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European Biophysics Journal : EBJ Apr 2018When a cell starts to divide, it forms a spindle, a micro-machine made of microtubules, which separates the duplicated chromosomes. The attachment of microtubules to... (Review)
Review
When a cell starts to divide, it forms a spindle, a micro-machine made of microtubules, which separates the duplicated chromosomes. The attachment of microtubules to chromosomes is mediated by kinetochores, protein complexes on the chromosome. Spindle microtubules can be divided into three major classes: kinetochore microtubules, which form k-fibers ending at the kinetochore; interpolar microtubules, which extend from the opposite sides of the spindle and interact in the middle; and astral microtubules, which extend towards the cell cortex. Recent work in human cells has shown a close relationship between interpolar and kinetochore microtubules, where interpolar bundles are attached laterally to kinetochore fibers almost all along their length, acting as a bridge between sister k-fibers. Most of the interpolar bundles are attached to a pair of sister kinetochore fibers and vice versa. Thus, the spindle is made of modules consisting of a pair of sister kinetochore fibers and a bundle of interpolar microtubules that connects them. These interpolar bundles, termed bridging fibers, balance the forces acting at kinetochores and support the rounded shape of the spindle during metaphase. This review discusses the structure, function, and formation of kinetochore fibers and interpolar bundles, with an emphasis on how they interact. Their connections have an impact on the force balance in the spindle and on chromosome movement during mitosis because the forces in interpolar bundles are transmitted to kinetochore fibers and hence to kinetochores through these connections.
Topics: Animals; Humans; Kinetochores; Microtubules; Spindle Apparatus; Stress, Mechanical
PubMed: 28725997
DOI: 10.1007/s00249-017-1244-4 -
International Journal of Molecular... Feb 2021The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular...
The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.
Topics: Actin Cytoskeleton; Cell Movement; Focal Adhesions; HaCaT Cells; Hemidesmosomes; Humans; Keratins; Microtubules; Models, Biological
PubMed: 33669958
DOI: 10.3390/ijms22042130 -
Nano Letters Sep 2020In nature, interactions between biopolymers and motor proteins give rise to biologically essential emergent behaviors. Besides cytoskeleton mechanics, active nematics...
In nature, interactions between biopolymers and motor proteins give rise to biologically essential emergent behaviors. Besides cytoskeleton mechanics, active nematics arise from such interactions. Here we present a study on 3D active nematics made of microtubules, kinesin motors, and depleting agent. It shows a rich behavior evolving from a nematically ordered space-filling distribution of microtubule bundles toward a flattened and contracted 2D ribbon that undergoes a wrinkling instability and subsequently transitions into a 3D active turbulent state. The wrinkle wavelength is independent of the ATP concentration and our theoretical model describes its relation with the appearance time. We compare the experimental results with a numerical simulation that confirms the key role of kinesin motors in cross-linking and sliding the microtubules. Our results on the active contraction of the network and the independence of wrinkle wavelength on ATP concentration are important steps forward for the understanding of these 3D systems.
Topics: Computer Simulation; Kinesins; Microtubules
PubMed: 32786934
DOI: 10.1021/acs.nanolett.0c01546 -
Current Biology : CB Feb 2019Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped...
Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped geometry despite microtubule turnover and internal stresses [1-6]. Regulation of microtubule dynamics, motor proteins, microtubule crosslinking, and chromatid cohesion can modulate spindle size and shape, and yet modulated spindles reach and hold a new steady state [7-11]. Here, we ask what maintains any spindle steady-state geometry. We report that clustering of microtubule ends by dynein and NuMA is essential for mammalian spindles to hold a steady-state shape. After dynein or NuMA deletion, the mitotic microtubule network is "turbulent"; microtubule bundles extend and bend against the cell cortex, constantly remodeling network shape. We find that spindle turbulence is driven by the homotetrameric kinesin-5 Eg5, and that acute Eg5 inhibition in turbulent spindles recovers spindle geometry and stability. Inspired by in vitro work on active turbulent gels of microtubules and kinesin [12, 13], we explore the kinematics of this in vivo turbulent network. We find that turbulent spindles display decreased nematic order and that motile asters distort the nematic director field. Finally, we see that turbulent spindles can drive both flow of cytoplasmic organelles and whole-cell movement-analogous to the autonomous motility displayed by droplet-encapsulated turbulent gels [12]. Thus, end-clustering by dynein and NuMA is required for mammalian spindles to reach a steady-state geometry, and in their absence Eg5 powers a turbulent microtubule network inside mitotic cells.
Topics: Cell Cycle Proteins; Cell Line; Dyneins; Humans; Microtubules; Spindle Apparatus
PubMed: 30744975
DOI: 10.1016/j.cub.2019.01.016 -
Biomolecules Jun 2023Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such...
Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in , with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.
Topics: Saccharomyces cerevisiae; Microscopy, Fluorescence; Microtubules; Software
PubMed: 37371519
DOI: 10.3390/biom13060939 -
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 -
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 -
Cytoskeleton (Hoboken, N.J.) Nov 2012The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the... (Review)
Review
The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the centralspindlin complex which is composed of two proteins, a kinesin-like protein, Mitotic kinesin-like protein 1, and a Rho GTPase activating protein (RhoGAP), CYK-4. Through its targeted localization to a narrow region of antiparallel microtubule overlap immediately following chromosome segregation, centralspindlin initiates central spindle assembly. Centralspindlin has several critical functions during cell division including positioning of the division plane, regulation of Rho family GTPases, as well as midbody assembly and abscission. In this review, we will examine the biochemistry of centralspindlin and its multiple functions during cell division. Remarkably, several of its critical functions are somewhat unexpected. Although endowed with motor domains, centralspindlin has an important role in generating stable, antiparallel microtubule bundles. Although it contains a Rho family GAP domain, it has a central role in the activation of RhoA during cytokinesis. Finally, centralspindlin functions as a motor protein complex, as a scaffold protein for key regulators of abscission and as a conventional RhoGAP. Because of these diverse functions, centralspindlin lies at the heart of the cytokinetic mechanism.
Topics: Animals; Cytokinesis; GTPase-Activating Proteins; Humans; Microtubule-Associated Proteins; Microtubules
PubMed: 22927365
DOI: 10.1002/cm.21065 -
The Plant Journal : For Cell and... Jul 2013Microtubules have a key role in plant morphogenesis, as they control the oriented deposition of cellulose in the cell wall, and thus growth anisotropy. The idea that... (Review)
Review
Microtubules have a key role in plant morphogenesis, as they control the oriented deposition of cellulose in the cell wall, and thus growth anisotropy. The idea that mechanical stress could be one of the main determinants behind the orientation of microtubules in plant cells emerged very soon after their discovery. The cause of mechanical stress in plant cells is turgor pressure, which can build up to 1 MPa and is restrained by cell wall stiffness. On the tissue scale, this can lead to regional patterns of tension, in particular in the epidermis of aerial organs, which resist the stress generated by cells in internal tissues. Here we summarize more than 50 years of work on the contribution of mechanical stress in guiding microtubule behavior, and the resulting impact on growth anisotropy and growth heterogeneity. We propose a conceptual model on microtubule dynamics and their ability to self-organize in bundles parallel to the direction of maximal stress, as well as a synthetic representation of the putative mechanotransducers at play.
Topics: Anisotropy; Biophysics; Cell Wall; Indoleacetic Acids; Microtubules; Models, Biological; Plant Cells; Plant Development; Signal Transduction; Stress, Mechanical
PubMed: 23551516
DOI: 10.1111/tpj.12188 -
BMC Biology Sep 2019Spindle microtubule organization, regulated by microtubule-associated proteins, is critical for cell division. Proper organization of kinetochore fiber (K-fiber),...
BACKGROUND
Spindle microtubule organization, regulated by microtubule-associated proteins, is critical for cell division. Proper organization of kinetochore fiber (K-fiber), connecting spindle poles and kinetochores, is a prerequisite for precise chromosomal alignment and faithful genetic material transmission. However, the mechanisms of K-fiber organization and dynamic maintenance are still not fully understood.
RESULTS
We reveal that two previously uncharacterized coiled-coil domain proteins CCDC74A and CCDC74B (CCDC74A/B) are spindle-localized proteins in mammalian cells. They bind directly to microtubules through two separate domains and bundle microtubules both in vivo and in vitro. These functions are required for K-fiber organization, bipolar spindle formation, and chromosomal alignment. Moreover, CCDC74A/B form homodimers in vivo, and their self-association activity is necessary for microtubule bundling and K-fiber formation.
CONCLUSIONS
We characterize CCDC74A and CCDC74B as microtubule-associated proteins that localize to spindles and are important K-fiber crosslinkers required for bipolar spindle formation and chromosome alignment.
Topics: Animals; COS Cells; Chlorocebus aethiops; HEK293 Cells; HeLa Cells; Humans; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 31521166
DOI: 10.1186/s12915-019-0694-9