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The Journal of Biological Chemistry Mar 2024The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed....
The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes.
Topics: Humans; HeLa Cells; Kinetochores; Microtubules; Mitosis; Spindle Apparatus; Tubulin; Animals; Chromosomes; Xenopus laevis
PubMed: 38272221
DOI: 10.1016/j.jbc.2024.105669 -
Current Biology : CB Jan 2024Actin-microtubule crosstalk diversifies cytoskeletal networks. A new study provides insight into how the microtubule polymerase CKAP5 mediates actin-microtubule...
Actin-microtubule crosstalk diversifies cytoskeletal networks. A new study provides insight into how the microtubule polymerase CKAP5 mediates actin-microtubule crosstalk. CKAP5 directs the assembly of stable actin bundles on dynamic microtubules; in turn, the actin bundles align growing microtubules along their length.
Topics: Actins; Cytoskeleton; Microtubules; Fungi; Nucleotidyltransferases
PubMed: 38262365
DOI: 10.1016/j.cub.2023.12.028 -
Frontiers in Pharmacology 2023Cancer cells evolve to be refractory to the intrinsic programmed cell death mechanisms, which ensure cellular tissue homeostasis in physiological conditions....
Cancer cells evolve to be refractory to the intrinsic programmed cell death mechanisms, which ensure cellular tissue homeostasis in physiological conditions. Chemotherapy using cytotoxic drugs seeks to eliminate cancer cells but spare non-cancerous host cells by exploring a likely subtle difference between malignant and benign cells. Presumably, chemotherapy agents achieve efficacy by triggering programmed cell death machineries in cancer cells. Currently, many major solid tumors are treated with chemotherapy composed of a combination of platinum agents and taxanes. Platinum agents, largely cis-platin, carboplatin, and oxaliplatin, are DNA damaging agents that covalently form DNA addicts, triggering DNA repair response pathways. Taxanes, including paclitaxel, docetaxel, and cabazitaxel, are microtubule stabilizing drugs which are often very effective in purging cancer cells in clinical settings. Generally, it is thought that the stabilization of microtubules by taxanes leads to mitotic arrest, mitotic catastrophe, and the triggering of apoptotic programmed cell death. However, the precise mechanism(s) of how mitotic arrest and catastrophe activate the caspase pathway has not been established. Here, we briefly review literature on the involvement of potential cell death mechanisms in cancer therapy. These include the classical caspase-mediated apoptotic programmed cell death, necroptosis mediated by MLKL, and pore forming mechanisms in immune cells, etc. In particular, we discuss a newly recognized mechanism of cell death in taxane-treatment of cancer cells that involves micronucleation and the irreversible rupture of the nuclear membrane. Since cancer cells are commonly retarded in responding to programmed cell death signaling, stabilized microtubule bundle-induced micronucleation and nuclear membrane rupture, rather than triggering apoptosis, may be a key mechanism accounting for the success of taxanes as anti-cancer agents.
PubMed: 38249350
DOI: 10.3389/fphar.2023.1338633 -
Proceedings of the National Academy of... Jan 2024Microtubules and molecular motors are essential components of the cellular cytoskeleton, driving fundamental processes in vivo, including chromosome segregation and...
Microtubules and molecular motors are essential components of the cellular cytoskeleton, driving fundamental processes in vivo, including chromosome segregation and cargo transport. When reconstituted in vitro, these cytoskeletal proteins serve as energy-consuming building blocks to study the self-organization of active matter. Cytoskeletal active gels display rich emergent dynamics, including extensile flows, locally contractile asters, and bulk contraction. However, it is unclear how the protein-protein interaction kinetics set their contractile or extensile nature. Here, we explore the origin of the transition from extensile bundles to contractile asters in a minimal reconstituted system composed of stabilized microtubules, depletant, adenosine 5'-triphosphate (ATP), and clusters of kinesin-1 motors. We show that the microtubule-binding and unbinding kinetics of highly processive motor clusters set their ability to end-accumulate, which can drive polarity sorting of the microtubules and aster formation. We further demonstrate that the microscopic time scale of end-accumulation sets the emergent time scale of aster formation. Finally, we show that biochemical regulation is insufficient to fully explain the transition as generic aligning interactions through depletion, cross-linking, or excluded volume interactions can drive bundle formation despite end-accumulating motors. The extensile-to-contractile transition is well captured by a simple self-assembly model where nematic and polar aligning interactions compete to form either bundles or asters. Starting from a five-dimensional organization phase space, we identify a single control parameter given by the ratio of the different component concentrations that dictates the material-scale organization. Overall, this work shows that the interplay of biochemical and mechanical tuning at the microscopic level controls the robust self-organization of active cytoskeletal materials.
Topics: Microtubules; Cytoskeleton; Kinesins; Cell Movement; Chromosome Segregation
PubMed: 38175870
DOI: 10.1073/pnas.2300174121 -
NPJ Parkinson's Disease Jan 2024Highly specialized microtubules in neurons are crucial to both health and disease of the nervous system, and their properties are strictly regulated by different...
Highly specialized microtubules in neurons are crucial to both health and disease of the nervous system, and their properties are strictly regulated by different post-translational modifications, including α-Tubulin acetylation. An imbalance in the levels of acetylated α-Tubulin has been reported in experimental models of Parkinson's disease (PD) whereas pharmacological or genetic modulation that leads to increased acetylated α-Tubulin successfully rescues axonal transport defects and inhibits α-Synuclein aggregation. However, the role of acetylation of α-Tubulin in the human nervous system is largely unknown as most studies are based on in vitro evidence. To capture the complexity of the pathological processes in vivo, we analysed post-mortem human brain of PD patients and control subjects. In the brain of PD patients at Braak stage 6, we found a redistribution of acetylated α-Tubulin, which accumulates in the neuronal cell bodies in subcortical structures but not in the cerebral cortex, and decreases in the axonal compartment, both in putamen bundles of fibres and in sudomotor fibres. High-resolution and 3D reconstruction analysis linked acetylated α-Tubulin redistribution to α-Synuclein oligomerization and to phosphorylated Ser 129 α-Synuclein, leading us to propose a model for Lewy body (LB) formation. Finally, in post-mortem human brain, we observed threadlike structures, resembling tunnelling nanotubes that contain α-Synuclein oligomers and are associated with acetylated α-Tubulin enriched neurons. In conclusion, we support the role of acetylated α-Tubulin in PD pathogenesis and LB formation.
PubMed: 38167511
DOI: 10.1038/s41531-023-00607-9 -
PLoS Genetics Dec 2023Oocyte meiotic spindles mediate the expulsion of ¾ of the genome into polar bodies to generate diploid zygotes in nearly all animal species. Failures in this process...
Oocyte meiotic spindles mediate the expulsion of ¾ of the genome into polar bodies to generate diploid zygotes in nearly all animal species. Failures in this process result in aneuploid or polyploid offspring that are typically inviable. Accurate meiotic chromosome segregation and polar body extrusion require the spindle to elongate while maintaining its structural integrity. Previous studies have implicated three hypothetical activities during this process, including microtubule crosslinking, microtubule sliding and microtubule polymerization. However, how these activities regulate spindle rigidity and elongation as well as the exact proteins involved in the activities remain unclear. We discovered that C. elegans meiotic anaphase spindle integrity is maintained through redundant microtubule crosslinking activities of the Kinesin-5 family motor BMK-1, the microtubule bundling protein SPD-1/PRC1, and the Kinesin-4 family motor, KLP-19. Using time-lapse imaging, we found that single depletion of KLP-19KIF4A, SPD-1PRC1 or BMK-1Eg5 had minimal effects on anaphase B spindle elongation velocity. In contrast, double depletion of SPD-1PRC1 and BMK-1Eg5 or double depletion of KLP-19KIF4A and BMK-1Eg5 resulted in spindles that elongated faster, bent in a myosin-dependent manner, and had a high rate of polar body extrusion errors. Bending spindles frequently extruded both sets of segregating chromosomes into two separate polar bodies. Normal anaphase B velocity was observed after double depletion of KLP-19KIF4A and SPD-1PRC1. These results suggest that KLP-19KIF4A and SPD-1PRC1 act in different pathways, each redundant with a separate BMK-1Eg5 pathway in regulating meiotic spindle elongation. Depletion of ZYG-8, a doublecortin-related microtubule binding protein, led to slower anaphase B spindle elongation. We found that ZYG-8DCLK1 acts by excluding SPD-1PRC1 from the spindle. Thus, three mechanistically distinct microtubule regulation modules, two based on crosslinking, and one based on exclusion of crosslinkers, power the mechanism that drives spindle elongation and structural integrity during anaphase B of C.elegans female meiosis.
Topics: Animals; Female; Caenorhabditis elegans; Kinesins; Diploidy; Caenorhabditis elegans Proteins; Microtubules; Spindle Apparatus; Meiosis; Oocytes
PubMed: 38150489
DOI: 10.1371/journal.pgen.1011090 -
ELife Dec 2023During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each...
During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes.
Topics: Spindle Apparatus; Kinetochores; Microtubules; Mitosis; Chromosome Segregation
PubMed: 38150374
DOI: 10.7554/eLife.89467 -
American Journal of Transplantation :... May 2024Immunotactoid deposition is a rare fibrillary deposition disease that is primarily seen in the kidney and is associated with paraproteinemia. Here, we report a case of...
Immunotactoid deposition is a rare fibrillary deposition disease that is primarily seen in the kidney and is associated with paraproteinemia. Here, we report a case of hepatic immunotactoid deposition in a 67-year-old male with a history of smoldering myeloma and chronic kidney disease who underwent liver transplantation for metabolic dysfunction-related cirrhosis. Immunotactoid deposition was first identified in the explanted liver and recurred in the allograft within only 7 weeks following transplantation, presenting as ascites with normal liver function tests. The patient's posttransplant course was complicated by proteinuria and renal failure requiring dialysis. Histologic examination of both native and allograft livers demonstrated pink amorphous material occupying sinusoidal spaces that were Congo-red negative and immunoglobulin M Kappa-restricted. Electron microscopy revealed characteristic deposits of electron-dense bundles of hollow microtubules with a 40 nm diameter within the sinusoids and space of Disse, consistent with immunotactoids. Therapy of the patient's underlying plasma-cell dyscrasia utilizing a daratumumab-based regimen showed decreased serum paraproteins, resolution of ascites, and improved kidney function, no longer requiring dialysis, without inducing rejection. The patient continues to respond to treatment 10 months posttransplant.
Topics: Humans; Male; Aged; Liver Transplantation; Recurrence; Prognosis; Liver Diseases; Postoperative Complications
PubMed: 38145784
DOI: 10.1016/j.ajt.2023.12.019 -
Plant Physiology and Biochemistry : PPB Jan 2024The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for...
The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for non-target organisms. The recent determination of ivermectin absorption and accumulation in tissues of higher plants and multiple shreds of evidence of its negative impact on plant physiology provide a basis for the search for ivermectin's molecular targets and mechanisms of action in plant cells. In this research, for the first time, the ivermectin effect on microtubules of Arabidopsis thaliana cells was studied. It was revealed that ivermectin (250 μg mL) disrupts the microtubule network, induces the loss of microtubule orientation, leads to microtubule curvature and shrinkage, and their longitudinal and cross-linked bundling in various cells of A. thaliana primary roots. Further, the previously proposed binding of ivermectin to the β1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana β1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana β-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization.
Topics: Humans; Animals; Tubulin; Ivermectin; Arabidopsis; Microtubules; Binding Sites
PubMed: 38141401
DOI: 10.1016/j.plaphy.2023.108296 -
The Journal of Cell Biology Feb 2024Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous...
Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous microtubule flux. NuSAP is a microtubule-stabilizing and -bundling protein that promotes chromosome-dependent spindle assembly. However, its function in spindle dynamics remains unclear. Here, we demonstrate that NuSAP regulates the metaphase spindle length control. Mechanistically, NuSAP facilitates kinetochore capture and spindle assembly by promoting Eg5 binding to microtubules. It also prevents excessive microtubule depolymerization through interaction with Kif2A, which reduces Kif2A spindle-pole localization. NuSAP is phosphorylated by Aurora A at Ser-240 during mitosis, and this phosphorylation promotes its interaction with Kif2A on the spindle body and reduces its localization with the spindle poles, thus maintaining proper spindle microtubule flux. NuSAP knockout resulted in the formation of shorter spindles with faster microtubule flux and chromosome misalignment. Taken together, we uncover that NuSAP participates in spindle assembly, dynamics, and metaphase spindle length control through the regulation of microtubule flux and Kif2A localization.
Topics: Humans; Chromosome Segregation; HeLa Cells; Kinesins; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 38117947
DOI: 10.1083/jcb.202108070