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Nature Communications Nov 2022Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how...
Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how these structures form remains poorly understood. Here we show that overlap bundles arise through a network-to-bundles transition driven by kinetochores and chromosomes. STED super-resolution microscopy reveals that PRC1-crosslinked microtubules initially form loose arrays, which become rearranged into bundles. Kinetochores promote microtubule bundling by lateral binding via CENP-E/kinesin-7 in an Aurora B-regulated manner. Steric interactions between the bundle-associated chromosomes at the spindle midplane drive bundle separation and spindle widening. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles leads to inefficient correction of erroneous kinetochore-microtubule attachments. Thus, kinetochores and chromosomes drive coarsening of a uniform microtubule array into overlap bundles, which promote not only spindle formation but also chromosome segregation fidelity.
Topics: Kinetochores; Microtubules; Chromosome Segregation; Kinesins
PubMed: 36435852
DOI: 10.1038/s41467-022-34957-4 -
ELife Nov 2022During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate...
During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al., 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 μm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.
Topics: Animals; Spindle Apparatus; Kinetochores; Microtubules; Cell Division; Mammals; Mitosis
PubMed: 36346735
DOI: 10.7554/eLife.79558 -
Proceedings of the National Academy of... Nov 2022Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled...
Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.
Topics: Tubulin; Axonal Transport; Kinesins; Microtubules; Mitochondria; Axons; Molecular Conformation
PubMed: 36322761
DOI: 10.1073/pnas.2203499119 -
Frontiers in Oncology 2022The mitotic kinesin Eg5 has emerged as a potential anti-mitotic target for the purposes of cancer chemotherapy. Whether clinical resistance to these inhibitors can arise...
The mitotic kinesin Eg5 has emerged as a potential anti-mitotic target for the purposes of cancer chemotherapy. Whether clinical resistance to these inhibitors can arise is unclear. We exploited HCT116 cancer cell line to select resistant clones to S-trityl-L-cysteine (STLC), an extensively studied Eg5 loop-L5 binding inhibitor. The STLC resistant clones differed in their resistance to other loop-L5 binding inhibitors but remained sensitive to the ATP class of competitive Eg5 specific inhibitors. Eg5 is still necessary for bipolar spindle formation in the resistant clones since the cells were sensitive to RNAi mediated depletion of Eg5. One clone expressing Eg5(T107N), a dominant point mutation in the P-loop of the ATP binding domain of the motor, appeared to be not only resistant but also dependent on the presence of STLC. Eg5(T107N) expression was associated also with resistance to the clinical relevant loop-L5 Eg5 inhibitors, Arry-520 and ispinesib. Ectopic expression of the Eg5(T107N) mutant in the absence of STLC was associated with strong non-exchangeable binding to microtubules causing them to bundle. Biochemical assays showed that in contrast to the wild type Eg5-STLC complex, the ATP binding site of the Eg5(T107N) is accessible for nucleotide exchange only when the inhibitor is present. We predict that resistance can be overcome by inhibitors that bind to other than the Eg5 loop-L5 binding site having different chemical scaffolds, and that allostery-dependent resistance to Eg5 inhibitors may also occur in cells and may have positive implications in chemotherapy since once diagnosed may be beneficial following cessation of the chemotherapeutic regimen.
PubMed: 36313676
DOI: 10.3389/fonc.2022.965455 -
ELife Oct 2022The human mitotic spindle is made of microtubules nucleated at centrosomes, at kinetochores, and from pre-existing microtubules by the augmin complex. However, it is...
The human mitotic spindle is made of microtubules nucleated at centrosomes, at kinetochores, and from pre-existing microtubules by the augmin complex. However, it is unknown how the augmin-mediated nucleation affects distinct microtubule classes and thereby mitotic fidelity. Here, we use superresolution microscopy to analyze the previously indistinguishable microtubule arrangements within the crowded metaphase plate area and demonstrate that augmin is vital for the formation of uniformly arranged parallel units consisting of sister kinetochore fibers connected by a bridging fiber. This ordered geometry helps both prevent and resolve merotelic attachments. Whereas augmin-nucleated bridging fibers prevent merotelic attachments by creating a nearly parallel and highly bundled microtubule arrangement unfavorable for creating additional attachments, augmin-nucleated k-fibers produce robust force required to resolve errors during anaphase. STED microscopy revealed that bridging fibers were impaired twice as much as k-fibers following augmin depletion. The complete absence of bridging fibers from a significant portion of kinetochore pairs, especially in the inner part of the spindle, resulted in the specific reduction of the interkinetochore distance. Taken together, we propose a model where augmin promotes mitotic fidelity by generating assemblies consisting of bridging and kinetochore fibers that align sister kinetochores to face opposite poles, thereby preventing erroneous attachments.
Topics: Humans; Kinetochores; Spindle Apparatus; Microtubules; Anaphase; Chromosome Segregation; Microtubule-Associated Proteins; Cell Cycle Proteins
PubMed: 36269126
DOI: 10.7554/eLife.83287 -
Cells Sep 2022Parkinson's disease is characterized by locomotion deficits, dopaminergic neuronal loss and alpha-synuclein (SYN) aggregates; the Tubulin Polymerization Promoting...
Parkinson's disease is characterized by locomotion deficits, dopaminergic neuronal loss and alpha-synuclein (SYN) aggregates; the Tubulin Polymerization Promoting Protein (TPPP/p25 or TPPP1) is also implicated in these processes. The moonlighting and chameleon TPPP1 modulates the dynamics/stability of the multifunctional microtubule network by promoting its acetylation and bundling. Previously, we identified the microtubule-associated TPPP3, a homologue of TPPP1 lacking its N-terminus; however, its involvement in physiological or pathological processes was not elucidated. In this work, we have shown the modulatory role of TPPP3, similarly to TPPP1, in microtubule organization, as well as its homo- and hetero-associations with TPPP1. TPPP3, in contrast to TPPP1, virtually does not bind to SYN; consequently, it does not promote SYN aggregation. Its anti-aggregative potency is achieved by counteracting the formation of the TPPP1-SYN pathological complex/aggregation leading to Parkinsonism. The interactions of TPPP3 have been determined and quantified in vitro with recombinant human proteins, cell extracts and in living human cells using different methods including bifunctional fluorescence complementation. The tight association of TPPP3 with TPPP1, but not with SYN, may ensure a unique mechanism for its inhibitory effect. TPPP3 or its selected fragments may become a leading agent for developing anti-Parkinson agents.
Topics: Cell Extracts; Cytoskeletal Proteins; Humans; Microtubules; Parkinson Disease; Recombinant Proteins; Tubulin; alpha-Synuclein
PubMed: 36230985
DOI: 10.3390/cells11193025 -
Molecular Biology of the Cell Dec 2022Although end-on microtubule-kinetochore attachments typically drive chromosome alignment, oocytes do not form these connections. Instead, microtubule bundles run...
Although end-on microtubule-kinetochore attachments typically drive chromosome alignment, oocytes do not form these connections. Instead, microtubule bundles run laterally alongside chromosomes and a ring-shaped protein complex facilitates congression (the "ring complex", RC). Here, we report new aspects of RC and chromosome structure that are required for congression and segregation. First, we found that in addition to encircling the outside of each homologous chromosome pair (bivalent), the RC also forms internal subloops that wrap around the domains where cohesion is lost during the first meiotic division; cohesin removal could therefore disengage these subloops in anaphase, enabling RC removal from chromosomes. Additionally, we discovered new features of chromosome organization that facilitate congression. Analysis of a mutant that forms bivalents with a fragile, unresolved homolog interface revealed that these bivalents are usually able to biorient on the spindle, with lateral microtubule bundles running alongside them and constraining the chromosome arms so that the two homologs are pointed to opposite spindle poles. This biorientation facilitates congression, as monooriented bivalents exhibited reduced polar ejection forces that resulted in congression defects. Thus, despite not forming end-on attachments, chromosome biorientation promotes congression in oocytes. Our work therefore reveals novel features of chromosome organization in oocytes and highlights the importance of proper chromosome structure for faithful segregation during meiotic divisions.
Topics: Animals; Caenorhabditis elegans; Kinetochores; Chromosome Segregation; Oocytes; Microtubules; Chromosomes; Spindle Apparatus; Meiosis
PubMed: 36222840
DOI: 10.1091/mbc.E22-07-0297 -
Current Biology : CB Nov 2022Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of...
Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.
Topics: Katanin; Optogenetics; Adenosine Triphosphatases; Microtubules; Mitosis
PubMed: 36174574
DOI: 10.1016/j.cub.2022.09.010 -
Cells Sep 2022To cope with continuous physiological and environmental stresses, cells of all sizes require an effective wound repair process to seal breaches to their cortex. Once a... (Review)
Review
To cope with continuous physiological and environmental stresses, cells of all sizes require an effective wound repair process to seal breaches to their cortex. Once a wound is recognized, the cell must rapidly plug the injury site, reorganize the cytoskeleton and the membrane to pull the wound closed, and finally remodel the cortex to return to homeostasis. Complementary studies using various model organisms have demonstrated the importance and complexity behind the formation and translocation of an actin ring at the wound periphery during the repair process. Proteins such as actin nucleators, actin bundling factors, actin-plasma membrane anchors, and disassembly factors are needed to regulate actin ring dynamics spatially and temporally. Notably, Rho family GTPases have been implicated throughout the repair process, whereas other proteins are required during specific phases. Interestingly, although different models share a similar set of recruited proteins, the way in which they use them to pull the wound closed can differ. Here, we describe what is currently known about the formation, translocation, and remodeling of the actin ring during the cell wound repair process in model organisms, as well as the overall impact of cell wound repair on daily events and its importance to our understanding of certain diseases and the development of therapeutic delivery modalities.
Topics: Actins; Cell Membrane; Cytoskeleton; Microtubules; rho GTP-Binding Proteins
PubMed: 36139352
DOI: 10.3390/cells11182777 -
Journal of the Royal Society, Interface Sep 2022We present a model of a system of elastic fibres which exhibits complex, coupled, nonlinear deformations via a connecting elastic spring network. This model can capture...
We present a model of a system of elastic fibres which exhibits complex, coupled, nonlinear deformations via a connecting elastic spring network. This model can capture physically observed deformations such as global buckling, pinching and internal collapse. We explore the transitions between these deformation modes numerically, using an energy minimization approach, highlighting how supported environments, or stiff outer sheath structures, favour internal structural collapse over global deformation. We then derive a novel analytic buckling criterion for the internal collapse of the system, a mode of structural collapse pertinent in many biological filament bundles such as the optic nerve bundle and microtubule bundles involved in cell abscission.
Topics: Cytoskeleton; Elastic Tissue
PubMed: 36128703
DOI: 10.1098/rsif.2022.0287