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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 -
Cell Structure and Function 2010Microtubules (MTs) play specialized roles in a wide variety of cellular events, e.g. molecular transport, cell motility, and cell division. Specialized MT architectures,... (Review)
Review
Microtubules (MTs) play specialized roles in a wide variety of cellular events, e.g. molecular transport, cell motility, and cell division. Specialized MT architectures, such as bundles, axonemes, and centrioles, underlie the function. The specialized function and highly organized structure depend on interactions with MT-binding proteins. MT-associated proteins (e.g. MAP1, MAP2, and tau), molecular motors (kinesin and dynein), plus-end tracking proteins (e.g. CLIP-170), and MT-severing proteins (e.g. katanin) interact with MTs. How can the MT-binding proteins know temporospatial information to associate with MTs and to properly play their roles? Post-translational modifications (PTMs) including detyrosination, polyglutamylation, and polyglycylation can provide molecular landmarks for the proteins. Recent efforts to identify modification-regulating enzymes (TTL, carboxypeptidase, polyglutamylase, polyglycylase) and to generate genetically manipulated animals enable us to understand the roles of the modifications. In this review, we present recent advances in understanding regulation of MT function, structure, and stability by PTMs.
Topics: Adenosine Triphosphatases; Katanin; Microtubule-Associated Proteins; Microtubules; Molecular Motor Proteins; Neoplasm Proteins; Peptides; Protein Processing, Post-Translational; Tyrosine
PubMed: 20190462
DOI: 10.1247/csf.09027 -
Cell Aug 2010Microtubules assemble into arrays of bundled filaments that are critical for multiple steps in cell division, including anaphase and cytokinesis. Recent structural and... (Review)
Review
Microtubules assemble into arrays of bundled filaments that are critical for multiple steps in cell division, including anaphase and cytokinesis. Recent structural and functional studies, including two papers in this issue of Cell (Bieling et al., 2010; Subramanian et al., 2010), demonstrate how the MAP65 protein PRC1 crosslinks microtubules and cooperates with kinesin motors to control the dynamics and size of bundled regions.
Topics: Animals; Cell Cycle Proteins; Kinesins; Microtubule-Associated Proteins; Microtubules; Plants
PubMed: 20691897
DOI: 10.1016/j.cell.2010.07.023 -
Molecular Biology of the Cell Feb 2018The cleavage furrow in zygotes is positioned by two large microtubule asters that grow out from the poles of the first mitotic spindle. Where these asters meet at the...
The cleavage furrow in zygotes is positioned by two large microtubule asters that grow out from the poles of the first mitotic spindle. Where these asters meet at the midplane, they assemble a disk-shaped interaction zone consisting of anti-parallel microtubule bundles coated with chromosome passenger complex (CPC) and centralspindlin that instructs the cleavage furrow. Here we investigate the mechanism that keeps the two asters separate and forms a distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1 and Kif4A. Prc1E, the egg orthologue of Prc1, and Kif4A were recruited to anti-parallel bundles at interaction zones between asters in egg extracts. Prc1E was required for Kif4A recruitment but not vice versa. Microtubule plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of large asters growing in isolation, apparently to compensate for the direction-randomizing influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with catastrophe factors, promote "anti-parallel pruning" that enforces radial organization within asters and generates boundaries to microtubule growth between asters.
Topics: Animals; Centrosome; Cleavage Stage, Ovum; Cytokinesis; DNA-Binding Proteins; Embryo, Nonmammalian; Embryonic Development; Kinesins; Microtubules; Nuclear Proteins; Spindle Apparatus; Xenopus Proteins; Xenopus laevis; Zygote
PubMed: 29187577
DOI: 10.1091/mbc.E17-09-0540 -
The EMBO Journal Jun 2022Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding....
Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.
Topics: Humans; Microtubules; Neurodegenerative Diseases; Neurons; Protein Binding; RNA; tau Proteins
PubMed: 35298090
DOI: 10.15252/embj.2021108882 -
ELife Oct 2018Motor and non-motor crosslinking proteins play critical roles in determining the size and stability of microtubule-based architectures. Currently, we have a limited...
Motor and non-motor crosslinking proteins play critical roles in determining the size and stability of microtubule-based architectures. Currently, we have a limited understanding of how geometrical properties of microtubule arrays, in turn, regulate the output of crosslinking proteins. Here we investigate this problem in the context of microtubule sliding by two interacting proteins: the non-motor crosslinker PRC1 and the kinesin Kif4A. The collective activity of PRC1 and Kif4A also results in their accumulation at microtubule plus-ends ('end-tag'). Sliding stalls when the end-tags on antiparallel microtubules collide, forming a stable overlap. Interestingly, we find that structural properties of the initial array regulate microtubule organization by PRC1-Kif4A. First, sliding velocity scales with initial microtubule-overlap length. Second, the width of the final overlap scales with microtubule lengths. Our analyses reveal how micron-scale geometrical features of antiparallel microtubules can regulate the activity of nanometer-sized proteins to define the structure and mechanics of microtubule-based architectures.
Topics: Cell Cycle Proteins; Humans; Kinesins; Microscopy, Fluorescence; Microtubules; Motion; Protein Binding
PubMed: 30353849
DOI: 10.7554/eLife.32595 -
Scientific Reports May 2015The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers...
The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts.
Topics: Actin Cytoskeleton; Actins; Binding Sites; Cross-Linking Reagents; Microtubules; Motion; Protein Binding; Protein Conformation; tau Proteins
PubMed: 25944224
DOI: 10.1038/srep09964 -
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 -
Neural Development Nov 2019Axons are the slender, cable-like, up to meter-long projections of neurons that electrically wire our brains and bodies. In spite of their challenging morphology, they... (Review)
Review
Axons are the slender, cable-like, up to meter-long projections of neurons that electrically wire our brains and bodies. In spite of their challenging morphology, they usually need to be maintained for an organism's lifetime. This makes them key lesion sites in pathological processes of ageing, injury and neurodegeneration. The morphology and physiology of axons crucially depends on the parallel bundles of microtubules (MTs), running all along to serve as their structural backbones and highways for life-sustaining cargo transport and organelle dynamics. Understanding how these bundles are formed and then maintained will provide important explanations for axon biology and pathology. Currently, much is known about MTs and the proteins that bind and regulate them, but very little about how these factors functionally integrate to regulate axon biology. As an attempt to bridge between molecular mechanisms and their cellular relevance, we explain here the model of local axon homeostasis, based on our own experiments in Drosophila and published data primarily from vertebrates/mammals as well as C. elegans. The model proposes that (1) the physical forces imposed by motor protein-driven transport and dynamics in the confined axonal space, are a life-sustaining necessity, but pose a strong bias for MT bundles to become disorganised. (2) To counterbalance this risk, MT-binding and -regulating proteins of different classes work together to maintain and protect MT bundles as necessary transport highways. Loss of balance between these two fundamental processes can explain the development of axonopathies, in particular those linking to MT-regulating proteins, motors and transport defects. With this perspective in mind, we hope that more researchers incorporate MTs into their work, thus enhancing our chances of deciphering the complex regulatory networks that underpin axon biology and pathology.
Topics: Animals; Axons; Homeostasis; Microtubules
PubMed: 31706327
DOI: 10.1186/s13064-019-0134-0 -
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