugate with a humanised anticlaudin 18.2 antibody linked to microtubule-disrupting agent monomethyl auristatin E. We aimed to assess the antitumour activity and safety of CMG901 in patients with advanced gastric or gastro-oesophageal junction cancer and other solid tumours. METHODS:KYM901 is a multicentre, open-label, single-arm, phase 1 trial consisting of dose-escalation and dose-expansion stages. Patients with advanced solid tumours, including gastric or gastro-oesophageal junction and pancreatic cancers, were recruited from 31 hospital sites in China. Eligible patients were aged 18 years or older, were refractory to standard therapy or had no available standard-of-care regimen, and had an Eastern Cooperative Oncology Group performance status score of 0-1, a life expectancy of at least 3 months, and at least one measurable lesion. Patients received intravenous CMG901 every 3 weeks (0·3-3·4 mg/kg in dose escalation and 2·2-3·0 mg/kg in dose expansion) until disease progression, unacceptable toxic effects, initiation of new antitumour therapy, study withdrawal, or death. Primary endpoints were adverse events and dose-limiting toxic effects in the dose-escalation phase, and objective response rate and recommended phase 2 dose in the dose-expansion phase. Confirmed objective response was defined as a partial or complete response that was verified by follow-up imaging at least 4 weeks after the initial assessment. Safety was assessed in all patients who received at least one dose of CMG901 with at least one post-dose safety evaluation. Antitumour activity was assessed in all patients who received at least one dose of CMG901 (full analysis set) and in all CMG901-treated patients with at least one post-dose imaging evaluation and no major protocol deviations (efficacy analysis set). Dose-expansion data for patients with pancreatic cancer will be published separately. Due to small sample sizes, results in patients with other solid tumours (n=2) are not planned for publication. This ongoing trial is registered with ClinicalTrials.gov, NCT04805307. FINDINGS:Between Dec 24, 2020, and Feb 23, 2023, 27 patients were enrolled in the dose-escalation phase (median age 57·0 years [IQR 48·0-63·0]; 14 [52%] male, 13 [48%] female) and 107 patients with gastric or gastro-oesophageal junction cancer in the dose-expansion phase (median age 56·0 years [44·0-64·0]; 57 [53%] male, 50 [47%] female). As of Feb 24, 2024, one dose-limiting toxic effect (grade 3 pancreatitis) occurred at 2·2 mg/kg, and the maximum tolerated dose was not reached in the dose-escalation phase. All 27 patients reported at least one treatment-emergent adverse event, most frequently vomiting (19 [70%]), decreased appetite (16 [59%]), proteinuria (16 [59%]), and anaemia (15 [56%]), and five (19%) had drug-related grade 3 or worse treatment-emergent adverse events. In 107 patients, grade 3 or worse treatment-emergent adverse events occurred in 73 (68%) patients and serious adverse events occurred in 54 (50%) patients in dose expansion. The most common grade 3-4 adverse events were neutrophil count decreased (22 [21%]), anaemia (15 [14%]), and vomiting (11 [10%]). One treatment-related death was reported. At median follow-up of 9·0 months (IQR 4·4-12·9), among 113 patients with gastric or gastro-oesophageal junction cancer in the 2·2-3·0 mg/kg cohort full analysis set across both the dose-escalation and dose-expansion phases, the confirmed objective response rate was 28% (95% CI 20-38; 32 of 113 patients). In the 109 patients included in the efficacy analysis set, the confirmed objective response rate was 29% (95% CI 21-39; 32 of 109 patients). Based on overall safety, activity, and pharmacokinetics of CMG901, 2·2 mg/kg was the proposed recommended phase 2 dose. INTERPRETATION:CMG901 showed a manageable safety profile and had promising antitumour activity in patients with advanced gastric or gastro-oesophageal junction cancer. FUNDING:KYM Biosciences.
10.1016/S1470-2045(24)00636-3
CAMSAP2 is required for bridging fiber assembly to ensure mitotic spindle assembly and chromosome segregation in human epithelial Caco-2 cells.
PloS one
In mammalian epithelial cells, cytoplasmic microtubules are mainly non-centrosomal, through the functions of the minus-end binding proteins CAMSAP2 and CAMSAP3. When cells enter mitosis, cytoplasmic microtubules are reorganized into the spindle composed of both centrosomal and non-centrosomal microtubules. The function of the CAMSAP proteins upon spindle assembly remains unknown, as these do not exhibit evident localization to spindle microtubules. Here, we demonstrate that CAMSAP2, but not CAMSAP3, is required for spindle assembly upon mitotic entry. CAMSAP2 knockout (KO) Caco-2 cells showed a delay in mitotic progression, whereas CAMSAP3 KO cells did not. The spindle in CAMSAP2 KO cells was short and displayed a reduced microtubule density, particularly around chromosomes. This indicated a loss of bridging fibers, which are known to assist alignment of sister kinetochores through interaction with kinetochore fibers. Consistent with this, live-cell imaging of CAMSAP2 KO cells captured slow elongation of the anaphase spindle and errors in chromosome segregation. Therefore, we propose that CAMSAP2 ensures efficient reorganization of cytoplasmic microtubules into the mitotic spindle through constructing bridging fibers that assist faithful segregation of sister chromatids.
10.1371/journal.pone.0308150
Targeting tubulin protein to combat fungal disease: Design, synthesis, and its new mechanistic insights of benzimidazole hydrazone derivatives.
International journal of biological macromolecules
As the vital the biomacromolecule in eukaryotic cells, tubulin protein is essential for preserving cell shape, facilitating cell division, and cell viability. Tubulin has been approved as promising target for anticancer, and antifungal therapy. However, there are still many gaps in tubulin-targeted fungicidal discovery. To expand the structural diversity of benzimidazoles and achieve the distinct interaction model, a series of novel benzimidazole hydrazone derivatives were therefore synthesized. Antifungal results showed that compound A was the most effective, achieving an EC value of 2.88 μg/mL in vitro against Colletotrichum sublineola. In vivo assay, compound A displayed encouraging efficacy, outperforming the reference agents ferimzone and tetramethylthiuram disulfide. Interestingly, mechanistic studies indicated that, compared with carbendazim, compound A might form stronger interactions with tubulin, exemplified by the presence of multiple hydrogen bonds and π-π interactions, leading to intracellular microtubule aggregation in compound A-treated cells. The significantly different interactions models between A-tubulin and carbendazim-tubulin complexes may endow to produce the low resistance risk. Additionally, compound A possessed low phytotoxicity and satisfactory ADME properties. This study not only provides a structural basis for the development of benzimidazole-based fungicides targeting tubulin but also offers new insights into the use of immunofluorescence assays in tubulin-targeting studies.
10.1016/j.ijbiomac.2025.140226
The Golgi complex governs natural killer cell lytic granule positioning to promote directionality in cytotoxicity.
Cell reports
Cytotoxic immune cells mediate precise attacks against diseased cells to maintain organismal health. Their operational unit of killing and host defense is lytic granules (LGs), which are specialized lysosomal-related organelles. Precision in cytotoxicity is achieved by converging the many LGs to the microtubule-organizing center (MTOC) and polarizing these to the diseased cell for secretion. We identify unappreciated intimate relationships between the Golgi, MTOC, and LGs after cytotoxic cell activation, as well as the trans-Golgin protein GCC2 on the LG surface. GCC2 serves to tether LGs to the Golgi following convergence, and both GCC2 and the Golgi are required for the persistence of convergence. GCC2 allows LGs to utilize the Golgi as a docking station preventing LG dispersion and innocent bystander killing in complex three-dimensional environments. We also identify GCC2 variants causing human natural killer cell deficiency, further emphasizing the importance of LG convergence and Golgi linkage in precision targeting for human immunity.
10.1016/j.celrep.2024.115156
WT161, a selective HDAC6 inhibitor, decreases growth, enhances chemosensitivity, promotes apoptosis, and suppresses motility of melanoma cells.
Cancer chemotherapy and pharmacology
PURPOSE:Histone deacetylase 6 (HDAC6) plays a critical role in tumorigenesis and tumor progression, contributing to proliferation, chemoresistance, and cell motility by regulating microtubule architecture. Despite its upregulation in melanoma tissues and cell lines, the specific biological roles of HDAC6 in melanoma are not well understood. This study aims to explore the functional effects and underlying mechanisms of WT161, a selective HDAC6 inhibitor, in melanoma cell lines. METHODS:Cell proliferation was assessed using both 2D and 3D cell culture systems, including MTT assays, spheroid growth analyses, and colony formation assays. The interaction between WT161 and the chemotherapeutic agents temozolomide (TMZ) or dacarbazine (DTIC) was evaluated using the Chou-Talalay method. Apoptotic cell death was analyzed through flow cytometry, while migration, adhesion, and invasion assays were conducted to evaluate the motility capacities of melanoma cells. Western blot assays quantified α-tubulin acetylation (Lys40), PARP cleavage, and protein levels of β-catenin and E-cadherin. RESULTS:WT161 significantly reduced cell growth in both 2D and 3D cultures, decreased clonogenic capacity, and showed synergistic interactions with TMZ and DTIC. The inhibitor also induced apoptotic cell death and enhanced TMZ-induced apoptosis. Additionally, WT161 reduced cell migration and invasion while increasing cell adhesion. These effects were linked to changes in β-catenin and E-cadherin levels, depending on the specific cell type evaluated. CONCLUSION:Our study underscores the pivotal role of HDAC6 in melanoma progression, establishing it as a promising therapeutic target. We provide the first comprehensive evidence of WT161's anti-melanoma effects, setting the stage for further research into HDAC6 inhibitors as a potential strategy for melanoma treatment.
10.1007/s00280-024-04731-y
Dynamic crosstalk between cytoskeletal filaments regulates dorsoventral cytoplasmic mechanics.
Journal of cell science
The cytoplasm exhibits viscoelastic properties, displaying both solid and liquid-like behaviour, and can actively regulate its mechanical attributes. The cytoskeleton is a major regulator among the numerous factors influencing cytoplasmic mechanics. We explore the interdependence of various cytoskeletal filaments and the impact of their density on cytoplasmic viscoelasticity. The heterogeneous distribution of these filaments gives rise to polarised mechanical properties of the cytoplasm along the dorsoventral axis. Actin filament disassembly softens the ventral cytoplasm while stiffening the mid cytoplasm, due to increased vimentin filament assembly. Disruption of microtubules or depletion of vimentin softens both the ventral and mid cytoplasm. Cytochalasin D (Cyto D) treatment results in a localised increase of vimentin assembly in the mid cytoplasm, which is dependent on the cytolinker plectin. Nocodazole treatment has a negligible effect on F-actin distribution but significantly alters the spatial arrangement of vimentin. We demonstrate that Cyto D treatment upregulates vimentin expression via reactive oxygen species-mediated activation of NF-κΒ. This article investigates how different cytoskeletal filaments influence the rheological characteristics of various cytoplasmic regions.
10.1242/jcs.263464
Global alignment and local curvature of microtubules in mouse fibroblasts are robust against perturbations of vimentin and actin.
Soft matter
The eukaryotic cytoskeleton is an intricate network of three types of mechanically distinct biopolymers - actin filaments, microtubules and intermediate filaments (IFs). These filamentous networks determine essential cellular functions and properties. Among them, microtubules are important for intracellular transport and establishing cell polarity during migration. Despite their intrinsic stiffness, they exhibit characteristic bending and buckling in cells due to nonthermal forces acting on them. Interactions between cytoskeletal filaments have been found but are complex and diverse with respect to their effect on the mechanical behavior of the filaments and the architecture of networks. We systematically study how actin and vimentin IFs influence the network structure and local bending of microtubules by analyzing fluorescence microscopy images of mouse fibroblasts on protein micropatterns. Our automated analysis averages over large amounts of data to mitigate the effect of the considerable natural variance in biological cell data. We find that the radial orientation of microtubules in circular cells is robust and is established independently of vimentin and actin networks. Observing the local curvature of microtubules, we find highly similar average bending of microtubules in the entire cell regardless of the cytoskeletal surrounding. Small systematic differences cannot be attributed directly to vimentin and actin densities. Our results suggest that, on average, microtubules in unpolarized mouse fibroblasts are unexpectedly independent of the rest of the cytoskeleton in their global network structure and their local curvature.
10.1039/d4sm01127a
Involvement of RBP-J interacting and tubulin-associated protein in the distribution of protein regulator of cytokinesis 1 in mitotic spindles.
Frontiers in cell and developmental biology
The protein regulator of cytokinesis 1 (PRC1) is a key regulator of microtubule crosslinking and bundling, which is crucial for spindle formation and cytokinesis. RITA, the BP-J nteracting and ubulin-ssociated protein, is a microtubule associated protein. We have reported that RITA localizes to mitotic spindles modulating microtubule dynamics and stability as well as to spindle poles affecting the activity of Aurora A. As defective chromosome congression and segregation are the most remarkable features of cells depleted of RITA, we aimed to explore further potential related mechanisms, using various cellular and molecular techniques, including clustered regularly interspaced short palindromic repeats technique/deactivated CRISPR-associated protein 9 (CRISPR/dCas9), mass spectrometry, confocal microscopy, immunofluorescence, immunoprecipitation and Western blot analysis. Here, we show that FLAG-RITA precipitates PRC1 and tubulin, and that these two proteins co-localize in the central region of the central spindle. Reduction of RITA enlarges the staining area of PRC1 in mitotic spindles as well as in the central spindle. Its suppression reduces the inter-centromere distance in metaphase cells. Interestingly, microtubule bundles of the central spindle are often less organized in a non-parallel pattern, as evidenced by increased angles, relative to corresponding separating chromosomes. These data suggest a novel role for RITA in mitotic distribution of PRC1 and that its deregulation may contribute to defective chromosome movement during mitosis. As both RITA and PRC1 are closely associated with malignant progression, further work is required to elucidate the detailed molecular mechanisms by which RITA acts as a modulator in central spindle formation and cytokinesis.
10.3389/fcell.2024.1472340
The GLP-1 agonist semaglutide ameliorates cognitive regression in P301S tauopathy mice model via autophagy/ACE2/SIRT1/FOXO1-Mediated Microglia Polarization.
European journal of pharmacology
Tau hyper-phosphorylation has been recognized as an essential contributor to neurodegeneration in Alzheimer's disease (AD) and related tauopathies. In the last decade, tau hyper-phosphorylation has gained considerable concern in AD therapeutic development. Tauopathies are manifested with a broad spectrum of symptoms, from dementia to cognitive decline and motor impairments. Tau undergoes conformational changes and abnormal phosphorylation that mediate its detaching from microtubules, forming neurofibrillary tangles (NFTs). In the current study, a widely used P301S transgenic mice model of tauopathy was employed to evaluate the possible neuroprotective effects of semaglutide as an autophagy regulator through modifications of the brain renin-angiotensin system (RAS). Mice were divided into two groups according to their genotypes (wild type (Wt) and P301S), which were further subdivided to receive either vehicle (saline) or semaglutide (25 nmol/kg, i. p.), once every 2 days for 28 days. Current data suggest that semaglutide ameliorated the hyperactive pattern and alleviated the cognitive decline of P301S mice. It also hastened the autophagic flux through augmenting angiotensin-converting enzyme 2/sirtuin 1/forkhead box protein O1 signaling. Semaglutide also hindered the expression of phosphorylated adenosine monophosphate-activated protein kinase and phosphorylated glycogen synthase kinase-3 beta at serine 9, reducing the propagation of neuroinflammatory cytokines and oxidative reactions. Finally, semaglutide protected against hippocampal degeneration and reduced the immunoreactivity for total tau and ionized calcium-binding adapter molecule. Semaglutide showed promising neuroprotective implications in alleviating tauopathy-related AD's molecular and behavioral deficits through controlling autophagy and brain RAS.
10.1016/j.ejphar.2025.177305
Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation.
Redox biology
Oocyte aging is closely related to a decline in female fertility, accompanied by increased reactive oxygen species levels and changes in protein posttranslational modifications. However, the role of protein palmitoylation in oocyte aging has not been investigated. In the present study, a new association between redox and palmitoylation in aging oocytes was found. We found that the protein level of palmitoyl-protein thioesterase 1 (PPT1), a depalmitoylation enzyme, was increased in maternally aged mice oocytes and follicular fluid of aged (age >35 years) patients with decreased ovarian reserve (DOR). Elevated PPT1 led to decreased S-palmitoylation levels in oocytes, which impaired oocyte maturation and spindle formation. Tubulin was identified as a critical palmitoylated protein regulated by PPT1, whose palmitoylation was also decreased by advanced age, accompanied by abnormalities in membrane localization and microtubule polymerization. Melatonin was found to down-regulate excessive PPT1 and rescue PPT1-induced damage in mouse oocytes, not only by regulating oxidative stress, but also by binding with PPT1 to regulate its lysosomal degradation. In summary, our data demonstrate that PPT1 participates in oocyte aging by regulating tubulin palmitoylation, providing evidence that oxidative stress regulates protein palmitoylation and revealing a novel mechanism of oocyte aging.
10.1016/j.redox.2025.103510
KIF18A Is a Novel Target of JNK1/c-Jun Signaling Pathway Involved in Cervical Tumorigenesis.
Journal of cellular physiology
Cervical cancer remains a significant global health concern. KIF18A, a kinesin motor protein regulating microtubule dynamics during mitosis, is frequently overexpressed in various cancers, but its regulatory mechanisms are poorly understood. This study investigates KIF18A's role in cervical cancer and its regulation by the JNK1/c-Jun signaling pathway. Cell growth was assessed in vitro using MTT and colony formation assays, and in vivo using a nude mouse xenograft model with KIF18A knockdown HeLa cells. The Genomic Data Commons (GDC) data portal was used to identify KIF18A-related protein kinases in cervical cancer. Western blot analysis was employed to analyze phosphor-c-Jun, c-Jun, and KIF18A expression levels following JNK1 inhibition, c-Jun knockdown/overexpression, and KIF18A knockdown in cervical cancer cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to assess c-Jun binding and transcriptional activity of the KIF18A promoter. KIF18A knockdown significantly impaired cervical cancer cell growth both in vitro and in vivo. A strong positive correlation was observed between JNK1 and KIF18A expression in cervical and other cancers. JNK1 inhibition decreased both KIF18A expression and c-Jun phosphorylation. c-Jun was found to directly bind to and activate the KIF18A promoter. Furthermore, c-Jun knockdown inhibited cervical cancer cell growth, and this effect was partially rescued by KIF18A overexpression. This study demonstrates that the JNK1/c-Jun pathway activates KIF18A expression, which is essential for cervical cancer cell growth. Targeting the JNK/c-Jun/KIF18A axis may represent a promising novel therapeutic strategy for cancer treatment.
10.1002/jcp.31516
Transforming an ATP-dependent enzyme into a dissipative, self-assembling system.
Nature chemical biology
Nucleoside triphosphate (NTP)-dependent protein assemblies such as microtubules and actin filaments have inspired the development of diverse chemically fueled molecular machines and active materials but their functional sophistication has yet to be matched by design. Given this challenge, we asked whether it is possible to transform a natural adenosine 5'-triphosphate (ATP)-dependent enzyme into a dissipative self-assembling system, thereby altering the structural and functional mode in which chemical energy is used. Here we report that FtsH (filamentous temperature-sensitive protease H), a hexameric ATPase involved in membrane protein degradation, can be readily engineered to form one-dimensional helical nanotubes. FtsH nanotubes require constant energy input to maintain their integrity and degrade over time with the concomitant hydrolysis of ATP, analogous to natural NTP-dependent cytoskeletal assemblies. Yet, in contrast to natural dissipative systems, ATP hydrolysis is catalyzed by free FtsH protomers and FtsH nanotubes serve to conserve ATP, leading to transient assemblies whose lifetimes can be tuned from days to minutes through the inclusion of external ATPases in solution.
10.1038/s41589-024-01811-1
Palmitoylation-mediated NLRP3 inflammasome activation in teleosts highlights evolutionary divergence in immune regulation.
Zoological research
NLRP3 inflammasome activation is pivotal for cytokine secretion and pyroptosis in response to diverse stimuli, playing a crucial role in innate immunity. While extensively studied in mammals, the regulatory mechanisms governing NLRP3 activation in non-mammalian vertebrates remain largely unexplored. Teleosts, as basal vertebrates, represent an ideal model for exploring the evolutionary trajectory of inflammasome regulation. In this study, ABE assays, confocal microscopy, and biochemical analyses were applied to systematically characterize the mechanisms underlying NLRP3 inflammasome in teleosts, using large yellow croakers ( , ) and zebrafish ( , ) as representative models. Our findings revealed a previously unrecognized palmitoylation-dependent regulatory mechanism essential for teleost NLRP3 activation. Specifically, zDHHC18-mediated palmitoylation at a teleost-specific cysteine residue (C946 in NLRP3, C1037 in NLRP3) was required for the translocation of NLRP3 to the dispersed trans-Golgi network, facilitating its subsequent recruitment to the microtubule-organizing center. This membrane trafficking was crucial for inflammasome assembly and downstream inflammatory responses. These findings provide new insights into the distinct regulatory mechanisms of NLRP3 activation in teleosts, highlighting an evolutionary divergence that contributes to innate immunity adaptation in early vertebrates.
10.24272/j.issn.2095-8137.2024.409
Protein phosphatase 4 is required for centrosome asymmetry in fly neural stem cells.
bioRxiv : the preprint server for biology
Asymmetric cell division is used by stem cells to create diverse cell types while self-renewing the stem cell population. Biased segregation of molecularly distinct centrosomes could provide a mechanism to maintain stem cell fate, induce cell differentiation or both. However, the molecular mechanisms generating molecular and functional asymmetric centrosomes remain incompletely understood. Here, we show that in asymmetrically dividing fly neural stem cells, Protein phosphatase 4 (Pp4) is necessary for correct centrosome asymmetry establishment during mitosis, and microtubule organizing center (MTOC) maintenance in interphase. Using live cell imaging we show that while wild type neural stem cells always maintain one active MTOC, mutant neuroblasts contain two inactive centrioles in interphase. Furthermore, centrosomes of mutant neural stem cells mature in mitosis but fail to correctly transfer the centriolar protein Centrobin (Cnb) from the mother to the daughter centriole. Using superresolution imaging, we find that phosphomimetic Centrobin fails to accurately relocalize in mitosis. We propose that Pp4 regulates the timely relocalization of Cnb in mitosis to establish two molecularly distinct centrosomes. In addition, Pp4 is also necessary to maintain MTOC activity in interphase, ensuring biased centrosome segregation. Mechanistically, Pp4 could regulate centrosome asymmetry by dephosphorylating both Cnb and gamma-Tubulin. SIGNIFICANCE STATEMENT:Asymmetric centrosome segregation occurs in stem cells and has been linked with cell fate decisions. Protein phosphatase 4 (Pp4), a conserved Serine/Threonine phosphatase, regulates centrosome asymmetry in neural stem cells by acting upon gamma tubulin and Centrobin. Pp4 regulates centrosome asymmetry establishment in mitosis and interphase, necessary for biased centrosome segregation.
10.1101/2025.01.15.633270
()-1-(3-(3-Hydroxy-4-Methoxyphenyl)-1-(3,4,5-Trimethoxyphenyl)allyl)-1-1,2,4-Triazole and Related Compounds: Their Synthesis and Biological Evaluation as Novel Antimitotic Agents Targeting Breast Cancer.
Pharmaceuticals (Basel, Switzerland)
The synthesis of ()-1-(1,3-diphenylallyl)-1-1,2,4-triazoles and related compounds as anti-mitotic agents with activity in breast cancer was investigated. These compounds were designed as hybrids of the microtubule-targeting chalcones, indanones, and the aromatase inhibitor letrozole. : A panel of 29 compounds was synthesized and examined by a preliminary screening in estrogen receptor (ER) and progesterone receptor (PR)-positive MCF-7 breast cancer cells together with cell cycle analysis and tubulin polymerization inhibition. : ()-5-(3-(1-1,2,4-triazol-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)-2-methoxyphenol was identified as a potent antiproliferative compound with an IC value of 0.39 mM in MCF-7 breast cancer cells, 0.77 mM in triple-negative MDA-MB-231 breast cancer cells, and 0.37 mM in leukemia HL-60 cells. In addition, compound demonstrated potent activity in the sub-micromolar range against the NCI 60 cancer cell line panel including prostate, melanoma, colon, leukemia, and non-small cell lung cancers. G/M phase cell cycle arrest and the induction of apoptosis in MCF-7 cells together with inhibition of tubulin polymerization were demonstrated. Immunofluorescence studies confirmed that compound targeted tubulin in MCF-7 cells, while computational docking studies predicted binding conformations for in the colchicine binding site of tubulin. Compound also selectively inhibited aromatase. : Based on the results obtained, these novel compounds are suitable candidates for further investigation as antiproliferative microtubule-targeting agents for breast cancer.
10.3390/ph18010118
Regulatory roles of eugenol in paraquat-altered SNCA/LZTS3/MAPT in the cerebellum of Wistar rats.
Laboratory animal research
BACKGROUND:The Microtubules-associated protein tau (MAPT), alpha-synuclein (SNCA), and leucine zipper tumor suppressor 3 (LZTS3) genes are implicated in neurodegeneration and tumor suppression, respectively. This study investigated the regulatory roles of eugenol on paraquat-altered genes. RESULTS:Forty male Wistar rats divided into five groups of eight rats were used. The control group received normal saline; the Paraquat (PQ)-untreated group received only Paraquat. The low dose of eugenol was 200 mg/kg, the medium dose of eugenol was 400 mg/kg, and the high dose of eugenol was 600 mg/kg. All groups except the control group received 10 mg/kg of PQ orally for 14 days at one-day intervals, allowing PQ in the rats for 28 days. Eugenol treatment started on the 29th and lasted 14 days. Motor impairments were determined using wire string and beam-walk; biomarkers were estimated using cerebellar homogenates, while frozen cerebellum was used to study LZTS3, MAPT, and SNCA gene expression. LZTS3 was significantly suppressed in the PQ-untreated group and highly expressed in the eugenol-treated group. The MAPT and SNCA genes were overexpressed in the PQ-untreated group compared to the control group. Eugenol significantly decreased the expression of these genes compared to that in the PQ-untreated group. Antioxidants were reduced considerably, and oxidative stress markers were increased significantly, which could have caused increased protein fibrillation and reduced limb functionality. Histology revealed that eugenol mitigated the alterations caused by Paraquat. CONCLUSIONS:PQ can enhance tumor expression in addition to causing neurotoxicity, which decreases limb functionality, while eugenol, an antioxidant, can mitigate the effects of PQ.
10.1186/s42826-025-00236-8
TCTEX1D2 is essential for sperm flagellum formation in mice.
Scientific reports
Flagella and cilia are widely conserved motile structures, in mammalian, sperm possess flagella. Large protein complexes called dynein, including cytoplasmic dynein 2 and axonemal dynein, play a role in the formation of cilia and flagella. The function of each subunit component of dynein complexes in sperm flagellum formation remains unclear. One such subunit is TCTEX1D2. Co-immunoprecipitation studies showed that TCTEX1D2 interacted with cytoplasmic dynein 2 subunits WDR34, WDR60, and DYNLT1 in the testes. Furthermore, TCTEX1D2 also interacted with WDR63 and WDR78, subunits of inner dynein arm, which is axonemal dynein. Tctex1d2 mice generated in this study exhibited male infertility due to flagellar dysplasia, and the axonemal structures were disrupted inside the flagella. Further, the localization of cytoplasmic dynein 2 subunits was abnormal in in Tctex1d2 mice. In contrast, the motile cilia of Tctex1d2 mice were normal. Overall, we revealed that TCTEX1D2 is important for the assembly of cytoplasmic dynein 2 and inner dynein arm and functions in two distinct dynein complexes during mouse sperm flagellum formation. This is only in sperm flagellum formation, not in cilia formation.
10.1038/s41598-024-83424-1
Respiratory failure as main presentation sign of MAPT-related disorder.
Journal of neurology
INTRODUCTION:The MAPT gene encodes Tau, a protein mainly expressed by neurons. Tau protein plays an important role in cerebral microtubule polymerization and stabilization, in axonal transport and synaptic plasticity. Heterozygous pathogenic variation in MAPT are involved in a spectrum of autosomal dominant neurodegenerative diseases known as taupathies, including Alzheimer's disease, Pick's disease, fronto-temporal dementia, cortico-basal degeneration and progressive supranuclear palsy. Taupathies are characterized by the constant presence of neuronal and/or glial aberrant Tau inclusions leading to atrophy and subsequent neuronal loss resulting in central nervous system degeneration. We report here two unrelated families in which segregates a MAPT-related neurodegenerative disorder marked by respiratory failure in the foreground. RESULTS:Nine individuals from two unrelated families were affected by a neurodegenerative disorder. Respiratory features were progressively worsening dyspnea-orthopnea with episodes of acute respiratory decompensation leading to hypercapnic coma or sudden death. A diaphragmatic paralysis was shown in three cases. Associated neurological signs were gait disturbances, bulbar signs including swallowing disorders and dysarthria, pyramidal signs, cognitive and behavioral disorders. ENMG inconstantly found signs of mild denervation. Post-mortem brain immuno-histochemical analysis in one patient revealed unusual composite neuronal Tau inclusions, significant neuronal loss and reactive gliosis, in cortical and subcortical regions, cranial nerves and anterior horn of spinal cord. The heterozygous missense variant c.2041C > T, p. (Pro681Ser) in MAPT was identified in both families by gene panel or exome sequencing. DISCUSSION:In the literature, four additional related patients carrying the same MAPT variant, in heterozygous state, also presented rapidly progressive respiratory failure and unusual composite neuronal Tau inclusions in anterior horn of spinal cord. CONCLUSION:Our observation allows to extend the phenotypic spectrum associated with MAPT variants describing a rapidly progressive respiratory failure, with episodes of exacerbations and premature death.
10.1007/s00415-024-12759-6
Brain-derived tau oligomer polymorphs: distinct aggregations, stability profiles, and biological activities.
Communications biology
Aggregation of microtubule-associated tau protein is a distinct hallmark of several neurodegenerative disorders such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and progressive supranuclear palsy (PSP). Tau oligomers are suggested to be the primary neurotoxic species that initiate aggregation and propagate prion-like structures. Furthermore, different diseases are shown to have distinct structural characteristics of aggregated tau, denoted as polymorphs. Here, we investigate the structural and functional differences of amplified brain-derived tau oligomers (aBDTOs) from AD, DLB, and PSP. Our results indicate that the aBDTOs possess different structural and morphological features that impact neuronal function, gene regulation, and ultimately disease progression. The distinct tau oligomeric polymorphs may thus contribute to the development of clinical phenotypes and shape the progression of diseases. Our results can provide insight into developing personalized therapy to target a specific neurotoxic tau polymorph.
10.1038/s42003-025-07499-w
Host factor DIAPH1 regulates pseudorabivirus replication by modulating the dynamics of cytoskeleton.
International journal of biological macromolecules
As obligate parasites, viruses exploit host cell organelles and molecular components to complete their life cycle. Among which, viruses firstly hijack the cytoskeleton of host cells to ensure their efficiently cell entry and replication. Although formin family members play a key role in both microfilament and microtubule cytoskeletal remodeling, few studies addressed the detailed function and mechanism of formins in the process of viral infection. Here, we showed that sus scrofa DIAPH1 was involved in the regulation of cytoskeletal dynamics during PRV replication. Firstly, we found that DIAPH1 showed significant changes in the expression level and intracellular localization during PRV infection of PK-15 cells. Next, inhibition of DIAPH1 by RNA interference or small molecular inhibitor SMIFH2 was found to diminish the outcome of PRV infection. Besides, DIAPH1 partially co-localized with actin and tubulin in PRV-infected cells. Cross-talk occurred between microfilaments and microfilaments, which also had an influence on the intracellular localization of DIAPH1. What's more, inhibition of DIAPH1 induced the reorganization of microfilament and the stability of microtubule. These results suggested that DIAPH1 regulated PRV infection by remodeling microfilament and microtubule cytoskeletal dynamics.
10.1016/j.ijbiomac.2025.140112
Effect of Cytoskeletal Linker Protein GAS2L1 on Oligodendrocyte and Myelin Development.
Glia
Oligodendrocytes (OLs), the myelin-forming cells of the central nervous system (CNS), develop from OL precursor cells (OPCs) through a complex process involving significant morphological changes that are critically dependent on the dynamic interactions between cytoskeletal networks. Growth arrest-specific 2-like protein 1 (GAS2L1) is a cytoskeletal linker protein that mediates the cross-talk between actin filaments and microtubules. However, its role in OL and myelin development remains unknown. Here, we report that GAS2L1 is expressed in both OPCs and mature OLs, and that overexpression or knockdown of Gas2l1 in cultured OPCs in vitro impaired or enhanced their differentiation, respectively, while both inhibited their proliferation. We generated a Gas2l1 mouse line and found that mice with conditional knockout of Gas2l1 in OL lineage cells (Olig1-Cre;Gas2l1 , cKO) showed an increased number of mature OLs and enhanced myelination, as well as a reduction in the branching complexity of OPCs. In addition, an alternative mouse line with postnatally induced Gas2l1 ablation specifically in OPCs (Pdfgra-CreER ;Gas2l1 , iKO) recapitulated the acceleration of OL and myelin development as well as the inhibition of OPC process branching. Furthermore, EdU tracking in Gas2l1 iKO mice in vivo and in their OPC cultures in vitro showed both a reduction in OPC proliferation and an increase in OL maturation. Finally, cultured OPCs from iKO mice showed an increase in filopodia extension. Taken together, our results demonstrate an effect of GAS2L1 on the regulation of OL/myelin development and may provide a novel potential therapeutic target for various diseases involving OL/myelin pathology.
10.1002/glia.24658
Altered cytoskeleton dynamics in patient-derived iPSC-based model of PCDH19 clustering epilepsy.
Frontiers in cell and developmental biology
Protocadherin 19 (PCDH19) is an adhesion molecule involved in cell-cell interaction whose mutations cause a drug-resistant form of epilepsy, named PCDH19-Clustering Epilepsy (PCDH19-CE, MIM 300088). The mechanism by which altered PCDH19 function drive pathogenesis is not yet fully understood. Our previous work showed that PCDH19 dysfunction is associated with altered orientation of the mitotic spindle and accelerated neurogenesis, suggesting a contribution of altered cytoskeleton organization in PCDH19-CE pathogenesis in the control of cell division and differentiation. Here, we evaluate the consequences of altered PCDH19 function on microfilaments and microtubules organization, using a disease model obtained from patient-derived induced pluripotent stem cells. We show that iPSC-derived cortical neurons are characterized by altered cytoskeletal dynamics, suggesting that this protocadherin has a role in modulating stability of MFs and MTs. Consistently, the levels of acetylated-tubulin, which is related with stable MTs, are significantly increased in cortical neurons derived from the patient's iPSCs compared to control cells, supporting the idea that the altered dynamics of the MTs depends on their increased stability. Finally, performing live-imaging experiments using fluorescence recovery after photobleaching and by monitoring GFP-tagged end binding protein 3 (EB3) "comets," we observe an impairment of the plus-end polymerization speed in PCDH19-mutated cortical neurons, therefore confirming the impaired MT dynamics. In addition to altering the mitotic spindle formation, the present data unveil that PCDH19 dysfunction leads to altered cytoskeletal rearrangement, providing therapeutic targets and pharmacological options to treat this disorder.
10.3389/fcell.2024.1518533
Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages.
eLife
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
10.7554/eLife.85998
The adaptor protein Miro1 modulates horizontal transfer of mitochondria in mouse melanoma models.
Cell reports
Recent research has shown that mtDNA-deficient cancer cells (ρ cells) acquire mitochondria from tumor stromal cells to restore respiration, facilitating tumor formation. We investigated the role of Miro1, an adaptor protein involved in movement of mitochondria along microtubules, in this phenomenon. Inducible Miro1 knockout (Miro1) mice markedly delayed tumor formation after grafting ρ cancer cells. Miro1 mice with fluorescently labeled mitochondria revealed that this delay was due to hindered mitochondrial transfer from the tumor stromal cells to grafted B16 ρ cells, which impeded recovery of mitochondrial respiration and tumor growth. Miro1 led to the perinuclear accumulation of mitochondria and impaired mobility of the mitochondrial network. In vitro experiments revealed decreased association of mitochondria with microtubules, compromising mitochondrial transfer via tunneling nanotubes (TNTs) in mesenchymal stromal cells. Here we show the role of Miro1 in horizontal mitochondrial transfer in mouse melanoma models in vivo and its involvement with TNTs.
10.1016/j.celrep.2024.115154
Plinabulin exerts an anti-proliferative effect via the PI3K/AKT/mTOR signaling pathways in glioblastoma.
Iranian journal of basic medical sciences
Objectives:Plinabulin, a marine-derived anticancer drug targeting microtubules, exhibits anti-cancer effects on glioblastoma cells. However, its therapeutic potential, specifically for glioblastoma treatment, remains underexplored. This study aims to elucidate the mechanisms by which plinabulin exerts its effects on glioblastoma cells. Materials and Methods:Using the SRB and colony formation assay to observe the effect of plinabulin on glioblastoma cell viability. Wound healing and transwell migration assay were used to test the effect of plinabulin on glioblastoma cell metastatic potential. Crucial target genes were identified through RNA sequencing and bioinformatics analysis. Protein levels were evaluated in a concentration-dependent manner using western blot analysis. Results:Plinabulin suppressed glioblastoma cell proliferation by causing cell cycle G2/M phase arrest and inhibited migration. The IC50 values were 22.20 nM in A172 cells and 20.55 nM in T98G cells. Plinabulin reduced AKT and mTOR phosphorylation. Combined with the AKT/mTOR inhibitors LY294002 and rapamycin, plinabulin decreased p-mTOR and EGFR protein levels and increased cleaved-PARP levels. Plinabulin induces autophagy, and using an autophagy inhibitor enhances plinabulin-induced cell apoptosis. This suggests that plinabulin might trigger cytoprotective autophagy in glioblastoma cells. These findings indicate that plinabulin hinders glioblastoma growth and induces protective autophagy via the PI3K/AKT/mTOR pathway. Additionally, plinabulin combined with erlotinib showed greater cytotoxic efficacy than either drug alone in glioblastoma cells . Conclusion:Our study provides new insights into the efficacy of plinabulin against glioblastoma and highlights the potential clinical utility of combining plinabulin with EGFR inhibitors as a chemotherapy strategy.
10.22038/ijbms.2024.79406.17200
Molecular switch of the dendrite-to-spine transport of TDP-43/FMRP-bound neuronal mRNAs and its impairment in ASD.
Cellular & molecular biology letters
BACKGROUND:Regulation of messenger RNA (mRNA) transport and translation in neurons is essential for dendritic plasticity and learning/memory development. The trafficking of mRNAs along the hippocampal neuron dendrites remains translationally silent until they are selectively transported into the spines upon glutamate-induced receptor activation. However, the molecular mechanism(s) behind the spine entry of dendritic mRNAs under metabotropic glutamate receptor (mGluR)-mediated neuroactivation and long-term depression (LTD) as well as the fate of these mRNAs inside the spines are still elusive. METHOD:Different molecular and imaging techniques, e.g., immunoprecipitation (IP), RNA-IP, Immunofluorescence (IF)/fluorescence in situ hybridization (FISH), live cell imaging, live cell tracking of RNA using beacon, and mouse model study are used to elucidate a novel mechanism regulating dendritic spine transport of mRNAs in mammalian neurons. RESULTS:We demonstrate here that brief mGluR1 activation-mediated dephosphorylation of pFMRP (S499) results in the dissociation of FMRP from TDP-43 and handover of TDP-43/Rac1 mRNA complex from the dendritic transport track on microtubules to myosin V track on the spine actin filaments. Rac1 mRNA thus enters the spines for translational reactivation and increases the mature spine density. In contrast, during mGluR1-mediated neuronal LTD, FMRP (S499) remains phosphorylated and the TDP-43/Rac1 mRNA complex, being associated with kinesin 1-FMRP/cortactin/drebrin, enters the spines owing to Ca-dependent microtubule invasion into spines, but without translational reactivation. In a VPA-ASD mouse model, this regulation become anomalous. CONCLUSIONS:This study, for the first time, highlights the importance of posttranslational modification of RBPs, such as the neurodevelopmental disease-related protein FMRP, as the molecular switch regulating the dendrite-to-spine transport of specific mRNAs under mGluR1-mediated neurotransmissions. The misregulation of this switch could contribute to the pathogenesis of FMRP-related neurodisorders including the autism spectrum disorder (ASD). It also could indicate a molecular connection between ASD and neurodegenerative disease-related protein TDP-43 and opens up a new perspective of research to elucidate TDP-43 proteinopathy among patients with ASD.
10.1186/s11658-024-00684-5
KIF1C activates and extends dynein movement through the FHF cargo adapter.
Nature structural & molecular biology
Cellular cargos move bidirectionally on microtubules by recruiting opposite polarity motors dynein and kinesin. These motors show codependence, where one requires the activity of the other, although the mechanism is unknown. Here we show that kinesin-3 KIF1C acts as both an activator and a processivity factor for dynein, using in vitro reconstitutions of human proteins. Activation requires only a fragment of the KIF1C nonmotor stalk binding the cargo adapter HOOK3. The interaction site is separate from the constitutive factors FTS and FHIP, which link HOOK3 to small G-proteins on cargos. We provide a structural model for the autoinhibited FTS-HOOK3-FHIP1B (an FHF complex) and explain how KIF1C relieves it. Collectively, we explain codependency by revealing how mutual activation of dynein and kinesin occurs through their shared adapter. Many adapters bind both dynein and kinesins, suggesting this mechanism could be generalized to other bidirectional complexes.
10.1038/s41594-024-01418-z
Non-nucleosomal (CENP-A/H4) - DNA complexes as a possible platform for centromere organization.
bioRxiv : the preprint server for biology
The centromere is a part of the chromosome that is essential for the even segregation of duplicated chromosomes during cell division. It is epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A associates specifically with a group of 16 proteins that form the centromere-associated network of proteins (CCAN). In mitosis, the kinetochore forms on the CCAN to connect the duplicated chromosomes to the microtubules protruding from the cell poles. Previous studies have shown that CENP-A replaces H3 in nucleosomes, and recently the structures of CENP-A-containing nucleosomes in complex with CCANs have been revealed, but they show only a limited interaction between CCANs and CENP-A. Here, we report the cryoEM structure of 2x(CENP-A/H4)-di-tetramers assembled on DNA in the absence of H2A/H2B histone dimer and speculate how (CENP-A/H4)-tetramers and -di-tetramers might serve as a platform for CCAN organization.
10.1101/2024.12.31.630874
Peri-centrosomal localization of small interfering RNAs in C. elegans.
Science China. Life sciences
The centrosome is the microtubule-organizing center and a crucial part of cell division. Centrosomal RNAs (cnRNAs) have been reported to enable precise spatiotemporal control of gene expression during cell division in many species. Whether and how cnRNAs exist in C. elegans are unclear. Here, using the nuclear RNAi Argonaute protein NRDE-3 as a reporter, we observed potential peri-centrosome localized small interfering (si)RNAs in C. elegans. NRDE-3 was previously shown to associate with pre-mRNAs and pre-rRNAs via a process involving the presence of complementary siRNAs. We generated a GFP-NRDE-3 knock-in transgene through CRISPR/Cas9 technology and observed that NRDE-3 formed peri-centrosomal foci neighboring the tubulin protein TBB-2, other centriole proteins and pericentriolar material (PCM) components in C. elegans embryos. The peri-centrosomal accumulation of NRDE-3 depends on RNA-dependent RNA polymerase (RdRP)-synthesized 22G siRNAs and the PAZ domain of NRDE-3, which is essential for siRNA binding. Mutation of eri-1, ergo-1, or drh-3 significantly increased the percentage of pericentrosome-enriched NRDE-3. At the metaphase of the cell cycle, NRDE-3 was enriched in both the peri-centrosomal region and the spindle. Moreover, the integrity of centriole proteins and pericentriolar material (PCM) components is also required for the peri-centrosomal accumulation of NRDE-3. Therefore, we concluded that siRNAs could accumulate in the pericentrosomal region in C. elegans and suggested that the peri-centrosomal region may also be a platform for RNAi-mediated gene regulation.
10.1007/s11427-024-2818-7
The Role of Autophagy in Copper-Induced Apoptosis and Developmental Neurotoxicity in SH-SY5Y Cells.
Toxics
Copper (Cu) is a global environmental pollutant that poses a serious threat to humans and ecosystems. Copper induces developmental neurotoxicity, but the underlying molecular mechanisms are unknown. Neurons are nonrenewable, and they are unable to mitigate the excessive accumulation of pathological proteins and organelles in cells, which can be ameliorated by autophagic degradation. In this study, we established an in vitro model of Cu-exposed (0, 15, 30, 60 and 120 μM) SH-SY5Y cells to explore the role of autophagy in copper-induced developmental neurotoxicity. The results showed that copper resulted in the reduction and shortening of neural synapses in differentiated cultured SH-SY5Y cells, a downregulated Wnt signaling pathway, and nuclear translocation of β-catenin. Exposure to Cu increased autophagosome accumulation and autophagic flux blockage in terms of increased sequestosome 1 (p62/SQSTM1) and microtubule-associated protein 1 light chain 3B (LC3B) II/LC3BI expressions and inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. Furthermore, copper induced apoptosis, characterized by increased expressions of Bcl2 X protein (Bax), caspase 3, and Poly (ADP-ribose) polymerase (PARP) and decreased expression of B-cell lymphoma 2 (Bcl2). Compared with the 120 μM Cu exposure group alone, autophagy activator rapamycin pretreatment increased expression of Wnt and β-catenin nuclear translocation, decreased expression of LC3BII/LC3BI and p62, as well as upregulated expression of Bcl2 and downregulated expressions of caspase 3 and PARP. In contrast, after autophagy inhibitor chloroquine pretreatment, expressions of Wnt and β-catenin nuclear translocation were decreased, expression levels of LC3BII/LC3BI and p62 were upregulated, expression of Bcl2 was decreased, while expression levels of caspase 3, Bax, and PARP were increased. In conclusion, the study demonstrated that autophagosome accumulation and autophagic flux blockage were associated with copper-induced developmental neurotoxicity via the Wnt signaling pathway, which might deepen the understanding of the developmental neurotoxicity mechanism of environmental copper exposure.
10.3390/toxics13010063
Structural insights into the role of the proline rich region in tau function.
Structure (London, England : 1993)
Tau plays an important role in modulating axonal microtubules in neurons, while intracellular tau aggregates are found in many neurodegenerative disorders. Tubulin binding sites are found in tau's proline-rich region (PRR), microtubule binding repeats (MTBRs), and pseudo-repeat (R'). Tau phosphorylation sites, which cluster with high frequency within the PRR, regulate tubulin interactions and correlates with disease. Here, we use fluorescence correlation spectroscopy and structural mass spectrometry techniques to characterize the impact of phosphomimic mutations in the PRR on tau function. We find that phosphomimics cumulatively diminish tubulin dimer binding and slow microtubule polymerization. Additionally, we map two ∼15 residue regions of the PRR as primary tubulin dimer binding sites and propose a model in which PRR enhances lateral interactions between tubulin dimers, complementing the longitudinal interactions observed for MTBR. Our study provides insight into the previously overlooked relevance of tau's PRR in functional interactions with tubulin dimers.
10.1016/j.str.2024.12.017
The kinesin Kar3 is required for endoplasmic reticulum-associated degradation.
Molecular biology of the cell
Degradation of aberrant, excess, and regulatory proteins at the endoplasmic reticulum (ER) is a conserved feature of eukaryotic cells, disruption of which contributes to disease. While remarkable progress has been made in recent years, mechanisms and genetic requirements for ER-associated degradation (ERAD) remain incompletely understood. We recently conducted a screen for genes required for turnover of a model ER translocon-associated substrate of the Hrd1 ubiquitin ligase in . This screen revealed loss of Kar3 impedes degradation of *-Sec62, which persistently and aberrantly engages the translocon. Kar3 is a microtubule-associated kinesin 14 family member that impacts multiple aspects of microtubule dynamics during cell division and karyogamy. We investigated involvement of Kar3 and its cofactors in ERAD. Loss of Kar3 hindered ERAD mediated by three ubiquitin ligases but did not impair turnover of a soluble nuclear protein. Further, deletion caused hypersensitivity to conditions associated with proteotoxic stress. Kar3's cytoplasmic cofactor Vik1 was also required for efficient degradation of *-Sec62. Our results reveal a profound and underappreciated role for microtubule-associated proteins in ERAD.
10.1091/mbc.E24-10-0437
A nucleotide code governs Lis1's ability to relieve dynein autoinhibition.
bioRxiv : the preprint server for biology
Dynein-1 is a microtubule motor responsible for the transport of cytoplasmic cargoes. Activation of motility requires it first overcome an autoinhibited state prior to its assembly with dynactin and a cargo adaptor. Studies suggest that Lis1 may relieve dynein's autoinhibited state. However, evidence for this mechanism is lacking. We first set out to determine the rules governing dynein-Lis1 binding, which reveals that their binding affinity is regulated by the nucleotide-bound states of each of three nucleotide-binding pockets within the dynein motor domain. We also find that distinct nucleotide 'codes' coordinate dynein-Lis1 binding stoichiometry by impacting binding affinity at two different sites within the dynein motor domain. Electron microscopy reveals that a 1 Lis1:1 dynein complex directly promotes an open, uninhibited conformational state of dynein, whereas a 2:1 complex resembles the autoinhibited state. Cryo-EM analysis reveals the structural basis for Lis1 opening dynein relies on interactions with the linker domain.
10.1101/2024.12.30.630615
Cytoplasmic flow is a cell size sensor that scales anaphase.
Nature cell biology
During early embryogenesis, fast mitotic cycles without interphase lead to a decrease in cell size, while scaling mechanisms must keep cellular structures proportional to cell size. For instance, as cells become smaller, if the position of nuclear envelope reformation (NER) did not adapt, NER would have to occur beyond the cell boundary. Here we found that NER position in anaphase scales with cell size via changes in chromosome motility, mediated by cytoplasmic flows that themselves scale with cell size. Flows are a consequence of friction between viscous cytoplasm and bulky cargo transported by dynein on astral microtubules. As an emerging property, confinement in cells of different sizes yields scaling of cytoplasmic flows. Thus, flows behave like a cell geometry sensor: astral microtubules approach the boundary causing flow velocity changes, which then affect the velocity of chromosome separation, thus scaling NER.
10.1038/s41556-024-01605-6
Classical cell cycle kinase limits tubulin polyglutamylation and cilium stability.
The Journal of cell biology
Tubulin polyglutamylation is essential for maintaining cilium stability and function, and defective tubulin polyglutamylation is associated with ciliopathies. However, the regulatory mechanism underlying proper axonemal polyglutamylation remains unclear. He et al. (https://doi.org/10.1083/jcb.202405170) discovered that Cdk7/Cdk6/FIP5 phosphorylation cascade controls the ciliary import of tubulin glutamylases, thereby modulating axoneme polyglutamylation and ciliary signaling.
10.1083/jcb.202412034
Cell adhesion and spreading on fluid membranes through microtubules-dependent mechanotransduction.
Nature communications
Integrin clusters facilitate mechanical force transmission (mechanotransduction) and regulate biochemical signaling during cell adhesion. However, most studies have focused on rigid substrates. On fluid substrates like supported lipid bilayers (SLBs), integrin ligands are mobile, and adhesive complexes are traditionally thought unable to anchor for cell spreading. Here, we demonstrate that cells spread on SLBs coated with Invasin, a high-affinity integrin ligand. Unlike SLBs functionalized with RGD peptides, integrin clusters on Invasin-SLBs grow in size and complexity comparable to those on glass. While actomyosin contraction dominates adhesion maturation on stiff substrates, we find that on fluid SLBs, integrin mechanotransduction and cell spreading rely on dynein pulling forces along microtubules perpendicular to the membranes and microtubules pushing on adhesive complexes, respectively. These forces, potentially present on non-deformable surfaces, are revealed in fluid substrate systems. Supported by a theoretical model, our findings demonstrate a mechanical role for microtubules in integrin clustering.
10.1038/s41467-025-56343-6
Tubulin detyrosination shapes cytoskeletal architecture and virulence.
Proceedings of the National Academy of Sciences of the United States of America
Tubulin detyrosination has been implicated in various human disorders and is important for regulating microtubule dynamics. While in most organisms this modification is restricted to α-tubulin, in trypanosomatid parasites, it occurs on both α- and β-tubulin. Here, we show that in , a single vasohibin (LmVASH) enzyme is responsible for differential kinetics of α- and β-tubulin detyrosination. LmVASH knockout parasites, which are completely devoid of detyrosination, show decreased levels of glutamylation and exhibit a strongly diminished pathogenicity in mice, correlating with decreased proliferation in macrophages. Reduced virulence is associated with altered morphogenesis and flagellum remodeling in detyrosination-deficient amastigotes. Flagellum shortening in the absence of detyrosination is caused by hyperactivity of a microtubule-depolymerizing Kinesin-13 homolog, demonstrating its function as a key reader of the trypanosomatid-tubulin code. Taken together, our work establishes the importance of tubulin detyrosination in remodeling the microtubule-based cytoskeleton required for efficient proliferation in the mammalian host. This highlights tubulin detyrosination as a potential target for therapeutic action against leishmaniasis.
10.1073/pnas.2415296122
BicD and MAP7 collaborate to activate homodimeric kinesin-1 by complementary mechanisms.
bioRxiv : the preprint server for biology
The folded auto-inhibited state of kinesin-1 is stabilized by multiple weak interactions and binds weakly to microtubules. Here we investigate the extent to which homodimeric kinesin-1 lacking light chains is activated by the dynein activating adaptor BicD. We show that one or two kinesins can bind to the central region of BicD (CC2), a region distinct from that which binds dynein-dynactin (CC1) and cargo-adaptor proteins (CC3). Kinesin light chain significantly reduces the amount of kinesin bound to BicD and thus regulates this interaction. Binding of kinesin to BicD increases the number of motors bound to the microtubule, the fraction moving processively and the run length, suggesting that BicD relieves kinesin auto-inhibition. In contrast, microtubule-associated protein 7 (MAP7) has minimal impact on the percentage of motors moving processively but enhances both kinesin-1 recruitment to microtubules and run length. BicD relieves auto-inhibition of kinesin, while MAP7 enables activated motors to engage productively with microtubules. When BicD and MAP7 are combined, the most robust activation of kinesin-1 occurs, highlighting the crosstalk between adaptors and microtubule associated proteins in regulating transport. These observations imply that when both dynein and kinesin-1 are simultaneously bound to BicD, the direction the complex moves on MTs will be influenced by MAP7 and the number of bound kinesins.
10.1101/2025.01.11.632512
Satellite DNA shapes dictate pericentromere packaging in female meiosis.
Nature
The abundance and sequence of satellite DNA at and around centromeres is evolving rapidly despite the highly conserved and essential process through which the centromere directs chromosome inheritance. The impact of such rapid evolution is unclear. Here we find that sequence-dependent DNA shape dictates packaging of pericentromeric satellites in female meiosis through a conserved DNA-shape-recognizing chromatin architectural protein, high mobility group AT-hook 1 (HMGA1). Pericentromeric heterochromatin in two closely related mouse species, M. musculus and M. spretus, forms on divergent satellites that differ by both density of narrow DNA minor grooves and HMGA1 recruitment. HMGA1 binds preferentially to M. musculus satellites, and depletion in M. musculus oocytes causes massive stretching of pericentromeric satellites, disruption of kinetochore organization and delays in bipolar spindle assembly. In M. musculus × spretus hybrid oocytes, HMGA1 depletion disproportionately impairs M. musculus pericentromeres and microtubule attachment to their kinetochores. Thus, DNA shape affects both pericentromere packaging and the segregation machinery. We propose that rapid evolution of centromere and pericentromere DNA does not disrupt these essential processes when the satellites adopt DNA shapes recognized by conserved architectural proteins (such as HMGA1). By packaging these satellites, architectural proteins become part of the centromeric and pericentromeric chromatin, suggesting an evolutionary strategy that lowers the cost of megabase-scale satellite expansion.
10.1038/s41586-024-08374-0
CLASP1/2 REGULATE IMMUNE SYNAPSE MATURATION IN NATURAL KILLER CELLS.
bioRxiv : the preprint server for biology
Natural killer (NK) cells are the first line of defense against viral infections and tumors. Their cytotoxic activity relies on the formation of an immune synapse (IS) with target cells. The lymphocyte function-associated antigen (LFA)-1 plays a central role in NK cell cytotoxicity by modulating NK-IS assembly and maturation. LFA-1 organization at the IS involves a Golgi-dependent mechanism, which has not been completely elucidated. CLIP-associating proteins (CLASP) 1/2 are microtubule plus-tip interacting proteins that control the dynamics of Golgi derived microtubules (GDMTs). In the present study, we found that CLASP1/2 depletion impaired LFA-1 organization at the IS and inhibited the polarization of the centrosome and the lytic granules towards the target cell. Our results also revealed the role of the Golgi apparatus as a microtubule organizing center (MTOC) in these cells. Furthermore, we found that, similarly to what was described in other cell types, NK cells require CLASP1/2 and AKAP350 for efficient nucleation of microtubules at the Golgi. Overall, this study uncovers the role of CLASP1/2 in the maturation of the lytic IS in NK cells, and presents evidence supporting the contribution of GDMTs in this process. Summary sentence:The Golgi apparatus (GA) functions as a microtubule-organizing center (MTOC) in NK cells. During the recognition of tumoral cells by NK cells, CLASP1/2-mediated stabilization of GA-derived microtubules (GDMTs) facilitates vesicular LFA-1 (LFA-1 ) trafficking toward the interaction surface, thereby promoting the immune synapse (IS) maturation.
10.1101/2025.01.20.633904
Structural diversity of axonemes across mammalian motile cilia.
Nature
Reproduction, development and homeostasis depend on motile cilia, whose rhythmic beating is powered by a microtubule-based molecular machine called the axoneme. Although an atomic model of the axoneme is available for the alga Chlamydomonas reinhardtii, structures of mammalian axonemes are incomplete. Furthermore, we do not fully understand how molecular structures of axonemes vary across motile-ciliated cell types in the body. Here we use cryoelectron microscopy, cryoelectron tomography and proteomics to resolve the 96-nm modular repeat of axonemal doublet microtubules (DMTs) from both sperm flagella and epithelial cilia of the oviduct, brain ventricles and respiratory tract. We find that sperm DMTs are the most specialized, with epithelial cilia having only minor differences across tissues. We build a model of the mammalian sperm DMT, defining the positions and interactions of 181 proteins including 34 newly identified proteins. We elucidate the composition of radial spoke 3 and uncover binding sites of kinases associated with regeneration of ATP and regulation of ciliary motility. We discover a sperm-specific, axoneme-tethered T-complex protein ring complex (TRiC) chaperone that may contribute to construction or maintenance of the long flagella of mammalian sperm. We resolve axonemal dyneins in their prestroke states, illuminating conformational changes that occur during ciliary movement. Our results illustrate how elements of chemical and mechanical regulation are embedded within the axoneme, providing valuable resources for understanding the aetiology of ciliopathy and infertility, and exemplifying the discovery power of modern structural biology.
10.1038/s41586-024-08337-5
Molecular Mechanisms of Alzheimer's Disease Induced by Amyloid-β and Tau Phosphorylation Along with RhoA Activity: Perspective of RhoA/Rho-Associated Protein Kinase Inhibitors for Neuronal Therapy.
Cells
Amyloid-β peptide (Aβ) is a critical cause of Alzheimer's disease (AD). It is generated from amyloid precursor protein (APP) through cleavages by β-secretase and γ-secretase. γ-Secretase, which includes presenilin, is regulated by several stimuli. Tau protein has also been identified as a significant factor in AD. In particular, Tau phosphorylation is crucial for neuronal impairment, as phosphorylated Tau detaches from microtubules, leading to the formation of neurofibrillary tangles and the destabilization of the microtubule structure. This instability in microtubules damages axons and dendrites, resulting in neuronal impairment. Notably, Aβ is linked to Tau phosphorylation. Another crucial factor in AD is neuroinflammation, primarily occurring in the microglia. Microglia possess several receptors that bind with Aβ, triggering the expression and release of an inflammatory factor, although their main physiological function is to phagocytose debris and pathogens in the brain. NF-κB activation plays a major role in neuroinflammation. Additionally, the production of reactive oxygen species (ROS) in the microglia contributes to this neuroinflammation. In microglia, superoxide is produced through NADPH oxidase, specifically NOX2. Rho GTPases play an essential role in regulating various cellular processes, including cytoskeletal rearrangement, morphology changes, migration, and transcription. The typical function of Rho GTPases involves regulating actin filament formation. Neurons, with their complex processes and synapse connections, rely on cytoskeletal dynamics for structural support. Other brain cells, such as astrocytes, microglia, and oligodendrocytes, also depend on specific cytoskeletal structures to maintain their unique cellular architectures. Thus, the aberrant regulation of Rho GTPases activity can disrupt actin filaments, leading to altered cell morphology, including changes in neuronal processes and synapses, and potentially contributing to brain diseases such as AD.
10.3390/cells14020089
Tau reduction impairs nephrocyte function in Drosophila.
BMB reports
Tau, a microtubule-associated protein, is known for its significant involvement in neurodegenerative diseases. While various molecular and immunohistochemical techniques have confirmed the presence of Tau in podocytes, its precise function within these cells remains elusive. In this study, we investigate the role of Tau in kidney podocytes using Drosophila pericardial nephrocytes as a model. We found that knockdown of Drosophila Tau in nephrocytes resulted in apoptotic cell death and the disruption of nephrocyte structure. Furthermore, we observed that decreased Tau levels induced genomic damage and abnormal distribution of γ-H2Av, altering nuclei architecture in nephrocytes, and affecting the nuclear membrane structure by interfering with lamin with aging. Additionally, Tau knockdown led to a reduction in lipid droplets in Drosophila fat body tissues, suggesting a potential role of Tau in inter-organ communication. These findings underscore the importance of Tau in the nephrocytes of Drosophila, and advocate further research to broaden our understanding of podocyte biology in kidney diseases.
A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth.
Nature structural & molecular biology
Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome. We show that CEP104, a protein with a tubulin-binding TOG domain, and its luminal partner CSPP1 inhibit microtubule growth and shortening. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics but act as scaffolds for their partners. Cryo-electron tomography demonstrated that, together, ciliary tip module members form plus-end-specific cork-like structures that reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.
10.1038/s41594-025-01483-y
Tubulin-Binding Region Modulates Cholesterol-Triggered Aggregation of Tau Proteins.
Journal of neurochemistry
A hallmark of Alzheimer disease (AD) and tauopathies, severe neurodegenerative diseases, is the progressive aggregation of Tau, also known as microtubule-associated Tau protein. Full-length Tau, also known as 2N4R, contains two N-terminal inserts that bind to tubulin. This facilitates the self-assembly of tubulin simultaneously enhancing stability of cell microtubules. Other Tau isoforms have one (1N4R) or zero (0N4R) N-terminal inserts, which makes 2N4R Tau more and 0N4R less effective in promoting microtubule self-assembly. A growing body of evidence indicates that lipids can alter the aggregation rate of Tau isoforms. However, the role of N-terminal inserts in Tau-lipid interactions remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which N-terminal inserts alter interactions of Tau isoforms with cholesterol, one of the most important lipids in plasma membranes. Our results showed that 2 N insert prevents amyloid-driven aggregation of Tau at the physiological concentration of cholesterol, while the absence of this N-terminal repeat (1N4R and 0N4R Tau) resulted in the self-assembly of Tau into toxic amyloid fibrils. We also found that the presence of cholesterol in the lipid bilayers caused a significant increase in the cytotoxicity of 1N4R and 0N4R Tau to neurons. This effect was not observed for 2N4R Tau fibrils formed in the presence of lipid membranes with low, physiological, and elevated concentrations of cholesterol. Using molecular assays, we found that Tau aggregates primarily exert cytotoxicity by damaging cell endosomes, endoplasmic reticulum, and mitochondria.
10.1111/jnc.16294
The KNL-1/Knl1 outer kinetochore protein caught regulating F-actin.
The Journal of cell biology
Kinetochores are multiprotein complexes that link chromosomes to microtubules and are essential for chromosome segregation during cell divisions. In this issue, Alves Domingos et al. (https://doi.org/10.1083/jcb.202311147) show that the conserved KNL-1/Knl1 protein of the Knl1/Mis12/Ndc80 (KMN) outer kinetochore complex postmitotically regulates F-actin to shape somatosensory dendrites.
10.1083/jcb.202412191
Transcriptome Analysis Suggests PKD3 Regulates Proliferative Glucose Metabolism, Calcium Homeostasis and Microtubule Dynamics After MEF Spontaneous Immortalization.
International journal of molecular sciences
Cell immortalization corresponds to a biologically relevant clinical feature that allows cells to acquire a high proliferative potential during carcinogenesis. In multiple cancer types, Protein Kinase D3 (PKD3) has often been reported as a dysregulated oncogenic kinase that promotes cell proliferation. Using mouse embryonic fibroblasts (MEFs), in a spontaneous immortalization model, PKD3 has been demonstrated as a critical regulator of cell proliferation after immortalization. However, the mechanisms by which PKD3 regulates proliferation in immortalized MEFs require further elucidation. Using a previously validated -deficient MEF model, we performed a poly-A transcriptomic analysis to identify putative -regulated biological processes and downstream targets in MEFs after spontaneous immortalization. To this end, differentially expressed genes (DEGs) were identified and further analyzed by gene ontology (GO) enrichment and protein-protein interaction (PPI) network analyses to identify potential hub genes. Our results suggest that modulates proliferation through the regulation of gene expression associated with glucose metabolism (, , , ), calcium homeostasis and transport ( and ) and microtubule dynamics ( and ). These candidate processes and associated genes represent potential mechanisms involved in -induced proliferation in spontaneously immortalized cells as well as clinical targets in several cancer types.
10.3390/ijms26020596
PLK2 disrupts autophagic flux to promote SNCA/α-synuclein pathology.
Autophagy
The aggregation and transmission of SNCA/α-synuclein (synuclein, alpha) is a hallmark pathology of Parkinson disease (PD). PLK2 (polo like kinase 2) is an evolutionarily conserved serine/threonine kinase that is more abundant in the brains of all family members, is highly expressed in PD, and is linked to SNCA deposition. However, in addition to its role in phosphorylating SNCA, the role of PLK2 in PD and the mechanisms involved in triggering neurodegeneration remain unclear. Here, we found that PLK2 regulated SNCA pathology independently of S129. Overexpression of PLK2 promoted SNCA preformed fibril (PFF)-induced aggregation of wild-type SNCA and mutant SNCA. Genetic or pharmacological inhibition of PLK2 attenuated SNCA deposition and neurotoxicity. Mechanistically, PLK2 exacerbated the propagation of SNCA pathology by impeding the clearance of SNCA aggregates by blocking macroautophagic/autophagic flux. We further showed that PLK2 phosphorylated S1098 of DCTN1 (dynactin 1), a protein that controls the movement of organelles, leading to impaired autophagosome-lysosome fusion. Furthermore, genetic suppression of PLK2 alleviated SNCA aggregation and motor dysfunction . Our findings suggest that PLK2 negatively regulates autophagy, promoting SNCA pathology, suggesting a role for PLK2 in PD.: AD: Alzheimer disease; AMPK: AMP-activated protein kinase; CASP3: caspase 3; DCTN1: dynactin 1; LBs: lewy bodies; LDH: lactate dehydrogenase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP2: microtubule associated protein 2; MTOR: mechanistic target of rapamycin kinase; NH4Cl: ammonium chloride; p-SNCA: phosphorylation of SNCA at S129; PD: Parkinson disease; PFF: preformed fibril; PI: propidium iodide; PLK2: polo like kinase 2; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit alpha; shRNA: short hairpin RNA; SNCA: synuclein, alpha; SQSTM1/p62: sequestosome 1; TH: tyrosine hydroxylase; TX: Triton X-100; ULK1: unc-51 like autophagy activating kinase 1.
10.1080/15548627.2024.2448914
The influence of the cytoskeleton on the development and behavior of viral factories in mammalian orthoreovirus.
Virology
Cytosolic viral factories (VFs) of mammalian orthoreovirus (MRV) are sites for viral genome replication and assembly of virus progeny. Despite advancements in reverse genetics, the formation and dynamics of VFs still need to be clarified. MRV exploits host cytoskeletal components like microtubules (MTs) throughout its life cycle, including cell entry, replication, and release. MRV VFs, membrane-less cytosolic inclusions, rely on the viral proteins μ2 and μNS for formation. Protein μ2 interacts and stabilizes MTs through acetylation, supporting VF formation and viral replication, while scaffold protein μNS influences cellular components to aid VF maturation. The disruption of the MT network reduces viral replication, underscoring its importance. Additionally, μ2 associates with MT-organizing centers, modulating the MT dynamics to favor viral replication. In summary, MRV subverts the cytoskeleton to facilitate VF dynamics and promote viral replication and assembly to promote VF dynamics, replication, and assembly, highlighting the critical role of the cytoskeleton in viral replication.
10.1016/j.virol.2025.110423
Quercetin inhibits hydrogen peroxide-induced cleavage of heat shock protein 90 to prevent glutathione peroxidase 4 degradation via chaperone-mediated autophagy.
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:Oxidative stress is caused by the accumulation of reactive oxygen species (ROS) and the depletion of free radical scavengers, which is closely related to ferroptosis in diseases. Quercetin, as a natural flavonoid compound, has been reported to have multiple pharmacological effects on the basis of its anti-oxidative and anti-ferroptotic activities. This study was designed to explore the specific mechanism of quercetin against ferroptosis induced by hydrogen peroxide (HO). METHODS:The HT22 cells (mouse hippocampal neuronal cells) treated with 40 μg·ml HO were used to investigate the role of ferroptosis in oxidative stress damage and the regulation of quercetin (7.5, 15, 30 μmol·l), as evidenced by assessments of cell viability, morphological damage, Fe accumulation, and the expressions of ferroptotic-related proteins. The changes in the expression levels of glutathione peroxidase 4 (GPX4), heat shock cognate protein 70 (HSC70), lysosomal-associated membrane protein 2a (LAMP-2a), and heat shock protein (HSP90) were assessed by qPCR, western blotting (WB) and immunofluorescence (IF) assays. Additionally, the interactions of GPX4, HSC70, LAMP-2a, and HSP90 were examined by co-immunoprecipitation (Co-IP) assay to elucidate the impact of quercetin on the degradation pathway of GPX4 and the CMA pathway. To further explore the regulatory mechanism of quercetin, the si-LAMP-2a and HSP90 mutant cells were conducted. RESULTS:Pretreatment with 30 μmol·l quercetin for 6 h significantly enhanced the survival rate (p < 0.05), maintained cell morphology, and inhibited Fe levels in HT22 cells exposed to HO (40 μg·ml). HT22 cells under oxidative stress showed lower expressions of GPX4 and ferritin heavy chain 1 (FTH1), and a higher level of Acyl-CoA synthetase long-chain family member 4 (ACSL4) (p < 0.05). And quercetin significantly reversed the expressions of these ferroptotic proteins (p < 0.05). Moreover, the autophagic lysosomal pathway inhibitor CQ effectively increased the expression of GPX4 in oxidative stress cell model. Further study showed that HO increased the activity of macroautophagy and chaperone-mediated autophagy (CMA), while quercetin notably suppressed the levels of microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ), LAMP-2a, and the activity of lysosomes (p < 0.01). Additionally, quercetin disrupted the interactions of GPX4, HSC70, and LAMP-2a, reduced cellular levels of CMA by decreasing the cleaved HSP90 (c-HSP90), and these effects were reversed in the R347 mutant HT22 cells. CONCLUSIONS:Quercetin has a significantly protective effect on oxidative stress cell model through the inhibition on ferroptosis, which is related to the degradation of GPX4 via CMA. And quercetin decreases the level of c-HSP90 induced by HO to reduce the activity of CMA by binding to R347 of HSP90.
10.1016/j.phymed.2024.156286
Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules.
Proceedings of the National Academy of Sciences of the United States of America
The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) organization and structures of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell-derived neurons, and pluripotent and differentiated P19 cells. We obtain in situ density maps of several lumenal particles from the respective cells and detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggest that a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggests a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.
10.1073/pnas.2404017121
[Effects of LncRNA SNHG20 on epithelial mesenchymal transition and microtubule formation in human oral squamous cell carcinoma cells through targeted regulation of the miR-520c-3p/ pathway].
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences
OBJECTIVE:To investigate the effects of LncRNA SNHG20 on epithelial mesenchymal transition (EMT) and microtubule formation in human oral squamous cell carcinoma (OSCC) cells through targeted regulation of the miR-520c-3p/ pathway. METHODS:After real-time fluorescence quantitative detection of LncRNA SNHG20, miR-520c-3p, mRNA expression levels in OSCC tissues and cells, dual luciferase reporter assay was used to detect the relationship between the three. OSCC cells were randomly separated into control group, sh-NC group, sh-SNHG20 group, sh-SNHG20+anti NC group, and sh-SNHG20+anti miR-520c-3p group. Western blotting was used to detect the expression of N-cadherin, vimentin, and E-cadherin proteins in the OSCC cells. The morphology of HSC-3 cells was observed under microscope. Changes in the number of microtubules formed were detected. The effect of LncRNA SNHG20 on the growth of OSCC tumors and the expression levels of LncRNA SNHG20, miR-520c-3p and RAB22 A in the transplanted tumors were detected by nude mice tumorigenesis experiment. RESULTS:LncRNA SNHG20 and mRNA were upregulated in the OSCC tissues and cells, while miR-520c-3p was downregulated ( < 0.05). There were binding sites between LncRNA SNHG20 and miR-520c-3p, RAB22A and miR-520c-3p, which had targeted regulation relationship. Compared with the sh-NC group, the sh-SNHG20 group had fewer stromal like cells, more epithelial like cells, incomplete microtubule structure, and fewer nodules. LncRNA SNHG20, RAB22A, N-Cadherin, and vimentin were downregulated, while miR-520c-3p and E-cadherin were upregulated ( < 0.05). Compared with the sh-SNHG20+anti-NC group, the sh-SNHG20+anti-miR-520c-3p group had a higher number of stromal like cells, a lower number of epithelioid cells, tighter microtubule arrangement, and more microtubule nodules. miR-520c-3p and E-cadherin were downregulated, while RAB22A, N-cadherin, and vimentin were upregulated ( < 0.05). The transplanted tumor of OSCC in sh-SNHG20 group was smaller and lower than that in sh-NC group. The expression levels of LncRNA SNHG20 and RAB22A in the transplanted tumor tissues were lower than those in sh-NC group, and the expression level of miR-520c-3p was higher than that in sh-NC group ( < 0.05). CONCLUSION:LncRNA SNHG20 promotes epithelial-mesenchymal transition and microtubule formation in human oral squamous cell carcinoma cells by targeting the miR-520c-3p/ pathway. Inhibiting the expression of LncRNA SNHG20 can target and regulate the miR-520c-3p/ pathway to inhibit EMT and microtubule formation in OSCC cells.
Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends.
The Journal of cell biology
Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4. We found that whereas CEP97 does not bind to microtubules directly, CP110 autonomously binds microtubule plus ends, blocks their growth, and inhibits depolymerization. Cryo-electron tomography revealed that CP110 associates with the luminal side of microtubule plus ends and suppresses protofilament flaring. CP110 directly interacts with CPAP, which acts as a microtubule polymerase that overcomes CP110-induced growth inhibition. Together, the two proteins impose extremely slow processive microtubule growth. Disruption of CP110-CPAP interaction in cells inhibits centriole elongation and increases incidence of centriole defects. Our findings reveal how two centriolar cap proteins with opposing activities regulate microtubule plus-end elongation and explain their antagonistic relationship during centriole formation.
10.1083/jcb.202406061
Aurora B controls microtubule stability to regulate abscission dynamics in stem cells.
Cell reports
Abscission is the last step of cell division. It separates the two sister cells and consists of cutting the cytoplasmic bridge. Abscission is mediated by the ESCRT membrane remodeling machinery, which also triggers the severing of a thick bundle of microtubules. Here, we show that rather than being passive actors in abscission, microtubules control abscission speed. Using mouse embryonic stem cells, which transition from slow to fast abscission during exit from naive pluripotency, we investigate the molecular mechanism for the regulation of abscission dynamics and identify crosstalk between Aurora B activity and microtubule stability. We demonstrate that naive stem cells maintain high Aurora B activity on the bridge after cytokinesis. This high Aurora B activity leads to transient microtubule stabilization that delays abscission by decreasing MCAK recruitment to the midbody. In turn, stable microtubules promote the activity of Aurora B. Overall, our data demonstrate that Aurora B-dependent microtubule stability controls abscission dynamics.
10.1016/j.celrep.2025.115238
Impact of Phosphorylation and O-GlcNAcylation on the Binding Affinity of R4 Tau Peptide to Microtubule and Its Conformational Preference upon Dissociation.
Journal of chemical information and modeling
Tau is a microtubule (MT)-associated protein that binds to and stabilizes the MTs of neurons. Due to its intrinsically disordered nature, it undergoes several post-translational modifications (PTMs) that are intricately linked to both the physiological and pathophysiological roles of Tau. Prior research has shown phosphorylation and O-GlcNAcylation to have contrasting effects on Tau aggregation; however, the precise molecular mechanisms and potential synergistic effects of these modifications remain elusive. In this article, we study the impact of phosphorylation at S352, and S356, as well as the phosphorylation of O-GlcNAcylation at S356, individually and in combination, on the binding of the R4 (336-367) peptide with MTs by performing classical molecular dynamics (MD) simulations. By analyzing the binding free energies of the Tau-MT complex, we found that both individual and combined phosphorylation at S352 and S356 sites decreased the affinity of the R4 peptide toward MT. Surprisingly, O-GlcNAcylation, a likely neuroprotective modification, at S356 also decreased the binding affinity of Tau to MT similar to the single phosphorylation systems (pS352 or pS356) but was observed to maintain major interactions with MT comparable to unmodified R4. Additionally, we investigated the impact of phosphorylation at both sites and the interplay between phosphorylation at S352 and O-GlcNAcylation at S356, which showed that the latter preserved the interactions and affinity of the Tau with MT better than dual phosphorylation, though still not as effectively as single phosphorylation. These findings suggest that O-GlcNAcylation at residue S356 has a moderate destabilizing effect. We also performed replica-exchange MD simulations of the R4 peptide to understand the changes in conformational preferences upon phosphorylation, O-GlcNAcylation, and a combination of both modifications. Both individual and combined phosphorylation of R4 peptide at S352, and S356, sites induced salt-bridge interactions with positively charged side chains of lysine and arginine amino acids. However, O-GlcNAcylation at S356 induced secondary structural changes on the R4 peptide, leading to the formation of a β-sheet structure, consistent with previous experimental observations. Interestingly, simultaneous phosphorylation at S352 and the phosphorylation of O-GlcNAcylation at S356 resulted in conformations promoting salt-bridges and β-sheets. Thus, our study provides atomistic insights into the impact of PTMs on the binding of Tau peptide to MT and its conformational preferences upon dissociation.
10.1021/acs.jcim.4c02109
NME7 maintains primary cilium assembly, ciliary microtubule stability, and Hedgehog signaling.
Life science alliance
NME7 (nucleoside diphosphate kinase 7), a lesser studied member of the non-metastatic expressed (NME) family, has been reported as a potential subunit of the γ-tubulin ring complex (γTuRC). However, its role in the cilium assembly and function remains unclear. Our research demonstrated that NME7 is located at the centrosome, including at the spindle poles during metaphase and at the basal bodies during cilium assembly. Notably, a small fraction of NME7 localizes within the cilium. Detailed analysis of cilium assembly after NME7 knockdown and knockout revealed that NME7 is required for this process. NME7 knockout cells exhibited sensitivity to nocodazole, indicating its role in ciliary microtubule stability. In addition, NME7 deficiency impacted the Hedgehog signaling pathway, evident from reduced smoothened (Smo) fluorescence within primary cilia. This role of NME7 in Hedgehog signaling may depend on its nucleoside diphosphate kinase activity and γTuRC association. In conclusion, these findings enhance our understanding of the γTuRC roles in primary cilia in mammalian cells, highlighting the importance of NME7 in ciliary functions and signaling pathways.
10.26508/lsa.202402933
Natural products against tau hyperphosphorylation-induced aggregates: Potential therapies for Alzheimer's disease.
Archiv der Pharmazie
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and memory impairments and is considered the most prevalent form of dementia. Among the contributing factors to AD lies the hyperphosphorylation of the microtubule-associated protein tau. Phosphorylated tau reduces its affinity for microtubules and triggers other posttranslational modifications that result in its aggregation and assembly into filaments. These structures progressively accumulate within neurons leading to neurodegeneration. While current AD medications often involve undesirable side effects, the exploration of natural products as a potential therapeutic alternative has gained considerable attention. Numerous compounds have shown potential capacity for reducing tau pathology through different mechanisms, such as inhibiting kinases to reduce tau hyperphosphorylation, enhancing phosphatase activity, and blocking fibril formation. Since tau hyperphosphorylation-induced aggregation is pivotal in AD onset, this review aims to elucidate the potential of natural products in modulating this crucial molecular mechanism.
10.1002/ardp.202400721
Pathogenic variants of TUBB8 cause oocyte spindle defects by disrupting with EB1/CAKP5 interactions and potential treatment targeting microtubule acetylation through HDAC6 inhibition.
Clinical and translational medicine
BACKGROUND:Numerous pathogenic variants causing human oocyte maturation arrest have been reported on the primate-specific TUBB8 gene. The main etiology is the dramatic reduction of tubulin α/β dimer, but still large numbers of variants remain unexplained. METHODS:Using microinjection mRNA and genome engineering to reintroduce the conserved pathogenic missense variants into oocytes or in generating TUBB8 variant knock-in mouse models, we investigated that the human deleterious variants alter microtubule nucleation and spindle assembly during meiosis. Live-cell imaging and immunofluorescence were utilised to track the dynamic expression of microtubule plus end-tracking proteins in vivo and analysed microtubule nucleation or spindle assembly in vitro, respectively. Immunoprecipitation-mass spectrometry and ultramicro-quantitative proteomics were performed to identify the differential abundance proteins and affected interactome of TUBB8 protein. RESULTS:First, we observed a significant depletion of the EB1 signal upon microinjection of mutated TUBB8 mRNA (including R262Q, M300I, and D417N missense variants), indicating disruption of microtubule nucleation caused by these introduced TUBB8 missense variants. Mechanically, we demonstrated that the in vivo TUBB8-D417N missense variant diminished the affinity of EB1 and microtubules. It also harmed the interaction between microtubules and CKAP5/TACC3, which are crucial for initiating microtubule nucleation. Attenuated Ran-GTP pathway was also found in TUBB8-D417N oocytes, leading to disrupted spindle assembly. Stable microtubule was largely abolished on the spindle of TUBB8-D417N oocytes, reflected by reduced tubulin acetylation and accumulated HDAC6. More importantly, selective inhibition of HDAC6 by culturing TUBB8-D417N oocytes with Tubacin or Tubastatin A showed morphologically normal spindle and drastically recovered polar-body extrusion rate. These rescue results shed light on the strategy to treat meiotic defects in a certain group of TUBB8 mutated patients. CONCLUSION:Our study provides a comprehensive mechanism elucidating how TUBB8 missense variants cause oocyte maturation arrest and offers new therapeutic avenues for treating female infertility in the clinic.
10.1002/ctm2.70193
A novel amino-pyrimidine inhibitor suppresses tumor growth via microtubule destabilization and Bmi-1 down-regulation.
Biochemical pharmacology
Colorectal cancer (CRC), one of the diseases posing a threat to global health, according to the latest data, is the third most common cancer globally and the second leading cause of cancer-related deaths. The development and refinement of novel structures of small molecular compounds play a crucial role in tumor treatment and overcoming drug resistance. In this study, our objective was to screen and characterize novel compounds for overcoming drug resistance via the B Lymphoma Mo-MLV insertion region 1 (Bmi-1) reporter screen assay. The stable cell line harboring the Bmi-1 reporter gene was utilized to screen 300 compounds, leading to the identification of an amino-pyrimidine compound, APD-94. In vitro, APD-94 markedly inhibited cancer cell proliferation and decreased Bmi-1 expression at both the RNA and protein levels. In vivo, APD-94 repressed the growth of HT29 cell xenografts in NOD/SCID mice without notable side effects. Flow cytometry results demonstrated that APD-94 induced G2/M phase arrest and apoptosis in cells. APD-94 was identified as a novel inhibitor of microtubule polymerization by directly targeting the tubulin. Furthermore, APD-94 was more effective in overcoming the resistance to paclitaxel in paclitaxel-resistant A549/Tax cells. This bifunctional inhibitor is a promising candidate drug for CRC treatment.
10.1016/j.bcp.2025.116783
Phase separation of a microtubule plus-end tracking protein into a fluid fractal network.
Nature communications
Microtubule plus-end tracking proteins (+TIPs) participate in nearly all microtubule-based cellular processes and have recently been proposed to function as liquid condensates. However, their formation and internal organization remain poorly understood. Here, we have study the phase separation of Bik1, a CLIP-170 family member and key +TIP involved in budding yeast cell division. Bik1 is a dimer with a rod-shaped conformation primarily defined by its central coiled-coil domain. Its liquid condensation likely involves the formation of higher-order oligomers that phase separate in a manner dependent on the protein's N-terminal CAP-Gly domain and C-terminal EEY/F-like motif. This process is accompanied by conformational rearrangements in Bik1, leading to at least a two-fold increase in multivalent interactions between its folded and disordered domains. Unlike classical liquids, Bik1 condensates exhibit a heterogeneous, fractal supramolecular structure with protein- and solvent-rich regions. This structural evidence supports recent percolation-based models of biomolecular condensates. Together, our findings offer insights into the structure, dynamic rearrangement, and organization of a complex, oligomeric, and multidomain protein in both dilute and condensed states. Our experimental framework can be applied to other biomolecular condensates, including more complex +TIP networks.
10.1038/s41467-025-56468-8
Tubulin tyrosination/detyrosination regulate the affinity and sorting of intraflagellar transport trains on axonemal microtubule doublets.
Nature communications
Cilia assembly and function rely on the bidirectional transport of components between the cell body and ciliary tip via Intraflagellar Transport (IFT) trains. Anterograde and retrograde IFT trains travel along the B- and A-tubules of microtubule doublets, respectively, ensuring smooth traffic flow. However, the mechanism underlying this segregation remains unclear. Here, we test whether tubulin detyrosination (enriched on B-tubules) and tyrosination (enriched on A-tubules) have a role in IFT logistics. We report that knockout of tubulin detyrosinase VashL in Chlamydomonas reinhardtii causes frequent IFT train stoppages and impaired ciliary growth. By reconstituting IFT train motility on de-membranated axonemes and synthetic microtubules, we show that anterograde and retrograde trains preferentially associate with detyrosinated and tyrosinated microtubules, respectively. We propose that tubulin tyrosination/detyrosination is crucial for spatial segregation and collision-free IFT train motion, highlighting the significance of the tubulin code in ciliary transport.
10.1038/s41467-025-56098-0
Data-driven equation-free dynamics applied to many-protein complexes: The microtubule tip relaxation.
Biophysical journal
Microtubules (MTs) constitute the largest components of the eukaryotic cytoskeleton and play crucial roles in various cellular processes, including mitosis and intracellular transport. The property allowing MTs to cater to such diverse roles is attributed to dynamic instability, which is coupled to the hydrolysis of guanosine-5'-triphosphate (GTP) to guanosine-5'-diphosphate (GDP) within the β-tubulin monomers. Understanding the dynamics and structural features of both GDP- and GTP-complexed MT tips, especially at an all-atom level, remains challenging for both experimental and computational methods because of their dynamic nature and the prohibitive computational demands of simulating large, many-protein systems. This study employs the "equation-free" multiscale computational method to accelerate the relaxation of all-atom simulations of MT tips toward their putative equilibrium conformation. Using large MT lattice systems (14 protofilaments × 8 heterodimers) comprising ∼21-38 million atoms, we applied this multiscale approach to leapfrog through time and nearly double the computational efficiency in realizing relaxed all-atom conformations of GDP- and GTP-complexed MT tips. Commencing from an initial 4 μs unbiased all-atom simulation, we interleave coarse-projective equation-free jumps with short bursts of all-atom molecular dynamics simulation to realize an additional effective simulation time of 1.875 μs. Our 5.875 μs of effective simulation trajectories for each system expose the subtle yet essential differences in the structures of MT tips as a function of whether β-tubulin monomer is complexed with GDP or GTP, as well as the lateral interactions within the MT tip, offering a refined understanding of features underlying MT dynamic instability. The approach presents a robust and generalizable framework for future explorations of large biomolecular systems at atomic resolution.
10.1016/j.bpj.2025.01.009
Characterization of multiple binding sites on microtubule associated protein 2c recognized by dimeric and monomeric 14-3-3ζ.
The FEBS journal
Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.
10.1111/febs.17405
Emerging roles for tubulin PTMs in neuronal function and neurodegenerative disease.
Current opinion in neurobiology
Neurons are equipped with microtubules of different stability with stable and dynamic domains often coexisting on the same microtubule. While dynamic microtubules undergo random transitions between disassembly and assembly, stable ones persist long enough to serve as platforms for tubulin-modifying enzymes (known as writers) that attach molecular components to the α- or β-tubulin subunits. The combination of these posttranslational modifications (PTMs) results in a "tubulin code," dictating the behavior of selected proteins (known as readers), some of which were shown to be crucial for neuronal function. Recent research has further highlighted that disturbances in tubulin PTMs can lead to neurodegeneration, sparking an emerging field of investigation with numerous questions such as whether and how tubulin PTMs can affect neurotransmission and synaptic plasticity and whether restoring balanced tubulin PTM levels could effectively prevent or mitigate neurodegenerative disease.
10.1016/j.conb.2025.102971
β3 accelerates microtubule plus end maturation through a divergent lateral interface.
Molecular biology of the cell
β-tubulin isotypes exhibit similar sequences but different activities, suggesting that limited sequence divergence is functionally important. We investigated this hypothesis for TUBB3/β3, a β-tubulin linked to aggressive cancers and chemoresistance in humans. We created mutant yeast strains with β-tubulin alleles that mimic variant residues in β3 and find that residues at the lateral interface are sufficient to alter microtubule dynamics and response to microtubule targeting agents. In HeLa cells, β3 overexpression decreases the lifetime of microtubule growth, and this requires residues at the lateral interface. These microtubules exhibit a shorter region of EB binding at the plus end, suggesting faster lattice maturation, and resist stabilization by paclitaxel. Resistance requires the H1-S2 and H2-S3 regions at the lateral interface of β3. Our results identify the mechanistic origins of the unique activity of β3 tubulin and suggest that tubulin isotype expression may tune the rate of lattice maturation at growing microtubule plus ends in cells.
10.1091/mbc.E24-08-0354
Hippocampal dendritic spines store-operated calcium entry and endoplasmic reticulum content is dynamic microtubule dependent.
Scientific reports
One of the mechanisms of calcium signalling in neurons is store-operated calcium entry (SOCE), which is activated when the calcium concentration in the smooth endoplasmic reticulum (ER) decreases and its protein-calcium sensor STIM (stromal interacting molecule) relocate to the endoplasmic reticulum and plasma membrane junctions, forms clusters and induces calcium entry. In electrically non-excitable cells, STIM1 is coupled with the positive end of a tubulin microtubule through interaction with EB1 (end-binding) protein, which controls its oligomerization, SOCE and participates in ER movement. STIM2 homologue, which is specific for mature hippocampal dendritic spines, is known to interact with EB3 protein, however, not much is known about the role of this interaction in STIM2 clustering or ER trafficking in neurons. Intriguingly, in neurons, reducing the expression of EB3 protein or disrupting the interaction of STIM2 protein with EB proteins results in decreased SOCE, in contrast to experiments with STIM1 in non-excitable cells. In this study, these two homologues are compared side-by-side in HEK-293T, and it is shown for the first time that their clustering and SOCE is oppositely regulated by dynamic tubulin microtubules. In particular, for STIM2, the interaction with dynamic microtubule cytoskeleton is required for clustering and is shown to potentiate SOCE, while for STIM1 this interaction restricts clustering, resulting in SOCE decrease. After store depletion in primary hippocampal neurons, the wild type STIM2 is redistributed from the necks to the heads of dendritic spines, while the STIM2 variant with a mutation that disrupts the interaction with EB proteins is excluded from dendritic spines. In addition, overexpression of the mutant variant leads to ER reorganization in neuronal soma and reduction of ER presence in spines. It also leads to a reduction in the number of spines containing the spine apparatus formed by ER cisternae, as well as a reduction in dendritic spines SOCE. These effects are opposite of those detected during overexpression of the wild type STIM2. Considered together, these findings underline the important role of dynamic microtubules in regulation of neuronal SOCE and ER morphology.
10.1038/s41598-024-85024-5
FSD1 inhibits glioblastoma diffuse infiltration through restriction of HDAC6-mediated microtubule deacetylation.
Science China. Life sciences
The infiltration of glioblastoma multiforme (GBM) is predominantly characterized by diffuse spread, contributing significantly to therapy resistance and recurrence of GBM. In this study, we reveal that microtubule deacetylation, mediated through the downregulation of fibronectin type III and SPRY domain-containing 1 (FSD1), plays a pivotal role in promoting GBM diffuse infiltration. FSD1 directly interacts with histone deacetylase 6 (HDAC6) at its second catalytic domain, thereby impeding its deacetylase activity on α-tubulin and preventing microtubule deacetylation and depolymerization. This inhibitory interaction is disrupted upon phosphorylation of FSD1 at its Ser317 and Ser324 residues by activated CDK5, leading to FSD1 dissociation from microtubules and facilitating HDAC6-mediated α-tubulin deacetylation. Furthermore, increased expression of FSD1 or interference with FSD1 phosphorylation reduces microtubule deacetylation, suppresses invasion of GBM stem cells, and ultimately mitigates tumor infiltration in orthotopic GBM xenografts. Importantly, GBM tissues exhibit diminished levels of FSD1 expression, correlating with microtubule deacetylation and unfavorable clinical outcomes in GBM patients. These findings elucidate the mechanistic involvement of microtubule deacetylation in driving GBM cell invasion and offer potential avenues for managing GBM infiltration.
10.1007/s11427-024-2616-7
The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.
Current biology : CB
Anaphase is tightly controlled spatiotemporally to ensure proper separation of chromosomes. The mitotic spindle, the self-organized microtubule structure driving chromosome segregation, scales in size with the available cytoplasm. Yet, the relationship between spindle size and chromosome movement remains poorly understood. Here, we address this relationship during the cleavage divisions of the Drosophila blastoderm. We show that the speed of chromosome separation gradually decreases during the four nuclear divisions of the blastoderm. This reduction in speed is accompanied by a similar reduction in spindle length, ensuring that these two quantities are tightly linked. Using a combination of genetic and quantitative imaging approaches, we find that two processes contribute to controlling the speed at which chromosomes move in anaphase: the activity of molecular motors important for microtubule depolymerization and sliding and the cell cycle oscillator. Specifically, we found that the levels of multiple kinesin-like proteins important for microtubule depolymerization, as well as kinesin-5, contribute to setting the speed of chromosome separation. This observation is further supported by the scaling of poleward flux rate with the length of the spindle. Perturbations of the cell cycle oscillator using heterozygous mutants of mitotic kinases and phosphatases revealed that the duration of anaphase increases during the blastoderm cycles and is the major regulator of chromosome velocity. Thus, our work suggests a link between the biochemical rate of mitotic exit and the forces exerted by the spindle. Collectively, we propose that the cell cycle oscillator and spindle length set the speed of chromosome separation in anaphase.
10.1016/j.cub.2024.11.046
Physical effects of crowdant size and concentration on collective microtubule polymerization.
Biophysical journal
The polymerization of cytoskeletal filaments is regulated by both biochemical pathways, as well as physical factors such as crowding. The effect of crowding in vivo emerges from the density of intracellular components. Due to the complexity of the intracellular environment, most studies are based on either in vitro reconstitution or theory. Crowding agent (crowdants) size has been shown to influence polymerization of both actin and microtubules (MTs). Previously, the elongation rates of MT dynamics observed at single filament scale were reported to decrease with increasing concentrations of small but not large crowdants, and this correlated with in vivo viscosity increases. However, the exact nature of the connection between viscosity, crowdant size, nucleation, and MT elongation has remained unclear. Here, we use in vitro reconstitution of bulk MT polymerization kinetics and microscopy to examine the collective effect of crowdant molecular weight, volume occupancy, and viscosity on elongation and spontaneous polymerization. We find MT elongation rates obtained from bulk polymerization decrease in the presence of multiple low-molecular weight (LMW) crowdants, while increasing with high-molecular weight (HMW) crowdants. Lattice Monte Carlo simulations of an effective model of collective polymerization demonstrate reduced polymerization rates arise due to decrease in monomer diffusion due to small-sized crowdants. However, MT polymerization in the absence of nucleators, de novo, shows a crowdant size independence of polymerization rate and critical concentration, depending solely on concentration of the crowdant. In microscopy, we find LMW crowdants result in short but many filaments, while HMW crowdants increase filament density, but have little effect on lengths. The effect of crowdant volume fraction ϕ and size in de novo polymerization match simulations, demonstrating crowdants affect elongation independent of nucleation. Thus, the effect of viscosity on collective MT dynamics, i.e., filament numbers and lengths, shows crowdant size dependence for elongation, but independence for de novo polymerization.
10.1016/j.bpj.2025.01.020
Comprehensive Biotechnological Strategies for Podophyllotoxin Production from Plant and Microbial Sources.
Planta medica
Podophyllotoxin is derived from plant sources and exhibits strong anticancer activity. However, limited natural availability and environmental impacts from traditional extraction methods drive the search for alternative production approaches. This review explores diverse strategies for sustainable podophyllotoxin synthesis, including biosynthesis, semi-synthesis, and biotransformation. Biosynthetic methods involve metabolic pathway engineering in plant or microbial cells, enabling increased yields by manipulating precursor availability and gene expression. Semi-synthetic approaches modify podophyllotoxin precursors or intermediates to enhance therapeutic effects, with derivatives like etoposide and teniposide showing clinical efficacy. Biotransformation, utilising organisms such as endophytic fungi or human hepatic enzymes, enables the transformation of substrates like deoxypodophyllotoxin into podophyllotoxin or its derivatives, yielding compounds with reduced environmental impact and improved purity. The anticancer efficacy of podophyllotoxin and its derivatives stems from multiple mechanisms. These compounds disrupt cell mitosis by inhibiting microtubule assembly, impairing nucleoside transport, and blocking topoisomerase II activity, leading to DNA cleavage and cancer cell apoptosis. Podophyllotoxin and its derivatives also exhibit anti-angiogenesis and anti-metastatic effects through signalling pathway modulation. Notably, derivatives like deoxypodophyllotoxin utilise advanced delivery systems, enhancing targeted efficacy and reducing side effects. Given the varied mechanisms and growing therapeutic applications, optimising biotransformation and delivery techniques remains essential for advancing podophyllotoxin-based therapies. This comprehensive review underscores the compound's potential as a robust anticancer agent and the need for continued research to maximise its production and clinical effectiveness.
10.1055/a-2504-3069
Pancreatic cancer cell-intrinsic transglutaminase-2 promotes T cell suppression through microtubule-dependent secretion of immunosuppressive cytokines.
Journal for immunotherapy of cancer
BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is mostly refractory to immunotherapy due to immunosuppression in the tumor microenvironment and cancer cell-intrinsic T cell tolerance mechanisms. PDAC is described as a "cold" tumor type with poor infiltration by T cells and factors leading to intratumoral T cell suppression have thus received less attention. Here, we identify a cancer cell-intrinsic mechanism that contributes to a T cell-resistant phenotype and describes potential combinatorial therapy. METHODS:We used an unbiased screening approach of T cell resistant and sensitive murine KPC ( ) PDAC cells in a three-dimensional co-culture platform with syngeneic antigen-educated T cells to identify potential cell-intrinsic drivers of T cell suppression in PDAC. Comparative transcriptomic analysis was performed to reveal promising candidates that mediate resistance to T cells. We investigated their contribution by shRNA-mediated knockdown and pharmacological inhibition in murine in vitro and in vivo studies, as well as in patient-derived organoids (PDOs). A combination of transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to validate the underlying T cell response phenotypes of PDAC cells. The action of TGM2 via interaction with tubulin and the impact of microtubule dynamics and vesicle trafficking were evaluated by protein analyses and live-cell imaging. Correlation analyses via TCGA data complemented the functional studies. RESULTS:We identified transglutaminase 2 (TGM2) as a mediator of T cell suppression in PDAC. We report that high levels of TGM2 expression in patients' tumors correlate with immunosuppressive signatures and poor overall survival. We found that TGM2 regulates vesicle trafficking by modulating microtubule network density and dynamics in pancreatic cancer cells, thus facilitating the secretion of immunosuppressive cytokines, which impair effector T cell functionality. In TGM2-expressing PDOs, pharmacological TGM2 inhibition or treatment with nocodazole increased T cell-mediated apoptosis. Also, pretreatment of TGM2 PDOs with sublethal doses of the spindle poisons paclitaxel or vincristine increased CD8+T cell activation and sensitized PDOs toward T cell-mediated cytotoxicity. CONCLUSIONS:These findings indicate that targeting microtubular function therapeutically may enhance antitumor T cell responses by impacting activity of immunosuppressive cytokines in the PDAC microenvironment.
10.1136/jitc-2024-010579
Cellular Uptake of Tau Aggregates Triggers Disulfide Bond Formation in Four-Repeat Tau Monomers.
ACS chemical neuroscience
Oxidative stress is an important driver of aging and has been linked to numerous neurodegenerative disorders, including Alzheimer's disease. A key pathological hallmark of Alzheimer's are filamentous inclusions made of the microtubule associated protein Tau. Based on alternative splicing, Tau protein can feature either three or four microtubule binding repeats. Distinctively, three-repeat Tau contains a single cysteine; four-repeat Tau contains two. Although there is evidence that the cysteines in pathological Tau filaments exist in the reduced form, very little is known about the alternative disulfide-bonded state. It is unclear whether it can exist nontransiently in the reducing environment of the cytosol. Such knowledge, however, is important as different redox states of Tau could modulate aggregation. To address this question, we transfected HEK293 cells expressing the P301S variant of four-repeat Tau with fibril seeds composed of compact, disulfide-bonded Tau monomers. In vitro, these fibrils are observed to recruit only compact Tau, but not Tau in which the cysteines are reduced or replaced by alanines or serines. In line with this characteristic, the fibrils dissociate when treated with a reducing agent. When offered to HEK293 cells, variant Tau protein is recruited to the seeds forming intracellular fibrils with the same seeding properties as the in vitro counterparts. Markedly, the proteins in these fibrils have a compact, disulfide-bonded configuration and dissociate upon reduction. These findings reveal that uptake of exogeneous fibril seeds triggers oxidation of Tau monomers, modulating intracellular aggregation.
10.1021/acschemneuro.4c00607
The Balbiani body is formed by microtubule-controlled molecular condensation of Buc in early oogenesis.
Current biology : CB
Vertebrate oocyte polarity has been observed for two centuries and is essential for embryonic axis formation and germline specification, yet its underlying mechanisms remain unknown. In oocyte polarization, critical RNA-protein (RNP) granules delivered to the oocyte's vegetal pole are stored by the Balbiani body (Bb), a membraneless organelle conserved across species from insects to humans. However, the mechanisms of Bb formation are still unclear. Here, we elucidate mechanisms of Bb formation in zebrafish through developmental biomolecular condensation. Using super-resolution microscopy, live imaging, biochemical, and genetic analyses in vivo, we demonstrate that Bb formation is driven by molecular condensation through phase separation of the essential intrinsically disordered protein Bucky ball (Buc). Live imaging, molecular analyses, and fluorescence recovery after photobleaching (FRAP) experiments in vivo reveal Buc-dependent changes in the Bb condensate's dynamics and apparent material properties, transitioning from liquid-like condensates to a solid-like stable compartment. Furthermore, we identify a multistep regulation by microtubules that controls Bb condensation: first through dynein-mediated trafficking of early condensing Buc granules, then by scaffolding condensed granules, likely through molecular crowding, and finally by caging the mature Bb to prevent overgrowth and maintain shape. These regulatory steps ensure the formation of a single intact Bb, which is considered essential for oocyte polarization and embryonic development. Our work offers insight into the long-standing question of the origins of embryonic polarity in non-mammalian vertebrates, supports a paradigm of cellular control over molecular condensation by microtubules, and highlights biomolecular condensation as a key process in female reproduction.
10.1016/j.cub.2024.11.056
Elongator is a microtubule polymerase selective for polyglutamylated tubulin.
The EMBO journal
Elongator is a tRNA-modifying complex that regulates protein translation. Recently, a moonlighting function of Elongator has been identified in regulating the polarization of the microtubule cytoskeleton during asymmetric cell division. Elongator induces symmetry breaking of the anaphase midzone by selectively stabilizing microtubules on one side of the spindle, contributing to the downstream polarized segregation of cell-fate determinants, and therefore to cell fate determination. Here, we investigate how Elongator controls microtubule dynamics. Elongator binds both to the tip of microtubules and to free GTP-tubulin heterodimers using two different subcomplexes, Elp123 and Elp456, respectively. We show that these activities must be coupled for Elongator to decrease the tubulin critical concentration for microtubule elongation. As a consequence, Elongator increases the growth speed and decreases the catastrophe rate of microtubules. Surprisingly, the Elp456 subcomplex binds to tubulin tails and has strong selectivity towards polyglutamylated tubulin. Hence, microtubules assembled by Elongator become selectively enriched with polyglutamylated tubulin, as observed in vitro, in mouse and Drosophila cell lines, as well as in vivo in Drosophila Sensory Organ Precursor cells. Therefore, Elongator rewrites the tubulin code of growing microtubules, placing it at the core of cytoskeletal dynamics and polarization during asymmetric cell division.
10.1038/s44318-024-00358-0
Diverse microtubule-binding repeats regulate TPX2 activities at distinct locations within the spindle.
The Journal of cell biology
TPX2 is an elongated molecule containing multiple α-helical repeats. It stabilizes microtubules (MTs), promotes MT nucleation, and is essential for spindle assembly. However, the molecular basis of how TPX2 performs these functions remains elusive. Here, we systematically characterized the MT-binding activities of all TPX2 modules individually and in combinations and investigated their respective contributions both in vitro and in cells. We show that TPX2 contains α-helical repeats with opposite preferences for "extended" and "compacted" tubulin dimer spacing, and their distinct combinations produce divergent outcomes, making TPX2 activity highly robust yet tunable. Importantly, a repeat group at the C terminus, R8-9, is the key determinant of the TPX2 function. It stabilizes MTs by promoting rescues in vitro and is critical in spindle assembly. We propose a model where TPX2 activities are spatially regulated via its diverse MT-binding repeats to accommodate its varied functions in distinct locations within the spindle. Furthermore, we reveal a synergy between TPX2 and HURP in stabilizing spindle MTs.
10.1083/jcb.202404025
Cannabidiol-Induced Autophagy Ameliorates Tau Protein Clearance.
Neurotoxicity research
Tau is a neuronal protein that confers stability to microtubules; however, its hyperphosphorylation and accumulation can lead to an impairment of protein degradation pathways, such as autophagy. Autophagy is a lysosomal catabolic process responsible for degrading cytosolic components, being essential for cellular homeostasis and survival. In this context, autophagy modulation has been postulated as a possible therapeutic target for the treatment of neurodegenerative diseases. Studies point to the modulatory and neuroprotective role of the cannabinoid system in neurodegenerative models and here it was investigated the effects of cannabidiol (CBD) on autophagy in a human neuroblastoma strain (SH-SY5Y) that overexpresses the EGFP-Tau WT (Wild Type) protein in an inducible Tet-On system way. The results demonstrated that CBD (100 nM and 10 µM) decreased the expression of AT8 and total tau proteins, activating autophagy, evidenced by increased expression of light chain 3-II (LC3-II) protein and formation of autophagosomes. Furthermore, the cannabinoid compounds CBD, ACEA (CB1 agonist) and GW-405,833 (CB2 agonist) decreased the fluorescence intensity of EGFP-Tau WT; and when chloroquine, an autophagic blocker, was used, there was a reversal in the fluorescence intensity of EGFP-Tau WT with CBD (1 and 10 µM) and GW-405,833 (2 µM), demonstrating the possible participation of autophagy in these groups. Thus, it was possible to conclude that CBD induced autophagy in EGFP-Tau WT cells which increased tau degradation, showing its possible neuroprotective role. Hence, this study may contribute to a better understanding of how cannabinoids can modulate autophagy and present a potential therapeutic target in a neurodegeneration model.
10.1007/s12640-025-00729-3
Tubulin Acetylation Enhances Microtubule Stability in Trabecular Meshwork Cells Under Mechanical Stress.
Investigative ophthalmology & visual science
Purpose:To study the roles of tubulin acetylation and cyclic mechanical stretch (CMS) in trabecular meshwork (TM) cells and their impact on outflow pathway physiology and pathology. Methods:Primary TM cell cultures were subjected to CMS (8% elongation, 24 hours), and acetylated α-tubulin at lysine 40 (Ac-TUBA4) was assessed by western blotting and immunofluorescence. Enzymes regulating tubulin acetylation were identified via siRNA-mediated knockdowns of ATAT1, HDAC6, and SIRT2. Ac-TUBA4 levels were compared between glaucomatous (GTM) and non-glaucomatous (NTM) TM cells and in frozen sections of human cadaver eyes. The effect of tubulin acetylation on substrate stiffness and cell contractility was evaluated by culturing cells on substrates with varying stiffness and by collagen gel contraction assays, respectively. Microtubule stability was examined by monitoring resistance to nocodazole-induced depolymerization. The in vivo effect on intraocular pressure (IOP) was evaluated following intracameral injections of tubacin in mice. Results:CMS induced tubulin acetylation in human TM cells by downregulating the deacetylase HDAC6. Elevated Ac-TUBA4 levels were observed in GTM compared NTM cells and tissues. Tubulin acetylation was not affected by substrate stiffness and did not show a direct effect on TM cell contractility. Tubulin acetylation was found to provide protection against microtubule destabilization induced by nocodazole. Importantly, intracameral injection of tubacin, an HDAC6 inhibitor, significantly lowered IOP in mice. Conclusions:Our study highlights a critical role of tubulin acetylation in TM cell response to mechanical stress and its potential impact on IOP regulation. Tubulin acetylation could represent a therapeutic target for glaucoma.
10.1167/iovs.66.1.43
Structural insights into the interplay between microtubule polymerases, γ-tubulin complexes and their receptors.
Nature communications
The γ-tubulin ring complex (γ-TuRC) is a structural template for controlled nucleation of microtubules from α/β-tubulin heterodimers. At the cytoplasmic side of the yeast spindle pole body, the CM1-containing receptor protein Spc72 promotes γ-TuRC assembly from seven γ-tubulin small complexes (γ-TuSCs) and recruits the microtubule polymerase Stu2, yet their molecular interplay remains unclear. Here, we determine the cryo-EM structure of the Candida albicans cytoplasmic nucleation unit at 3.6 Å resolution, revealing how the γ-TuRC is assembled and conformationally primed for microtubule nucleation by the dimerised Spc72 CM1 motif. Two coiled-coil regions of Spc72 interact with the conserved C-terminal α-helix of Stu2 and thereby position the α/β-tubulin-binding TOG domains of Stu2 in the vicinity of the microtubule assembly site. Collectively, we reveal the function of CM1 motifs in γ-TuSC oligomerisation and the recruitment of microtubule polymerases to the γ-TuRC.
10.1038/s41467-024-55778-7
The Use of APC/C Antagonists to Promote Mitotic Catastrophe in Cancer Cells.
Methods in molecular biology (Clifton, N.J.)
The multiprotein subunit E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) plays a key role in the control of mitosis progression. APC/C is the ultimate effector of the Spindle Assembly Checkpoint (SAC), the signaling system of higher organisms including the human that monitors the proper attachment of chromosomes to microtubules during cell division. Defects in this process result in genome instability, aneuploidy, premature aging, and cancer. APC/C roles in the SAC require its activation by the protein Cdc20. Interfering with APC/C activation by Cdc20 impairs APC/C substrate recognition, resulting in a delayed mitotic exit and eventually inducing cell death. This may be advantageous for the treatment of cancer and malignancies associated with SAC dysregulation. Here we describe a protocol to interfere with mitotic exit through the use of commercially available (Apcin, proTAME) as well as innovative small molecules we have developed that function as antagonists of APC/C activation by Cdc20. We show that the use of these molecules alone and in combination is effective to promote mitotic catastrophe and suppress cell expansion in 2D and 3D (spheroids) cancer cells of different tissue origin, including breast, cervical, and ovarian cancer.
10.1007/978-1-0716-4236-8_17
A Two-Heads-Bound State Drives KIF1A Superprocessivity.
bioRxiv : the preprint server for biology
KIF1A, a neuron-specific Kinesin-3 motor, is indispensable for long-distance axonal transport and nuclear migration, processes vital for neuronal function. Using MINFLUX tracking, we reveal that KIF1A predominantly adopts a two-heads-bound state, even under ATP-limiting conditions, challenging prior models proposing a one-head-bound rate-limiting step. This two-heads-bound conformation, stabilized by interactions between the positively charged K-loop and negatively charged tubulin tails, enhances microtubule affinity and minimizes detachment. The shorter neck linker facilitates inter-head tension, keeping the heads out of phase and enabling highly coordinated stepping. In contrast, Kinesin-1 (KIF5B) transitions to a one-head-bound state under similar conditions, limiting its processivity. Perturbing KIF1A's mechanochemical cycle by prolonging its one-head-bound state significantly reduces processivity, underscoring the critical role of the two-heads-bound state in motility. These findings establish a mechanistic framework for understanding KIF1A's adaptations for neuronal transport and dysfunction in neurological diseases.
10.1101/2025.01.14.632505
Diverse microtubule-destabilizing drugs induce equivalent molecular pathway responses in endothelial cells.
bioRxiv : the preprint server for biology
Drugs that modulate microtubule (MT) dynamics are well-characterized at the molecular level, yet the mechanisms linking these molecular effects to their distinct clinical outcomes remain unclear. Several MT-destabilizing drugs, including vinblastine, combretastatin A4, and plinabulin, are widely used, or are under evaluation for cancer treatment. Although all three depolymerize MTs, they do so through distinct biochemical mechanisms. Furthermore, their clinical profiles and therapeutic uses differ considerably. To investigate whether differential modulation of molecular pathways might account for clinical differences, we compared gene expression and signaling pathway responses in human pulmonary microvascular endothelial cells (HPMECs), alongside the MT-stabilizing drug docetaxel and the pro-inflammatory cytokine TNF-α. RNA-sequencing and phosphoproteomics revealed that all three MT destabilizers triggered equivalent molecular responses. The substantial changes in gene expression caused by MT destabilization were completely dependent on Rho family GTPase activation. These findings suggest that the distinct clinical profiles of the destabilizing drugs depend on differences in pharmacokinetics (PK) and tissue distribution rather than molecular actions. The washout rate of the three drugs differed, which likely translates to PK differences. Our data provide insights into how MT destabilization triggers signaling changes, potentially explaining how these drugs induce cell cycle re-entry in quiescent cells and how plinabulin ameliorates chemotherapy-induced neutropenia.
10.1101/2025.01.22.632572
Dysregulation of alternative splicing is a transcriptomic feature of patient-derived fibroblasts from CAG repeat expansion spinocerebellar ataxias.
Human molecular genetics
The spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of rare dominantly inherited neurodegenerative diseases characterized by progressive ataxia. The most common mutation seen across the SCAs is a CAG repeat expansion, causative for SCA1, 2, 3, 6, 7, 12 and 17. We recently identified dysregulation of alternative splicing as a novel, presymptomatic transcriptomic hallmark in mouse models of SCAs 1, 3 and 7. In order to understand if dysregulation of alternative splicing is a transcriptomic feature of patient-derived cell models of CAG SCAs, we performed RNA sequencing and transcriptomic analysis in patient-derived fibroblast cell lines of SCAs 1, 3 and 7. We identified widespread and robust dysregulation of alternative splicing across all CAG expansion SCA lines investigated, with disease relevant pathways affected, such as microtubule-based processes, transcriptional regulation, and DNA damage and repair. Novel disease-relevant alternative splicing events were validated across patient-derived fibroblast lines from multiple CAG SCAs and CAG containing reporter cell lines. Together this study demonstrates that dysregulation of alternative splicing represents a novel and shared pathogenic process in CAG expansion SCA1, 3 and 7 and can potentially be used as a biomarker across patient models of this group of devastating neurodegenerative diseases.
10.1093/hmg/ddae174
Arginylation of ⍺-tubulin at E77 regulates microtubule dynamics via MAP1S.
The Journal of cell biology
Arginylation is the posttranslational addition of arginine to a protein by arginyltransferase-1 (ATE1). Previous studies have found that ATE1 targets multiple cytoskeletal proteins, and Ate1 deletion causes cytoskeletal defects, including reduced cell motility and adhesion. Some of these defects have been linked to actin arginylation, but the role of other arginylated cytoskeletal proteins has not been studied. Here, we characterize tubulin arginylation and its role in the microtubule cytoskeleton. We identify ATE1-dependent arginylation of ⍺-tubulin at E77. Ate1-/- cells and cells overexpressing non-arginylatable ⍺-tubulinE77A both show a reduced microtubule growth rate and increased microtubule stability. Additionally, they show an increase in the fraction of the stabilizing protein MAP1S associated with microtubules, suggesting that E77 arginylation directly regulates MAP1S binding. Knockdown of Map1s is sufficient to rescue microtubule growth rate and stability to wild-type levels. Together, these results demonstrate a new type of tubulin regulation by posttranslational arginylation, which modulates microtubule growth rate and stability through the microtubule-associated protein, MAP1S.
10.1083/jcb.202406099
Exploration of the cytotoxic and microtubule disruption potential of novel imidazo[1,5-]pyridine-based chalcones.
RSC medicinal chemistry
In continuation of our efforts to develop new anticancer compounds, a new series of imidazo[1,5-]pyridine-chalcone derivatives was designed, synthesized, characterized, and evaluated for its cytotoxicity against five human cancer cell lines, , breast (MDA-MB-231), colon (RKO), bone (Mg-63), prostate (PC-3), and liver (HepG2) cell lines, as well as a normal cell line (HEK). Among the synthesized compounds, two exhibited promising cytotoxicity against the MDA-MB-231 cell line with IC values of 4.23 ± 0.25 μM and 3.26 ± 0.56 μM. We also studied apoptotic induction of the compounds using annexin V-FITC/PI staining, and ROS-mediated mitochondrial damage was elucidated using DCFDA, followed by JC-1 staining. The potential activity of the compounds was further confirmed by immuno-fluorescence and molecular docking studies, which revealed the anticancer activity of active compounds through binding and microtubule disruption.
10.1039/d4md00838c
Visualizing Cartwheel Disassembly Process During Mitosis in Fixed and Live Cells by Fluorescence Microscope.
Methods in molecular biology (Clifton, N.J.)
Centrosome is an evolutionarily conserved organelle that comprises two barrel-shaped centrioles surrounded by pericentriolar material (PCM). It functions as the major microtubule-organizing center (MTOC) to regulate cell polarity, motility, intracellular material transport during interphase, and bipolar spindle assembly during mitosis. Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis at early S phase. In human cells, cartwheel is a transient scaffolding structure that is disassembled during procentriole maturation at mitotic exit. This chapter describes methods for synchronizing mammalian cells into mitosis by small molecule inhibitor treatment and visualizing cartwheel disassembly process during mitosis in fixed and live cells by fluorescence microscope.
10.1007/978-1-0716-4236-8_7
Osmotic stress influences microtubule drug response via WNK1 kinase signaling.
Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy
Ion homeostasis is critical for numerous cellular processes, and disturbances in ionic balance underlie diverse pathological conditions, including cancer progression. Targeting ion homeostasis is even considered as a strategy to treat cancer. However, very little is known about how ion homeostasis may influence anticancer drug response. In a genome-wide CRISPR-Cas9 resistance drug screen, we identified and validated the master osmostress regulator WNK1 kinase as a modulator of the response to the mitotic inhibitor rigosertib. Osmotic stress and WNK1 inactivation lead to an altered response not only to rigosertib treatment but also to other microtubule-related drugs, minimizing the prototypical mitotic arrest produced by these compounds. This effect is due to an alteration in microtubule stability and polymerization dynamics, likely maintained by fluctuations in intracellular molecular crowding upon WNK1 inactivation. This promotes resistance to microtubule depolymerizing compounds, and increased sensitivity to microtubule stabilizing drugs. In summary, our data proposes WNK1 osmoregulation activity as an important modulator for microtubule-associated chemotherapy response.
10.1016/j.drup.2025.101203
Genetics of human handedness: microtubules and beyond.
Trends in genetics : TIG
Handedness (i.e., the preference to use either the left or the right hand for fine motor tasks) is a widely investigated trait. Handedness heritability is consistently estimated to be 25%. After decades of research, recent large-scale genome-wide association and exome sequencing studies have identified multiple genes associated with handedness and highlighted tubulin genes. Tubulin genes play a role in several processes during brain development that may be relevant for handedness ontogenesis, including axon guidance, axon growth, and forming the inner structure of motile cilia. Moreover, tubulin genes are associated with several psychiatric disorders. This finding therefore may offer insights into biological pathways mediating the link between handedness, brain asymmetries, and psychiatric traits.
10.1016/j.tig.2025.01.006
Insights into the role of phosphorylation on microtubule cross-linking by PRC1.
Molecular biology of the cell
The mitotic spindle is composed of distinct networks of microtubules, including interpolar bundles that can bridge sister kinetochore fibers and bundles that organize the spindle midzone in anaphase. The cross-linking protein PRC1 can mediate such bundling interactions between antiparallel microtubules. PRC1 is a substrate of mitotic kinases including CDK/cyclin-B, suggesting that it can be phosphorylated in metaphase and dephosphorylated in anaphase. How these biochemical changes to specific residues regulate its function and ability to organize bundles has been unclear. Here, we perform biophysical analyses on microtubule networks cross-linked by two PRC1 constructs, one a wild-type reflecting a dephosphorylated state, and one phosphomimetic construct with two threonine to glutamic acid substitutions near PRC1's microtubule binding domain. We find that the wild-type construct builds longer and larger bundles that form more rapidly and are much more resistant to mechanical disruption than the phosphomimetic PRC1. Interestingly, microtubule pairs organized by both constructs behave similarly within the same assays. Our results suggest that phosphorylation of PRC1 in metaphase could tune the protein to stabilize smaller and more flexible bundles, while removal of these post-translational modifications in anaphase would promote the assembly of larger, more mechanically robust bundles to resist chromosome and pole separation forces at the spindle midzone.
10.1091/mbc.E24-12-0565
LC3B-regulated autophagy mitigates zinc oxide nanoparticle-induced epithelial cell dysfunction and acute lung injury.
Toxicological sciences : an official journal of the Society of Toxicology
Zinc oxide nanoparticles (ZnONPs) are widely utilized across various industries, raising concerns about their potential toxicity, especially in the respiratory system. This study explores the role of autophagy, regulated by microtubule-associated protein 1A/1B-light chain 3B (LC3B), in ZnONPs-induced toxicity using both in vivo (LC3B knockout mice) and in vitro (BEAS-2B cells) models. Our findings demonstrate that LC3B-regulated autophagy mitigates ZnONPs-induced epithelial cell dysfunction and acute lung injury. In the absence of LC3B, oxidative stress, inflammation, and intracellular zinc accumulation are exacerbated, resulting in mitochondrial dysfunction and epithelial cell death. In vitro, LC3B knockdown disrupted zinc ion transporter expression and impaired mitophagic flux in BEAS-2B cells. Treatment with zinc ion chelators alleviated these toxic effects, confirming that free zinc ions play a critical role in driving ZnONPs toxicity. These findings highlight that targeting autophagy and maintaining zinc homeostasis could offer therapeutic strategies to reduce ZnONPs-induced lung damage.
10.1093/toxsci/kfae146
Stem cell mechanoadaptation. II. Microtubule stabilization and substrate compliance effects on cytoskeletal remodeling.
APL bioengineering
Stem cells adapt to their local mechanical environment by rearranging their cytoskeleton, which underpins the evolution of their shape and fate as well as the emergence of tissue structure and function. Here, in the second part of a two-part experimental series, we aimed to elucidate spatiotemporal cytoskeletal remodeling and resulting changes in morphology and mechanical properties of cells and their nuclei. Akin to mechanical testing of the most basic living and adapting unit of life, i.e., the cell, in model tissue templates, we probed native and microtubule-stabilized (via Paclitaxel, PAX, exposure) stem cells' cytoskeletal adaptation capacity on substrates of increasing compliance (exerting local tension on cells) and with increased target seeding densities (exerting local compression on cells). On 10 and 100 kPa gels, cells seeded at 5000 cells/cm and cells proliferated to 15 000 cells/cm exhibited bulk moduli that nearly matched those of their respective substrates; hence, they exhibited a greater increase in Young's Modulus after microtubule stabilization than cells cultured on glass. Culture on compliant substrates also reduced microtubule-stabilized cells' F-actin, and microtubule concentration increases compared to cells seeded on glass. On gels, F-actin alignment decreased as more randomly oriented, short actin crosslinks were observed, representing emergent adaptation to the compliant substrate, mediated through myosin II contractility. We conclude that stem cell adaptation to compliant substrates facilitates the accommodation of larger loads from the PAX-stabilized polymerizing microtubule, which, in turn, exerts a larger effect in determining cells' capacity to stiffen and remodel the cytoskeleton. Taken as a whole, these studies establish correlations between cytoskeleton and physical and mechanical parameters of stem cells. Hence, the studies progress our understanding of the dynamic cytoskeleton as well as shape changes in cells and their nuclei, culminating in emergent tissue development and healing.
10.1063/5.0231287
Klp2-mediated Rsp1-Mto1 colocalization inhibits microtubule-dependent microtubule assembly in fission yeast.
Science advances
Microtubule assembly takes place at the centrosome and noncentrosomal microtubule-organizing centers (MTOCs). However, the mechanisms controlling the activity of noncentrosomal MTOCs are poorly understood. Here, using the fission yeast as a model organism, we demonstrate that the kinesin-14 motor Klp2 interacts with the J-domain Hsp70/Ssa1 cochaperone Rsp1, an inhibitory factor of microtubule assembly, and that Klp2 is required for the proper localization of Rsp1 to microtubules. In addition, we demonstrate that Klp2 is not required for the localization of Mto1, a factor promoting microtubule assembly, to microtubules. Moreover, Rsp1-Ssa1 inhibits the interaction of Mto1-Mto2 with the gamma-tubulin small complex. The absence of Klp2 reduces the colocalization of Rsp1 and Mto1 foci on preexisting microtubules, resulting in an increased microtubule-dependent microtubule assembly. Our results suggest that Klp2 regulates the activity of noncentrosomal MTOCs by targeting Rsp1 to the sites of Mto1 activity and reveal a mechanism for the inhibition of noncentrosomal microtubule assembly by a kinesin-14 motor.
10.1126/sciadv.adq0670
The microtubule-severing enzyme spastin regulates spindle dynamics to promote chromosome segregation in .
bioRxiv : the preprint server for biology
Microtubule-severing enzymes play essential roles in regulating diverse cellular processes, including mitosis and cytokinesis, by modulating microtubule dynamics. In the early branching protozoan parasite , microtubule-severing enzymes are involved in cytokinesis and flagellum length control during different life cycle stages, but none of them have been found to regulate mitosis in any life cycle form. Here, we report the biochemical and functional characterization of the microtubule-severing enzyme spastin in the procyclic form of . We demonstrate that spastin catalyzes microtubule severing and ectopic overexpression of spastin disrupts spindle microtubules in trypanosome cells, leading to defective chromosome segregation. Knockdown of spastin impairs spindle integrity and disrupts chromosome alignment in metaphase and chromosome segregation in anaphase. We further show that the function of spastin requires the catalytic AAA-ATPase domain, the microtubule-binding domain, and the microtubule interacting and trafficking domain, and that the association of spastin with spindle depends on the microtubule-binding domain. Together, these results uncover an essential role for spastin in chromosome segregation by regulating spindle dynamics in this unicellular eukaryote.
10.1101/2025.01.03.631140
CRMP/UNC-33 maintains neuronal microtubule arrays by promoting individual microtubule rescue.
Current biology : CB
Microtubules (MTs) are intrinsically dynamic polymers. In neurons, staggered individual microtubules form stable, polarized acentrosomal MT arrays spanning the axon and dendrite to support long-distance intracellular transport. How the stability and polarity of these arrays are maintained when individual MTs remain highly dynamic is still an open question. Here, we visualize MT arrays in vivo in C. elegans neurons with single MT resolution. We find that the CRMP family homolog UNC-33 is essential for the stability and polarity of MT arrays in neurites. In unc-33 mutants, MTs exhibit dramatically reduced rescue after catastrophe, develop gaps in coverage, and lose their polarity, leading to trafficking defects. UNC-33 is stably anchored on the cortical cytoskeleton and forms patch-like structures along the dendritic shaft. These discrete and stable UNC-33 patches concentrate free tubulins and correlate with MT rescue sites. In vitro, purified UNC-33 preferentially associates with MT tips and increases MT rescue frequency. Together, we propose that UNC-33 functions as a microtubule-associated protein (MAP) to promote individual MT rescue locally. Through this activity, UNC-33 prevents the loss of individual MTs, thereby maintaining the coverage and polarity of MT arrays throughout the lifetime of neurons.
10.1016/j.cub.2024.12.030
Stem cell mechanoadaptation. I. Effect of microtubule stabilization and volume changing stresses on cytoskeletal remodeling.
APL bioengineering
Here, we report on the first part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling, which underpins the evolution of stem cell shape and fate, and the emergence of tissue structure and function. In Part I of these studies, we first develop protocols to stabilize microtubules exogenously using paclitaxel (PAX) in a standardized model murine embryonic stem cell line (C3H/10T1/2) to maximize comparability with previously published studies. We then probe native and microtubule-stabilized stem cells' capacity to adapt to volume changing stresses effected by seeding at increasing cell densities, which emulates local compression and tissue template formation during development. Within the concentration range of 1-100 nM, microtubule-stabilized stem cells maintain viability and reduce proliferation. PAX stabilization of microtubules is associated with increased cell volume as well as flattening of the cell and nucleus. Compared to control cells, microtubule-stabilized cells exhibit thick, bundled microtubules and highly aligned, thicker and longer F-actin fibers, corresponding to an increase in the Young's modulus of the cell. Both F-actin and microtubule concentration increase with increasing PAX concentration, whereby the increase in F-actin is more prominent in the basal region of the cell. The corresponding increase in microtubule is observed more globally across the apical and basal region of the cell. Seeding at increasing target densities induces local compression on cells. This increase in local compression modulates cell volume and concomitant increases in F-actin and microtubule concentration to a greater degree than microtubule stabilization via PAX. Cells seeded at high density exhibit higher bulk modulus than corresponding cells seeded at low density. These data demonstrate the capacity of stem cells to adapt to an interplay of mechanical and chemical cues, i.e., respective compression and exogenous microtubule stabilization; the resulting cytoskeletal remodeling manifests as evolution of mechanical properties relevant to development of multicellular tissue constructs.
10.1063/5.0231273
Proteasome inhibition induces microtubule-dependent changes in nuclear morphology.
iScience
Cancers and neurodegenerative disorders are associated with both disrupted proteostasis and altered nuclear morphology. Determining if changes in nuclear morphology contribute to pathology requires an understanding of the underlying mechanisms, which are difficult to elucidate in cells where pleiotropic effects of altering proteostasis might indirectly influence nuclear morphology. To investigate direct effects, we studied nuclei assembled in egg extract where potentially confounding effects of transcription, translation, cell cycle progression, and actin dynamics are absent. We report that proteasome inhibition causes acute microtubule-dependent changes in nuclear morphology and stability and altered microtubule dynamics and organization. Proteomic analysis of proteasome-inhibited extracts identified an increased abundance of microtubule nucleator TubGCP6, and TubGCP6 depletion partially rescued nuclear morphology. Key results were confirmed in HeLa cells. We propose that accumulation of TubGCP6 leads to altered microtubule dynamics proximal to the nucleus, producing forces that deform the nucleus and impact nuclear morphology and integrity.
10.1016/j.isci.2024.111550
Selective regulation of kinesin-5 function by β-tubulin carboxy-terminal tails.
The Journal of cell biology
The tubulin code hypothesis predicts that tubulin tails create programs for selective regulation of microtubule-binding proteins, including kinesin motors. However, the molecular mechanisms that determine selective regulation and their relevance in cells are poorly understood. We report selective regulation of budding yeast kinesin-5 motors by the β-tubulin tail. Cin8, but not Kip1, requires the β-tubulin tail for recruitment to the mitotic spindle, creating a balance of both motors in the spindle and efficient mitotic progression. We identify a negatively charged patch in the β-tubulin tail that mediates interaction with Cin8. Using in vitro reconstitution with genetically modified yeast tubulin, we demonstrate that the charged patch of β-tubulin tail increases Cin8 plus-end-directed velocity and processivity. Finally, we determine that the positively charged amino-terminal extension of Cin8 coordinates interactions with the β-tubulin tail. Our work identifies a molecular mechanism underlying selective regulation of closely related kinesin motors by tubulin tails and how this regulation promotes proper function of the mitotic spindle.
10.1083/jcb.202405115
Reprogramming metabolic microenvironment for nerve regeneration via waterborne polylactic acid-polyurethane copolymer scaffolds.
Biomaterials
Cell metabolism, as the key driver of inflammation, revascularization and even subsequent tissue regeneration, is controlled by and also conversely influenced by signal transduction. Incorporation of cell metabolism into tissue engineering research holds immense potential for in-situ treatment repair and further understanding of the host-biomaterial cues in body response. In this study, an anti-inflammatory waterborne polyurethane scaffold incorporated with poly-l-lactic acid (PLLA) block was served to repair nerve injuries (LAx-WPU). Lactate was released through the degradation of LAx-WPU scaffolds, and the content increased with the addition of PLLA block over the degradation times. Thenceforth, the production of adenosine triphosphate (ATP) in primary neurons and neuronal axon growth were achieved by taking up lactate through monocarboxylate transporters (MCT2) for energy metabolism under glucose-free environment treated with LAx-WPU degradation solution. After LAx-WPU was implanted to repair brain nerve defects in rats, filamentous neurons elongation, rapid vascularization, and nerve tissue regeneration were realized up to 28 days with the positive expression of microtubule-associated protein (MAP2), β-tubulin (Tuj1), and platelet endothelial cell adhesion molecule (CD31) in the scaffolds. Results highlighted that the LAx-WPU scaffolds up-regulated not only the ATP-ADP-AMP purine metabolism compounds to mainly bridge neuroactive ligand-receptor interaction genes, cAMP pathway genes, and calcium pathway genes for neurocytes but also the ATP-GMP purine metabolism to angiogenesis in Gene Ontology (GO) analysis. Further analysis in reverse showed axonal regeneration is restrained by the inhibition of MCT2, proving LAx-WPU promoted nerve repair depended on lactate for energy. Therefore, LAx-WPU scaffolds construct an expected way to modulate the metabolic microenvironment for inducing nerve regeneration by intrinsic biomaterial metabolism cues without any bioactive factors.
10.1016/j.biomaterials.2024.122942
A Method for Analyzing Acentrosomal Mitotic Spindles in Human Cells.
Methods in molecular biology (Clifton, N.J.)
Centrosomes, the major microtubule organizing centers, facilitate mitotic spindle formation. However, recent studies have revealed that some cancer cells lack centrosomes. These findings suggest that certain types of cancer cells drive centrosome-independent mechanisms for the assembly of mitotic spindles. Therefore, analyzing the systems of acentrosomal spindle formation is beneficial for understanding the divergence of mitotic processes in cancer cells. Here, we focus on a method for generating acentrosomal cells using the PLK4 inhibitor centrinone and describe the proper conditions, spindle pole staining markers, and microscopy settings for observing acentrosomal spindle formation in human cells.
10.1007/978-1-0716-4224-5_15
Design, synthesis and anti-tumor evaluation of novel pyrimidine and quinazoline analogues.
European journal of medicinal chemistry
Disrupting microtubule dynamics has emerged as a promising strategy for cancer therapy. Novel trimethoxyanilino-substituted pyrimidine and quinazoline derivatives were designed and synthesized to serve as potent microtubule-inhibiting agents with anti-proliferative activity. Compound 2k demonstrates high efficacy against B16-F10 cancer cells at low nanomolar concentrations, with an IC of 0.098 ± 0.006 μM, which is comparable to colchicine. Mechanistic studies have revealed that 2k has the ability to inhibit microtubule protein polymerization in vitro, resulting in cell cycle arrest and apoptosis. Furthermore, 2k inhibits tumor cell migration and exhibits significant anti-tumor efficacy in a melanoma tumor model without causing obvious toxicity. In summary, the pyrimidine derivative 2k exhibits excellent anticancer activity and provides a new scaffold for the development of novel microtubule inhibitors, which deserves further in-depth research.
10.1016/j.ejmech.2024.117057
17β-Estradiol Counteracts Pathological Microtubule Remodeling To Enhance Cardiac Function.
bioRxiv : the preprint server for biology
The female-predominate sex hormone 17β-estradiol exerts cardioprotective effects via multiple mechanisms. Available data demonstrate 17β-estradiol modulates microtubule dynamics , but its effects on pathogenic microtubule remodeling in pressure-overloaded cardiomyocytes are unexplored. Here, we show 17β-estradiol directly blunts microtubule polymerization , counteracts endothelin-mediated microtubule remodeling in iPSC-cardiomyocytes, and mitigates microtubule stabilization in pulmonary artery banded right ventricular cardiomyocytes. 17β-estradiol treatment blunts cardiomyocyte and nuclear hypertrophy, restores t-tubule architecture, and prevents mislocalization of connexin-43 in RV cardiomyocytes of pulmonary artery banded rats. These cellular phenotypes are paired with significant improvements in RV function. Thus, we propose 17β-estradiol exerts cardioprotective effects via direct modulation of microtubules in addition to its well ascribed signaling functions.
10.1101/2025.01.22.634271
Fibrous corona is reduced in cancer cell lines that attenuate microtubule nucleation from kinetochores.
Cancer science
Most cancer cells show increased chromosome missegregation, known as chromosomal instability (CIN), which promotes cancer progression and drug resistance. The underlying causes of CIN in cancer cells are not fully understood. Here we found that breast cancer cell lines show a reduced kinetochore localization of ROD, ZW10, and Zwilch, components of the fibrous corona, compared with non-transformed breast epithelial cell lines. The fibrous corona is a structure formed on kinetochores before their end-on attachment to microtubules and plays a role in efficient kinetochore capture and the spindle assembly checkpoint. The reduction in the fibrous corona was not due to reduced expression levels of the fibrous corona components or to a reduction in outer kinetochore components. Kinetochore localization of Bub1 and CENP-E, which play a role in the recruitment of the fibrous corona to kinetochores, was reduced in cancer cell lines, presumably due to reduced activity of Mps1, which is required for their recruitment to kinetochores through phosphorylating KNL1. Increasing kinetochore localization of Bub1 and CENP-E in cancer cells restored the level of the fibrous corona. Cancer cell lines showed a reduced capacity to nucleate microtubules from kinetochores, which was recently shown to be dependent on the fibrous corona, and increasing kinetochore localization of Bub1 and CENP-E restored the microtubule nucleation capacity on kinetochores. Our study revealed a distinct feature of cancer cell lines that may be related to CIN.
10.1111/cas.16406
High-Resolution Imaging of Spindle Orientation Dynamics in 3D Intestinal Organoids.
Methods in molecular biology (Clifton, N.J.)
Live imaging of microtubules (MTs) allows obtaining mechanistic insights into cell division. In literature, mitotic spindle dynamics have been investigated in mammalian systems largely focusing on established cell lines. Here, we describe a detailed protocol that investigates MT dynamics during cell division in a 3D mouse intestinal organoid model that more accurately captures the in vivo system.
10.1007/978-1-0716-4224-5_18
Synthetic Engineering of Cortical Polarity During Mitosis Using Designed Proteins.
Methods in molecular biology (Clifton, N.J.)
During asymmetric cell division, cortical polarity cues drive the polarization of the microtubule cytoskeleton to ensure the generation of two daughter cells with different fates. While this a critical process for development and tissue homeostasis, the underlying molecular mechanisms orchestrating those processes are not completely understood, especially in mammals. Here, we present an assay that allows the study of the molecular mechanisms driving mammalian asymmetric cell division in a high-throughput manner by capitalizing on protein design to engineer cortical polarity of virtually any protein of interest in otherwise unpolarized mammalian culture cells.
10.1007/978-1-0716-4224-5_17
Visualizing the Submolecular Organization of αβ-Tubulin Subunits on the Microtubule Inner Surface Using Atomic Force Microscopy.
Nano letters
Microtubules (MTs) are dynamic cytoskeletal polymers essential for mediating fundamental cellular processes, including cell division, intracellular transport, and cell shape maintenance. Understanding the arrangement of tubulin heterodimers within MTs is key to their function. Using frequency modulation atomic force microscopy (FM-AFM) and simulations, we revealed the submolecular arrangement of α- and β-tubulin subunits on the inner MT surface. We observed an undulating molecular arrangement of protofilaments (PFs) with alternating height variations, attributed to different structural orientations and the confirmation of αβ-tubulin heterodimers in adjacent PFs, forming bimodal lateral contacts, as confirmed by AFM simulations. Structural defects resulting from missing tubulin units were directly identified. This detailed structural information provides critical insight into the MT functional properties. Our findings highlight the potential of FM-AFM in liquid as a powerful tool for elucidating the complex interactions among MTs, MT-associated proteins, and other molecules, which are essential for understanding MT dynamics in the cellular context.
10.1021/acs.nanolett.4c04294
Microtubule acetylation is not required for HIV-1 infection or TRIM69-mediated restriction of HIV-1 infection.
Journal of virology
Microtubule acetylation, a post-translational modification catalyzing the addition of acetyl groups to lysine residues on alpha tubulin, confers mechanical resilience to microtubules and influences intracellular cargo transport. Despite its known cellular functions, its role in viral infections remains poorly understood. The goal of this study was to determine the role of microtubule acetylation in both HIV-1 infection and TRIM69-mediated restriction. To this end, we generated CRIPSR/Cas9 vectors to disrupt alpha-tubulin acetyltransferase (αTAT1), the main enzyme responsible for microtubule acetylation. We assessed the role of acetylation in HIV-1 infectivity and the degree to which TRIM69 relies on microtubule acetylation for its ability to restrict HIV-1. We determined that microtubule acetylation is not required for HIV-1 infection and that preventing microtubule acetylation actually leads to a modest increase in HIV-1 infection. We also determined that TRIM69 can restrict a diverse range of viruses and that its restriction of HIV-1 does not rely on microtubule acetylation. IMPORTANCE:Although microtubule acetylation is a well-studied post-translational modification in the context of cellular processes, its role during viral infections remains underexplored. Existing studies often rely on various protein and drug perturbations to indirectly examine microtubule acetylation. In this study, we directly target the enzyme responsible for microtubule acetylation to delineate its role in both HIV-1 infection and TRIM69-mediated restriction.
10.1128/jvi.01026-24
Spatial Statistics of Three-Dimensional Growth Dynamics of Spindle Microtubules.
Methods in molecular biology (Clifton, N.J.)
The latest high-resolution 3D live-cell imaging technology, lattice light-sheet microscopy (LLSM), has successfully tracked the dynamics of microtubule growth throughout the entire mitotic spindle with unparalleled precision. By using green fluorescent protein-labeled end-binding protein 1 (EB1-GFP) as a marker for growing microtubule ends, LLSM has generated an extensive collection of multidimensional datasets mapping the positions and trajectories of these growing microtubule ends. Processing this data requires statistical analysis in three-dimensional space. This chapter describes the spatial statistical methods developed for this purpose, illustrated with practical examples. Finally, we discuss future prospects for analyzing complex, large-scale image data.
10.1007/978-1-0716-4224-5_4
Super-Resolution Imaging of Mitotic Spindle Microtubules Using STED Microscopy.
Methods in molecular biology (Clifton, N.J.)
Stimulated emission depletion (STED) microscopy is a powerful super-resolution imaging technique that only recently entered the field of mitosis, where it proved to be invaluable for studying various microtubule classes, kinetochore-microtubule attachments and chromosome segregation errors. Here, we describe immunofluorescence combined with STED microscopy as a method for analyzing microtubules and kinetochore-microtubule attachments in human mitotic spindles. We also describe live-cell STED microscopy as a method for single-plane short-term imaging of transient processes in crowded spindle areas. Finally, we outline image analysis approaches for the quantitative assessment of microtubule bundles within the spindle.
10.1007/978-1-0716-4224-5_1
Modulation of microtubule dynamics by monovalent ions.
PNAS nexus
The microtubule cytoskeleton is a dynamic network essential for many cellular processes, influenced by physicochemical factor, such as temperature, pH, dimer concentration, and ionic environment. In this study, we used in vitro reconstitution assays to examine the effects of four monovalent ions (Na, K, Cl, and Ac) on microtubule dynamics, uncovering distinct effects for each ion. Na was found to increase microtubule dynamicity by raising catastrophe frequency, polymerization and depolymerization speeds, and ultimately reducing microtubule lifetime by 80%. Conversely, Ac boosts microtubule nucleation and stabilizes microtubules by increasing rescue frequency and preventing breakages, resulting in longer microtubules with extended lifetimes. Cl appeared to potentiate the effects of Na, while K had minimal impact on microtubule dynamic parameters. These findings demonstrate that Na and Ac have opposing effects on microtubule dynamics, with Na destabilizing and Ac stabilizing the microtubule structure. This ionic impact is mainly through modulation of tubulin-tubulin interactions rather than affecting the hydrolysis rate. In conclusion, ion identity plays a crucial role in modulating microtubule dynamics. Understanding the ionic environment is essential for microtubule-related research, as it significantly influences microtubule behavior, stability, and interactions with other proteins.
10.1093/pnasnexus/pgae544
Structure of the ciliary tip central pair reveals the unique role of the microtubule-seam binding protein SPEF1.
bioRxiv : the preprint server for biology
Motile cilia are unique organelles with the ability to autonomously move. Force generated by beating cilia propels cells and moves fluids. The ciliary skeleton is made of peripheral doublet microtubules and a central pair (CP) with a distinct structure at the tip. In this study, we present a high-resolution structure of the CP in the ciliary tip of the ciliate and identify several tip proteins that bind and form unique patterns on both microtubules of the tip CP. Two of those proteins that contain tubulin polymerization-promoting protein (TPPP)-like domains, TLP1 and TLP2, bind to high curvature regions of the microtubule. TLP2, which contains two TPPP-like domains, is an unusually long protein that wraps laterally around half a microtubule and forms the bridge between the two microtubules. Moreover, we found that the conserved protein SPEF1 binds to both microtubule seams. , human SPEF1 not only binds to the microtubule seam but also crosslinks two parallel microtubules. Single-molecule microtubule dynamics assays indicate that SPEF1 stabilizes microtubules . Together, these data show that the proteins in the tip CP maintain stable microtubule structure and probably play important roles in maintaining the integrity of the axoneme.
10.1101/2024.12.02.626492
The disordered effector RipAO of Ralstonia solanacearum destabilizes microtubule networks in Nicotiana benthamiana cells.
Molecules and cells
Ralstonia solanacearum causes bacterial wilt, a devastating disease in solanaceous crops. The pathogenicity of R. solanacearum depends on its type III secretion system, which delivers a suite of type III effectors into plant cells. The disordered core effector RipAO is conserved across R. solanacearum species and affects plant immune responses when transiently expressed in Nicotiana benthamiana. Specifically, RipAO impairs pathogen-associated molecular pattern-triggered reactive oxygen species production, an essential plant defense mechanism. RipAO fused to yellow fluorescent protein initially localizes to filamentous structures, resembling the cytoskeleton, before forming large punctate aggregates around the nucleus. Consistent with these findings, tubulin alpha 6 (TUA6) and tubulin beta-1, building blocks of microtubules, were identified as putative targets of RipAO in immunoprecipitation and mass spectrometry analyses. In the presence of RipAO, TUA6-labeled microtubules fragmented into puncta, mimicking the effects of oryzalin, a microtubule polymerization inhibitor. These effects were corroborated in a N. benthamiana transgenic line constitutively expressing green fluorescent protein-labeled TUA6, where RipAO reduced microtubule density and stability at an accumulation level that did not induce aggregation. Moreover, oryzalin treatment further enhanced RipAO's impairment of reactive oxygen species production, suggesting that RipAO disrupts microtubule networks via its association with tubulins, leading to immune suppression. Further research into RipAO's interaction with the microtubule network will enhance our understanding of bacterial strategies to subvert plant immunity.
10.1016/j.mocell.2024.100167
Bi-allelic variants in WDR47 cause a complex neurodevelopmental syndrome.
EMBO molecular medicine
Brain development requires the coordinated growth of structures and cues that are essential for forming neural circuits and cognitive functions. The corpus callosum, the largest interhemispheric connection, is formed by the axons of callosal projection neurons through a series of tightly regulated cellular events, including neuronal specification, migration, axon extension and branching. Defects in any of those steps can lead to a range of disorders known as syndromic corpus callosum dysgenesis (CCD). We report five unrelated families carrying bi-allelic variants in WDR47 presenting with CCD together with other neuroanatomical phenotypes such as microcephaly and enlarged ventricles. Using in vitro and in vivo mouse models and complementation assays, we show that WDR47 is required for survival of callosal neurons by contributing to the maintenance of mitochondrial and microtubule homeostasis. We further propose that severity of the CCD phenotype is determined by the degree of the loss of function caused by the human variants. Taken together, we identify WDR47 as a causative gene of a new neurodevelopmental syndrome characterized by corpus callosum abnormalities and other neuroanatomical malformations.
10.1038/s44321-024-00178-z
The kinetochore protein KNL-1 regulates the actin cytoskeleton to control dendrite branching.
The Journal of cell biology
The function of the nervous system is intimately tied to its complex and highly interconnected architecture. Precise control of dendritic branching in individual neurons is central to building the complex structure of the nervous system. Here, we show that the kinetochore protein KNL-1 and its associated KMN (Knl1/Mis12/Ndc80 complex) network partners, typically known for their role in chromosome-microtubule coupling during mitosis, control dendrite branching in the Caenorhabditis elegans mechanosensory PVD neuron. KNL-1 restrains excess dendritic branching and promotes contact-dependent repulsion events, ensuring robust sensory behavior and preventing premature neurodegeneration. Unexpectedly, KNL-1 loss resulted in significant alterations of the actin cytoskeleton alongside changes in microtubule dynamics within dendrites. We show that KNL-1 modulates F-actin dynamics to generate proper dendrite architecture and that its N-terminus can initiate F-actin assembly. These findings reveal that the postmitotic neuronal KMN network acts to shape the developing nervous system by regulating the actin cytoskeleton and provide new insight into the mechanisms controlling dendrite architecture.
10.1083/jcb.202311147
Tracking Single Kinesin in Live Cells Using MINFLUX.
Methods in molecular biology (Clifton, N.J.)
MINFLUX is a super-resolution fluorescence microscopy technique that enables single-molecule tracking in live cells at a single-nanometer spatial and sub-millisecond temporal resolution. This chapter describes a method for tracking fluorescently labeled human kinesin-1 in live cells using MINFLUX and analyzing kinesin stepping dynamics.
10.1007/978-1-0716-4280-1_5
Isolation of Mitotic Centrosomes from Cultured Human Cells.
Methods in molecular biology (Clifton, N.J.)
The centrosome plays a crucial role in facilitating mitotic spindle assembly through its microtubule organizing capacities. Analyzing the composition, structure, and functions of mitotic centrosomes is essential for understanding the mechanisms underlying cell division and centrosome-associated diseases. Isolating centrosomes is an effective method to gain comprehensive information about them while minimizing interference from other cellular components. In this chapter, we describe a protocol for isolating mitotic centrosomes from cultured human cells. This protocol includes cell synchronization and centrosome isolation through ultracentrifugation with a sucrose gradient. We also describe a method for conducting a microtubule nucleation assay to assess the functionality of isolated centrosomes.
10.1007/978-1-0716-4224-5_16
Measurements of neurite extension and nucleokinesis in an iPSC-derived model system following microtubule perturbation.
Molecular biology of the cell
In neurons, patterns of different microtubule types are essential for neurite extension and nucleokinesis. Cellular model systems such as rodent primary cultures and induced pluripotent stem cells (iPSC)-derived neurons have provided key insights into how these patterns are created and maintained through the action of microtubule-associated proteins, motor proteins, and regulatory enzymes. iPSC-derived models show tremendous promise but lack benchmarking and validation relative to rodent primary cultures. Here we have characterized a recent iPSC-derived model, in which doxycycline-induced expression of Neurogenin-2 drives consistent transdifferentiation into the neuronal state (EBiSC-NEUR1 neurons, referred to as NGN2 neurons below). We developed a suite of open-access, semiautomated methods to measure neurite extension and nucleokinesis of NGN2 neurons, which compare favorably to published data from other models. Then, we challenged NGN2 neurons with a panel of drugs that perturb microtubule physiology. NGN2 neurons extension and nucleokinesis were significantly perturbed by two microtubule-targeting drugs, namely a taxane (paclitaxel) and a vinca alkaloid (DZ-2384). In contrast, inhibition of microtubule severing (spastazoline) or of deacetylation (trichostatin A) had a limited effect on nucleokinesis only. Our results support the primary importance of microtubule dynamics in neuronal development and demonstrate the power of NGN2 neurons as a model system.
10.1091/mbc.E24-02-0061
Tumor microtubes: A new potential therapeutic target for high-grade gliomas.
Journal of neuropathology and experimental neurology
High-grade infiltrating gliomas are highly aggressive and fatal brain tumors that present significant challenges for research and treatment due to their complex microenvironment and tissue structure. Recent discovery of tumor microtubes (TMs) has provided new insights into how high-grade gliomas develop in the brain and resist treatment. TMs are unique, ultra-long, and highly functional membrane protrusions that form multicellular networks and play crucial roles in glioma invasiveness, drug resistance, recurrence, and heterogeneity. This review focuses on the different roles that TMs play in glioma cell communication, material transport, and tumor cell behavior. Specifically, non-connecting TMs primarily promote glioma invasiveness, likely related to their role in enhancing cell motility. On the other hand, interconnecting TMs form functional and communication networks by connecting with surrounding astrocytes and neurons, thereby promoting glioma malignancy. We summarize the factors that influence the formation of TMs in gliomas and current strategies targeting TMs. As the understanding of TMs advances, we are closer to uncovering whether they might be the long-sought Achilles' heel of treatment-resistant gliomas. By delving deeper into TMs research, we hope to develop more effective therapeutic strategies for patients with malignant gliomas.
10.1093/jnen/nlae119
Proteostasis as a fundamental principle of Tau immunotherapy.
Brain : a journal of neurology
The microtubule-associated protein Tau is a driver of neuronal dysfunction in Alzheimer's disease and other tauopathies. In this process, Tau initially undergoes subtle changes to its abundance, subcellular localization and a vast array of post-translational modifications including phosphorylation that progressively result in the protein's somatodendritic accumulation and dysregulation of multiple Tau-dependent cellular processes. Given the various loss- and gain-of-functions of Tau in disease and the brain-wide changes in the proteome that characterize tauopathies, we asked whether targeting Tau would restore the alterations in proteostasis observed in disease. Therefore, by phage display, we generated a novel pan-Tau antibody, RNJ1, that preferentially binds human Tau and neutralizes proteopathic seeding activity in multiple cell lines and benchmarked it against a clinically tested pan-Tau antibody, HJ8.5 (murine version of tilavonemab). We then evaluated both antibodies, alone and in combination, in the K3 tauopathy mouse model, showing reduced Tau pathology and improvements in neuronal function following 14 weekly treatments, without obtaining synergy for the combination. These effects were more pronounced in female mice. To investigate the molecular mechanisms contributing to improvements in neuronal function, we employed quantitative proteomics, phosphoproteomics and kinase prediction analysis to first establish alterations in K3 mice relative to wild-type controls at the proteome level. In female K3 mice, we found 342 differentially abundant proteins, which are predominantly involved in metabolic and microtubule-associated processes, strengthening previously reported findings of defects in several functional domains in multiple tauopathy models. We next asked whether antibody-mediated Tau target engagement indirectly affects levels of deregulated proteins in the K3 model. Importantly, both immunotherapies, in particular RNJ1, induced abundance shifts towards a restoration to wild-type levels (proteostasis). A total of 257 of 342 (∼75%) proteins altered in K3 were closer in abundance to wild-type levels after RNJ1 treatment, and 73% after HJ8.5 treatment. However, the magnitude of these changes was less pronounced than that observed with RNJ1. Furthermore, analysis of the phosphoproteome showed an even stronger restoration effect with RNJ1, with ∼82% of altered phosphopeptides in K3 showing a shift to wild-type levels, and 75% with HJ8.5. Gene set over-representation analysis further confirmed that proteins undergoing restoration are involved in biological pathways affected in K3 mice. Together, our study suggests that a Tau immunotherapy-induced restoration of proteostasis links target engagement and treatment efficacy.
10.1093/brain/awae254
CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation.
Life science alliance
Establishing the correct kinetochore-microtubule attachment is crucial for faithful chromosome segregation. The kinetochore has various regulatory mechanisms for establishing correct bipolar attachment. However, how the regulations are coupled is not fully understood. Here, we demonstrate a regulatory loop between the kinetochore protein CENP-C and Aurora B kinase, which is critical for the error correction of kinetochore-microtubule attachment. This regulatory loop is mediated through the binding of CENP-C to the outer kinetochore Mis12 complex (Mis12C). Although the Mis12C-binding region of CENP-C is dispensable for mouse development and proliferation in human RPE-1 cells, those cells lacking this region display increased mitotic defects. The CENP-C-Mis12C interaction facilitates the centromeric recruitment of Aurora B and the mitotic error correction in human cells. Given that Aurora B reinforces the CENP-C-Mis12C interaction, our findings reveal a positive regulatory loop between Aurora B recruitment and the CENP-C-Mis12C interaction, which ensures chromosome biorientation for accurate chromosome segregation.
10.26508/lsa.202402927
Non-canonical CDK6 activity promotes cilia disassembly by suppressing axoneme polyglutamylation.
The Journal of cell biology
Tubulin polyglutamylation is a posttranslational modification that occurs primarily along the axoneme of cilia. Defective axoneme polyglutamylation impairs cilia function and has been correlated with ciliopathies, including Joubert Syndrome (JBTS). However, the precise mechanisms regulating proper axoneme polyglutamylation remain vague. Here, we show that cyclin-dependent kinase 6 (CDK6), but not its paralog CDK4, localizes to the cilia base and suppresses axoneme polyglutamylation by phosphorylating RAB11 family interacting protein 5 (FIP5) at site S641, a critical regulator of cilia import of glutamylases. S641 phosphorylation disrupts the ciliary recruitment of FIP5 and its association with RAB11, thereby reducing the ciliary import of glutamylases. Encouragingly, the FDA-approved CDK4/6 inhibitor Abemaciclib can effectively restore cilia function in JBTS cells with defective glutamylation. In summary, our study elucidates the regulatory mechanisms governing axoneme polyglutamylation and suggests that developing CDK6-specific inhibitors could be a promising therapeutic strategy to enhance cilia function in ciliopathy patients.
10.1083/jcb.202405170
Tubulin sequence divergence is associated with the use of distinct microtubule regulators.
Current biology : CB
Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it difficult to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria expresses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule-binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule-binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners.
10.1016/j.cub.2024.11.022
Microtubule-Targeting NAP Peptide-Ru(II)-polypyridyl Conjugate As a Bimodal Therapeutic Agent for Triple Negative Breast Carcinoma.
Journal of the American Chemical Society
Triple-negative breast cancer (TNBC) poses significant treatment challenges due to its high metastasis, heterogeneity, and poor biomarker expression. The N-terminus of an octapeptide NAPVSIPQ () was covalently coupled to a carboxylic acid derivative of Ru(2,2'-bipy) () to synthesize an N-stapled short peptide-Rubpy conjugate (). This photosensitizer (PS) was utilized to treat TNBC through microtubule (MT) targeted chemotherapy and photodynamic therapy (PDT). formed more elaborate molecular aggregates with fibrillar morphology as compared to . A much higher binding affinity of over toward β-tubulin (: (6.8 ± 0.55) × 10 M; : (8.2 ± 1.1) × 10 M) was observed due to stronger electrostatic interactions between the MT with an average linear charge density of ∼85 e/nm and the cationic part of . This was also supported by docking, simulation, and appropriate imaging studies. promoted serum stability, specific binding of to the E-site of the β-tubulin followed by the disruption of the MT network, and effective singlet oxygen generation in TNBC cells (MDA-MB-231), causing cell cycle arrest in the G2/M phase and triggering apoptosis. Remarkably, MDA-MB-231 cells were more sensitive to compared to noncancerous human embryonic kidney (HEK293 cells) when exposed to light (IC[HEK293]: 17.2 ± 2.5 μM, compared to IC[MDA-MB-231]: 32.5 ± 7.8 nM, IC[HEK293]: > 80 μM, compared to IC[MDA-MB-231]: 2.9 ± 0.5 μM). also effectively inhibited tumor growth in MDA-MB-231 xenograft models in nude mice. Our findings provide strong evidence that has a potential therapeutic role in TNBC treatment.
10.1021/jacs.4c11820
The journey of antibody-drug conjugates for revolutionizing cancer therapy: A review.
Bioorganic & medicinal chemistry
Antibody-drug conjugates (ADCs) represent a powerful class of targeted cancer therapies that harness the specificity of monoclonal antibodies to deliver cytotoxic payloads directly to tumor cells, minimizing off-target effects. This review explores the advancements in ADC technologies, focusing on advancing next-generation ADCs with novel payloads, conjugation strategies, and enhanced pharmacokinetic profiles. In particular, we highlight innovative payloads, including microtubule inhibitors, spliceosome modulators, and RNA polymerase inhibitors, that offer new mechanisms of cytotoxicity beyond traditional apoptosis induction. Additionally, the introduction of sophisticated conjugation techniques, such as site-specific conjugation using engineered cysteines, enzymatic methods, and integration of non-natural amino acids, has greatly improved the homogeneity, efficacy, and safety of ADCs. Furthermore, the review delves into the mechanistic insights into ADC action, detailing the intracellular pathways that facilitate drug release and cell death, and discussing the significance of bioconjugation methods in optimizing drug-antibody ratios (DARs). The establishment of comprehensive databases like ADCdb, which catalog vital pharmacological and biological data for ADCs, is also explored as a critical resource for advancing ADC research and clinical application. Finally, the clinical landscape of ADCs is examined, with a focus on the evolution of FDA-approved ADCs, such as Gemtuzumab Ozogamicin and Trastuzumab Emtansine, as well as emerging candidates in ongoing trials. As ADCs continue to evolve, their potential to revolutionize cancer therapy remains immense, offering new hope for more effective and personalized treatment options. ADCs also offer a significant advancement in targeted cancer therapy by merging the specificity of monoclonal antibodies with cytotoxic potency of chemotherapeutic agents. Hence, this dual mechanism intensifies tumor selectivity while minimizing systemic toxicity, paving the way for more effective and safer cancer treatments.
10.1016/j.bmc.2024.118010
Mechanistic basis of temperature adaptation in microtubule dynamics across frog species.
Current biology : CB
Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking, as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures. We determine the microtubule structure across all three species at between 3.0 and 3.6 Å resolution by cryo-electron microscopy and find small differences at the β-tubulin lateral interactions. Using in vitro reconstitution assays and quantitative biochemistry, we show that tubulin's free energy scales inversely with temperature. The observed weakening of lateral contacts and the low apparent activation energy for tubulin incorporation provide an explanation for the overall stability and higher growth rates of microtubules in cold-adapted frog species. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches.
10.1016/j.cub.2024.12.022
Interplay of force and local mechanisms in axonal plasticity and beyond.
Biochimica et biophysica acta. Molecular cell research
The interactions between mechanical forces and neuronal dynamics have long intrigued researchers. Several studies revealed that force plays a pivotal role in shaping axonal outgrowth. However, the molecular mechanisms underpinning force-driven axonal plasticity remain not completely elucidated. This review explores the relationship between force and axonal plasticity, with a focus on local mechanisms, including local translation and axonal transport, and the emerging concept of force-driven cross-talk, a dialogue in which local dynamics are tightly regulated. Recent experimental evidence suggests that microtubules may serve as key mediators of this cross-talk, orchestrating the coordination between local mechanisms and facilitating mass addition.
10.1016/j.bbamcr.2024.119874
Axonemal microtubule dynamics in the assembly and disassembly of cilia.
Biochemical Society transactions
Cilia and eukaryotic flagella (exchangeable terms) function in cell motility and signaling, which are pivotal for development and physiology. Cilia dysfunction can lead to ciliopathies. Cilia are usually assembled in quiescent and/or differentiated cells and undergo disassembly when cells enter cell cycle or in response to environmental stresses. Cilia contain a microtubule-based structure termed axoneme that comprises nine outer doublet microtubules with or without a pair of central microtubules, which is ensheathed by the ciliary membrane. Regulation of the axonemal microtubule dynamics is tightly associated with ciliary assembly and disassembly. In this short review, we discuss recent findings on the regulation of axonemal microtubules by microtubule-binding proteins and microtubule modulating kinesins during ciliary assembly and disassembly.
10.1042/BST20240688
A polarized multicomponent foundation upholds ciliary central microtubules.
Journal of molecular cell biology
Cilia's back-and-forth beat pattern requires a central pair (CP) of microtubules. However, the mechanism by which the CP is upheld above the transition zone (TZ) remains unclear. Here, we showed that a rod-like substructure marked by Cep131 and ciliary Centrin serves as a polarized CP-supporting foundation. This CP-foundation (CPF) was assembled independently of the CP during ciliogenesis in mouse ependymal cells. It protruded from the distal end of the basal body out of the TZ to enwrap the proximal end of the CP. Through proximity labeling, we identified 26 potential CPF components, among which Ccdc148 specifically localized at the proximal region of Centrin-decorated CPF and was complementary to the Cep131-enriched distal region. Cep131 deficiency abolished the CPF, resulting in CP penetration into the TZ. Consequently, cilia became prone to ultrastructural abnormality and paralysis, and Cep131-deficient mice were susceptible to late-onset hydrocephalus. In addition to Centrin, phylogenetic analysis also indicated conservations of Ccdc131 and Ccdc148 from protists to mammals, suggesting that the CPF is an evolutionarily conserved multicomponent CP-supporting platform in cilia.
10.1093/jmcb/mjae031
Evolution and functional divergence of the Fidgetin family.
Biochimica et biophysica acta. Molecular cell research
The Fidgetin (FIGN) family, which comprises FIGN, Fidgetin-like 1 (FIGNL1), and Fidgetin-like 2 (FIGNL2), is a vital group of microtubule-severing proteins. These proteins feature a conserved AAA+ domain essential for ATPase activity and a hexameric assembly. This review provides an in-depth analysis of the evolution and functional divergence of the FIGN family members, highlighting their role in the dynamic organization of the cytoskeleton. We further explore their broader biological functions across various species, systems, and subcellular localization. Although the FIGN family is conserved, each member exhibits unique structural characteristics and functions that reflect their evolutionary adaptations. FIGNL1 is found across animal species, while FIGNL2 is specific to vertebrates, thereby indicating its more recent evolutionary origin. Moreover, synteny analysis has revealed that FIGN is located in a more conserved genomic region compared to FIGNL2, which has undergone substantial evolutionary changes. The expression patterns of the FIGN members also vary across organisms and tissues. For example, FIGNL2 shows a notably reduced expression in the mammalian nervous system compared to that in lower vertebrates. The FIGN family members have distinct roles in microtubule severing, cell division, and DNA repair. Specifically, FIGN is involved in cell division and neuronal regeneration, FIGNL1 in axonal growth and DNA repair, and FIGNL2 in cell migration and vascular development. Their involvement in these processes underscores their role as potential biomarkers for certain cancers as well as therapeutic targets for diseases affecting the nervous system and cardiovascular development. All these evolutionary insights and functional distinctions of the FIGN family offer a comprehensive framework for understanding cytoskeletal regulation and its implications in health and disease.
10.1016/j.bbamcr.2024.119870
Recent advances of selenized tubulin inhibitors in cancer therapy.
Bioorganic & medicinal chemistry letters
Cancer treatment always a huge challenge amidst the resistance and relapse caused by the various treatments. Inhibitors targeting mitosis have been considered as promising therapeutic drugs in clinic, of which tubulins play an important role. Selenium (Se) as an essential microelement in humans and animals, playing a crucial role in the formation of anti-oxidase (glutathione peroxidase) and selenoprotein, also attracted broad attention in cancer therapy. Because the introduction of Se atom could change the length and angle of chemical bond and alter their functional properties, regulating selenized chemotherapeutics has become one of the hot spots. However, little attention has been paid to studying the combination of Se and tubulin inhibitors. Herein, we review the latest research results of selenized tubulin inhibitors in cancer therapy, including its mechanisms, categories and biological activities, providing a theoretical basis for different selenized microtubules inhibitors therapies.
10.1016/j.bmcl.2024.130037
B-type Plexins Regulate Mitosis via RanGTPase.
Molecular cancer research : MCR
Aberrant mitosis can result in aneuploidy and cancer. The small GTPase, Ras-related nuclear protein (Ran), is a key regulator of mitosis. B-type plexins regulate Ran activity by acting as RanGTPase-activating proteins and have been implicated in cancer progression. However, whether B-type plexins have a role in mitosis has not so far been investigated. We show here that Plexin B1 functions in the control of mitosis. Depletion of Plexin B1 affects mitotic spindle assembly, significantly delaying anaphase. This leads to mitotic catastrophe in some cells and prolonged application of the spindle assembly checkpoint. Plexin B1 depletion also promoted acentrosomal microtubule nucleation and defects in spindle pole refocusing and increased the number of cells with multipolar or aberrant mitotic spindles. An increase in lagging chromosomes or chromosomal bridges at anaphase was also found upon Plexin B1 depletion. Plexin B1 localizes to the mitotic spindle in dividing cells. The mitotic defects observed upon Plexin B1 depletion were rescued by an RCC1 inhibitor, indicating that Plexin B1 signals, via Ran, to affect mitosis. These errors in mitosis generated multinucleate cells and nuclei of altered morphology and abnormal karyotype. Furthermore, semaphorin 4D treatment increased the percentage of cells with micronuclei, precursors of chromothripsis. Implications: Defects in B-type plexins may contribute to the well-established role of plexins in cancer progression by inducing chromosomal instability.
10.1158/1541-7786.MCR-23-0836
Systematic Identification of Microtubule Posttranslational Modification "Readers" by Quantitative Proteomics.
Methods in molecular biology (Clifton, N.J.)
Microtubules, dynamic polymers assembled from α, β-tubulin dimers, contribute to myriad cellular processes. This is largely attributed to microtubule-associated proteins (MAPs). How MAPs selectively bind microtubules to carry out various functions is not known. The "Tubulin Code" theory proposes that posttranslational modifications (PTMs) of microtubules serve as signs that can be read by specific MAPs, thereby conferring specific functional properties to the microtubules. In support of this hypothesis, "reader" MAPs have been identified for various tubulin PTMs, but, until recently, no systematic screening had been performed to identify readers in an unbiased manner. We addressed this by developing a reader identification pipeline that uses quantitative mass spectrometry to interrogate the microtubule proteome of cells programmed to express specific PTMs. This pipeline can be used to identify readers for any tubulin PTM from various cell types as long as the writer enzymes are known. We also provide an alternative, complementary approach to obtain modified microtubules using a generic writer enzyme in vitro.
10.1007/978-1-0716-4224-5_13
Looking Under the Hood at the Cytoskeletal Engine of Platelet Production.
Arteriosclerosis, thrombosis, and vascular biology
Blood platelets are anucleate cells essential for normal blood hemostasis. To maintain a normal platelet count of 150 000 to 400 000 per μL of blood, 10 platelets must be released each day from precursor cells called megakaryocytes. In this review, we aim to provide an overview of platelet production and evaluate the proposed mechanisms of platelet generation. We will discuss novel cytoskeletal mechanisms of platelet production, including microtubule and actin-based systems. We present new evidence that supports a cytoplasmic trigger for platelet production, discuss centrosome clustering as a new mechanism to trigger proplatelet production, and review new data supporting the bone marrow as the major location of platelet production.
10.1161/ATVBAHA.124.320392
Kinetochores grip microtubules with directionally asymmetric strength.
The Journal of cell biology
For accurate mitosis, all chromosomes must achieve "biorientation," with replicated sister chromatids coupled via kinetochores to the plus ends of opposing microtubules. However, kinetochores first bind the sides of microtubules and subsequently find plus ends through a trial-and-error process; accurate biorientation depends on the selective release of erroneous attachments. Proposed mechanisms for error-correction have focused mainly on plus-end attachments. Whether erroneous side attachments are distinguished from correct side attachments is unknown. Here, we show that side-attached kinetochores are very sensitive to microtubule polarity, gripping sixfold more strongly when pulled toward plus versus minus ends. This directionally asymmetric grip is conserved in human and yeast subcomplexes, and it correlates with changes in the axial arrangement of subcomplexes within the kinetochore, suggesting that internal architecture dictates attachment strength. We propose that the kinetochore's directional grip promotes accuracy during early mitosis by stabilizing correct attachments even before both sisters have found plus ends.
10.1083/jcb.202405176
Functional genetics reveals modulators of antimicrotubule drug sensitivity.
The Journal of cell biology
Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to antimicrotubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug paclitaxel. We further conducted a focused secondary screen to test drug sensitivity for ∼1,400 gene targets across two distinct human cell lines and to additionally test sensitivity to the KIF11 inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to antimicrotubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.
10.1083/jcb.202403065
Triplet Energy Migration in Cytoskeletal Polymers.
The journal of physical chemistry. B
Dexter energy transfer (DET) of triplet electronic states is used to direct energy in photovoltaics, quench reactive singlet oxygen species in biological systems, and generate them in photodynamic therapy. However, the extent to which repeated DET between aromatic residues can lead to triplet energy migration in proteins has not been investigated. Here, we computationally describe DET rates in microtubules, actin filaments and the intermediate filament, vimentin. We discover instances where interaromatic residue Dexter couplings within individual protein subunits of these polymers are similar those of small molecules used for organic electronics. However, interaromatic residue coupling is mostly weak (<10 eV), limiting triplet energy diffusion lengths to 6.1, 0.5 and 1.0 Å in microtubules, actin filaments and vimentin, respectively. On the other hand, repeated förster resonance energy transfer (FRET) between aromatic residues leads to singlet energy diffusion lengths of 12.4 Å for actin filaments and about 8.6 Å for both microtubules and vimentin filaments. Our work shows that singlet energy migration dominates over triplet energy migration in cytoskeletal polymers.
10.1021/acs.jpcb.4c06748
Structural response of microtubule and actin cytoskeletons to direct intracellular load.
The Journal of cell biology
Microtubule and actin are the two major cytoskeletal polymers that form organized functional structures in the interior of eukaryotic cells. Although the structural mechanics of the cytoskeleton has been extensively studied by direct manipulations in in vitro reconstitution systems, such unambiguous characterizations inside the living cell are sparse. Here, we report a comprehensive analysis of how the microtubule and actin cytoskeletons structurally respond to direct intracellular load. Ferrofluid-based intracellular magnetic tweezers reveal rheological properties of the microtubule complex primarily determined by filamentous actin. The strain fields of the microtubule complex and actin meshwork follow the same scaling, suggesting that the two cytoskeletal systems behave as an integrated elastic body. The structural responses of single microtubules to contact and remote forces further evidence that the individual microtubules are enclosed by the elastic medium of actin. These results, directly characterizing the microtubule and actin cytoskeletons as an interacting continuum throughout the cytoplasm, serve as a cornerstone for the physical understanding of intracellular organization.
10.1083/jcb.202403136
Tubulin/HDAC dual-target inhibitors: Insights from design strategies, SARs, and therapeutic potential.
European journal of medicinal chemistry
Microtubules, one of the cytoskeletons in eukaryotic cells, maintain the proper operation of several cellular functions. Additionally, they are regulated by the acetylation of HDAC6 and SIRT2 which affects microtubule dynamics. Given the fact that tubulin and HDAC inhibitors play a synergistic effect in the treatment of many cancers, the development of tubulin/HDAC dual-target inhibitors is conducive to addressing multiple limitations including drug resistance, dose toxicity, and unpredictable pharmacokinetic properties. At present, tubulin/HDAC dual-target inhibitors have been obtained in three main ways: uncleavable linked pharmacophores, cleavable linked pharmacophores, and modification of single-target drugs. Their therapeutic efficacy has been verified in vivo and in vitro assays. In this article, we reviewed the research progress of tubulin/HDAC dual inhibitors from design strategies, SARs, and biological activities, which may provide help for the discovery of novel tubulin/HDAC dual inhibitors.
10.1016/j.ejmech.2024.117022
StableMARK-decorated microtubules in cells have expanded lattices.
The Journal of cell biology
Microtubules are crucial in cells and are regulated by various mechanisms like posttranslational modifications, microtubule-associated proteins, and tubulin isoforms. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but it has remained unclear to what extent different lattices co-exist within the cell. Using cryo-electron tomography, we find that, while most microtubules have a compacted lattice (∼41 Å monomer spacing), approximately a quarter of the microtubules displayed more expanded lattice spacings. The addition of the microtubule-stabilizing agent Taxol increased the lattice spacing of all microtubules, consistent with results on reconstituted microtubules. Furthermore, correlative cryo-light and electron microscopy revealed that the stable subset of microtubules labeled by StableMARK, a marker for stable microtubules, predominantly displayed a more expanded lattice spacing (∼41.9 Å), further suggesting a close connection between lattice expansion and microtubule stability. The coexistence of different lattices and their correlation with stability implicate lattice spacing as an important factor in establishing specific microtubule subsets.
10.1083/jcb.202206143
Cellular and Nuclear Forces: An Overview.
Methods in molecular biology (Clifton, N.J.)
Biological cells sample their surrounding microenvironments using nanoscale force sensors on the cell surfaces. These surface-based force and stress sensors generate physical and chemical responses inside the cell. The inherently well-connected cytoskeleton and its physical contacts with the force elements on the nuclear membrane lead these physicochemical responses to cascade all the way inside the cell nucleus, physically altering the nuclear state. These physical alterations of the cell nucleus, through yet-unknown complex steps, elicit physical and functional responses from the chromatin in the form of altered gene expression profiles. This mechanism of force/stress sensing by the cell and then its nuclear response has been shown to play a vital role in maintaining robust cellular homeostasis, controlling gene expression profiles during developmental phases as well as cell differentiation. In the last few years, there has been appreciable progress toward the identification of the molecular players responsible for force sensing. However, the actual sensing mechanism of cell surface-bound force sensors and more importantly cascading of the signals, both physical (via cytosolic force sensing elements such as microtubule and actin framework) as well as chemical (cascade of biochemical signaling from cell surface to nuclear surface and further to the chromatin), inside the cell is poorly understood. In this chapter, we present a review of the currently known molecular players in cellular as well as nuclear force sensing repertoire and their possible mechanistic aspects. We also introduce various biophysical concepts and review some frequently used techniques that are used to describe the force/stress sensing and response of a cell. We hope that this will help in asking clearer questions and designing pointed experiments for better understanding of the force-dependent design principles of the cell surface, nuclear surface, and gene expression.
10.1007/978-1-0716-4280-1_1
Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation.
Life science alliance
Actin and microtubules are essential cytoskeletal components and coordinate their dynamics through multiple coupling and decoupling mechanisms. However, how actin and microtubule dynamics are decoupled remains incompletely understood. Here, we identified TBC1D3C as a new regulator that can decouple actin filament assembly from microtubule disassembly. We showed that TBC1D3C induces the release of GEF-H1 from microtubules into the cytosol without perturbing microtubule arrays, leading to RhoA activation and actin filament assembly. Mechanistically, we found that TBC1D3C directly binds to GEF-H1, disrupting its interaction with the Tctex-DIC-14-3-3 complex and thereby displacing GEF-H1 from microtubules independently of microtubule disassembly. Super-resolution microscopy and live-cell imaging further confirmed that TBC1D3C triggers GEF-H1 release and actin filament assembly while maintaining microtubule integrity. Therefore, our findings demonstrated that TBC1D3C functions as a direct GEF activator and a novel regulator in decoupling actin assembly from microtubule disassembly, providing new insights into cytoskeletal regulation.
10.26508/lsa.202402585
Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates.
Autophagy
TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes, resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an ATG8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling. ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2: calcium binding and coiled-coil domain 2; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; IFN: interferon; IκB: inhibitor of nuclear factor kappa B; IKK: IκB kinase; IRF: interferon regulatory factor; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; NFKB: nuclear factor kappa B; OPTN: optineurin; Poly(I:C): polyinosinic-polycytidylic acid; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SDD-AGE: semi-denaturing detergent-agarose gel electrophoresis; SeV: Sendai virus; SLR: SQSTM1-like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF: TNF receptor associated factor; VSV: vesicular stomatitis virus; ZnF: zinc finger.
10.1080/15548627.2024.2394306
Fidgetin binds spastin to attenuate the microtubule-severing activity.
Biochimica et biophysica acta. Molecular cell research
Microtubule-severing enzymes such as spastin, katanin, and fidgetin, characterized by their AAA ATPase domains, are pivotal in modulating microtubule dynamics and behavior across various cellular processes. While spastin and katanin are recognized for their predominant and robust severing of stable microtubules, thereby enhancing microtubule turnover, fidgetin exhibits comparatively weaker severing activity and selectively targets labile microtubules. The interplay among these enzymes and their mutual regulatory mechanisms remains inadequately understood. In this study, we elucidate the functional interaction between spastin and fidgetin, focusing on their roles in microtubule severing and neurite outgrowth. Our findings demonstrate that fidgetin serves as a negative regulator of spastin's severing activity. Co-expression assays revealed that fidgetin significantly attenuates spastin's severing efficiency, as confirmed by fluorescence-based microtubule polymerization assays and quantitative imaging of microtubule dynamics. Co-immunoprecipitation and Förster Resonance Energy Transfer (FRET) analyses further established a direct interaction between fidgetin and spastin, suggesting that fidgetin modulates spastin's activity through direct binding, possibly contributing to forming the hetero-hexmeric ring for their severing activities. Functionally, spastin overexpression in neuronal cells enhances neurite outgrowth, an effect that is suppressed upon co-expression with fidgetin, indicating that fidgetin counterbalances spastin's activity to regulate neurite extension. Therefore, this study uncovers a previously unrecognized mechanism by which fidgetin modulates spastin's function, providing critical insights into the intricate regulation of microtubule severing. These findings have significant implications for therapeutic strategies targeting microtubule-severing activities, particularly in neurodevelopmental and neurodegenerative disorders where microtubule dysregulation is a hallmark.
10.1016/j.bbamcr.2024.119890
Mitochondrial bioenergetics stimulates autophagy for pathological MAPT/Tau clearance in tauopathy neurons.
Autophagy
Hyperphosphorylation and aggregation of MAPT (microtubule-associated protein tau) is a pathogenic hallmark of tauopathies and a defining feature of Alzheimer disease (AD). Pathological MAPT/tau is targeted by macroautophagy/autophagy for clearance after being sequestered within autophagosomes, but autophagy dysfunction is indicated in tauopathy. While mitochondrial bioenergetic deficits have been shown to precede MAPT/tau pathology in tauopathy brains, it is unclear whether energy metabolism deficiency is involved in the pathogenesis of autophagy defects. Here, we reveal that stimulation of anaplerotic metabolism restores defective oxidative phosphorylation (OXPHOS) in tauopathy neurons which, strikingly, leads to pronounced MAPT/tau clearance by boosting autophagy functionality through enhancements of mitochondrial biosynthesis and supply of phosphatidylethanolamine for autophagosome biogenesis. Furthermore, early anaplerotic stimulation of OXPHOS elevates autophagy activity and attenuates MAPT/tau pathology, thereby counteracting memory impairment in tauopathy mice. Taken together, our study sheds light on a pivotal role of mitochondrial bioenergetic deficiency in tauopathy-related autophagy defects and suggests a new therapeutic strategy to prevent the buildup of pathological MAPT/tau in AD and other tauopathy diseases.: AA: antimycin A; AD, Alzheimer disease; ATP, adenosine triphosphate; AV, autophagosome/autophagic vacuole; AZ, active zone; Baf-A1: bafilomycin A; CHX, cycloheximide; COX, cytochrome c oxidase; DIV, days ; DRG, dorsal root ganglion; ETN, ethanolamine; FRET, Förster/fluorescence resonance energy transfer; FTD, frontotemporal dementia; Gln, glutamine; HA: hydroxylamine; HsMAPT/Tau, human MAPT; IMM, inner mitochondrial membrane; LAMP1, lysosomal-associated membrane protein 1; LIs, lysosomal inhibitors; MDAV, mitochondria-derived autophagic vacuole; MmMAPT/Tau, murine MAPT; NFT, neurofibrillary tangle; OCR, oxygen consumption rate; Omy: oligomycin; OXPHOS, oxidative phosphorylation; PPARGC1A/PGC-1alpha: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PE, phosphatidylethanolamine; phospho-MAPT/tau, hyperphosphorylated MAPT; PS, phosphatidylserine; PISD, phosphatidylserine decarboxylase;SQSTM1/p62, sequestosome 1; STX1, syntaxin 1; SYP, synaptophysin; Tg, transgenic; TCA, tricarboxylic acid; TEM, transmission electron microscopy.
10.1080/15548627.2024.2392408
Tubulin glutamylation regulates axon guidance via the selective tuning of microtubule-severing enzymes.
The EMBO journal
The microtubule cytoskeleton is a major driving force of neuronal circuit development. Fine-tuned remodelling of this network by selective activation of microtubule-regulating proteins, including microtubule-severing enzymes, has emerged as a central process in neuronal wiring. Tubulin posttranslational modifications control both microtubule properties and the activities of their interacting proteins. However, whether and how tubulin posttranslational modifications may contribute to neuronal connectivity has not yet been addressed. Here we show that the microtubule-severing proteins p60-katanin and spastin play specific roles in axon guidance during zebrafish embryogenesis and identify a key role for tubulin polyglutamylation in their functional specificity. Furthermore, our work reveals that polyglutamylases with undistinguishable activities in vitro, TTLL6 and TTLL11, play exclusive roles in motor circuit wiring by selectively tuning p60-katanin- and spastin-driven motor axon guidance. We confirm the selectivity of TTLL11 towards spastin regulation in mouse cortical neurons and establish its relevance in preventing axonal degeneration triggered by spastin haploinsufficiency. Our work thus provides mechanistic insight into the control of microtubule-driven neuronal development and homeostasis and opens new avenues for developing therapeutic strategies in spastin-associated hereditary spastic paraplegia.
10.1038/s44318-024-00307-x
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