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BioRxiv : the Preprint Server For... Oct 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.
PubMed: 37905093
DOI: 10.1101/2023.06.29.547092 -
Cell Jun 2024Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional...
Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.
Topics: Animals; Humans; Mice; Cell Cycle Proteins; Centromere; Chickens; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Cohesins; Kinetochores; Microtubules; Mitosis; Spindle Apparatus
PubMed: 38744280
DOI: 10.1016/j.cell.2024.04.014 -
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 -
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 -
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 -
Journal of Cell Science Nov 2023The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the...
The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.
Topics: Actins; Microtubules; Actin Cytoskeleton; Cell Polarity; Pseudopodia
PubMed: 37870087
DOI: 10.1242/jcs.261667 -
Genetics Aug 2023Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature...
Gamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. Here, we show that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. We show that Mob4 is required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. Our study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization.
Topics: Animals; Female; Humans; Male; Adaptor Proteins, Signal Transducing; Drosophila; Drosophila melanogaster; Drosophila Proteins; Infertility, Male; Nerve Tissue Proteins; Semen; Spermatids; Spermatogenesis; Testis
PubMed: 37259670
DOI: 10.1093/genetics/iyad104 -
Nucleic Acids Research Oct 2023Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are...
Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. We identify Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69. TRIM69 redistributes MST2 to the perinuclear cytoskeleton, promotes its association with Polo-like kinase 1 (PLK1) and stimulates MST2 phosphorylation at S15 (a known PLK1 phosphorylation site that is critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation that requires PRC1 and DYNEIN. Taken together, we identify TRIM69 as a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis.
Topics: Cell Cycle Proteins; Centrosome; Chromosome Segregation; Mitosis; Phosphorylation; Signal Transduction; Spindle Apparatus
PubMed: 37739411
DOI: 10.1093/nar/gkad766 -
Cytoskeleton (Hoboken, N.J.) 2023Microtubule-associated proteins (MAPs) regulate assembly and stability of microtubules (MTs) during cell cytokinesis, cell migration, neuronal growth, axon guidance, and...
Microtubule-associated proteins (MAPs) regulate assembly and stability of microtubules (MTs) during cell cytokinesis, cell migration, neuronal growth, axon guidance, and synapse formation. Using data mining of the Human Protein Atlas database and experimental screening, we identified nucleosome assembly protein 1 like 1 (NAP1L1) as a new MAP. The Human Protein Atlas and PubMed database screening identified 99 potential new MAPs. Twenty candidate proteins that highly co-localized with MTs were exogenously expressed with green fluorescent protein (GFP) or hemagglutinin (HA) tags in tissue culture cells and MTs were co-stained for immunofluorescent microscopy. We found that NAP1L1 is mainly localized in the cytosol with MTs during interphase. Using bacterially expressed recombinant NAP1L1 fragments and purified MTs, we biochemically mapped the MT-binding site on the N-terminal region (1-72aa) and the central region (164-269aa) of NAP1L1. NAP1L1 dimerizes through the long helix region (73-163aa), and full-length NAP1L1 induces the formation of thick MTs, indicating that NAP1L1 has the ability to bundle MTs in cells. Analysis of publicly available RNA-seq data of NAP1L1 depleted cells suggested that NAP1L1 is involved in cell adhesion and migration in agreement with the function of NAP1L1 as a MAP.
PubMed: 37098731
DOI: 10.1002/cm.21761 -
Journal of Veterinary Diagnostic... Nov 2023An 11-y-old hembra alpaca was admitted because of cerebellar and vestibular signs, dysphagia, and aspiration pneumonia; without clinical improvement following empirical...
An 11-y-old hembra alpaca was admitted because of cerebellar and vestibular signs, dysphagia, and aspiration pneumonia; without clinical improvement following empirical therapy, the patient was euthanized. On autopsy, a neoplasm was found incorporating the right vestibulocochlear nerve at the level of the acoustic meatus. Histologically, the mass was composed of a multiphasic primitive cell population associated with a dense fibrous stroma and enveloping a remnant ganglion and nerve bundles. Patterns included dense ribbons and cords of embryonal neuroepithelial cells admixed with loosely defined interlacing spindle cells. The embryonal cells had angular cell profiles with variable amounts of lightly basophilic cytoplasm, ovoid-to-irregular nuclei, and an open chromatin pattern with a typically inapparent nucleolus. Necrosis was not evident, and there was 1 mitotic figure per 2.37 mm. The entire mass was infiltrated by small numbers of lymphocytes and plasma cells. Immunohistochemistry (IHC) revealed strong and diffuse cytoplasmic immunolabeling for vimentin, microtubule-associated protein-2, protein gene product 9.5, and synaptophysin; ~50% immunolabeling for cytokeratin AE1/3; sporadic OLIG2 and S100 immunolabeling; and absent glial fibrillary acidic protein immunolabeling. Based on the histologic pattern and the IHC results, our diagnosis was a poorly differentiated embryonal tumor with ependymal differentiation associated with the vestibulocochlear nerve.
Topics: Animals; Camelids, New World; Neoplasms, Germ Cell and Embryonal
PubMed: 37638696
DOI: 10.1177/10406387231195611