How Do Kinetochores CLASP Dynamic Microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizesto the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

How do kinetochores CLASP dynamic microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dynamics

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.     

The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function

Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

Identification of a phragmoplast-associated kinesin-related protein in higher plants

The phragmoplast executes cytokinesis in higher plants. The major components of the phragmoplast are microtubules, which are arranged in two mirror-image arrays perpendicular to the division plane [1]. The plus ends of these microtubules are located near the site of the future cell plate. Golgi-derived vesicles are transported along microtubules towards the plus ends to deliver materials bound for the cell plate [2] [3]. During cell division, rapid microtubule reorganization in the phragmoplast requires the orchestrated activities of microtubule motor proteins such as kinesins. We isolated an Arabidopsis cDNA clone of a gene encoding an amino-terminal motor kinesin, AtPAKRP1, and have determined the partial sequence of its rice homolog. Immunofluorescence experiments with two sets of specific antibodies revealed consistent localization of AtPAKRP1 and its homolog in Arabidopsis and rice cells undergoing anaphase, telophase and cytokinesis. AtPAKRP1 started to accumulate along microtubules towards the spindle midzone during late anaphase. Once the phragmoplast microtubule array was established, AtPAKRP1 conspicuously localized to microtubules near the future cell plate. Our results provide evidence that AtPAKRP1 is a hitherto unknown motor that may take part in the establishment and/or maintenance of the phragmoplast microtubule array.     

CLASP modulates microtubule-cortex interaction during self-organization of acentrosomal microtubules

CLASP proteins associate with either the plus ends or sidewalls of microtubules depending on the subcellular location and cell type. In plant cells, CLASP's distribution along the full length of microtubules corresponds with the uniform anchorage of microtubules to the cell cortex. Using live cell imaging, we show here that loss of CLASP in Arabidopsis thaliana results in partial detachment of microtubules from the cortex. The detached portions undergo extensive waving, distortion, and changes in orientation, particularly when exposed to the forces of cytoplasmic streaming. These deviations from the normal linear polymerization trajectories increase the likelihood of intermicrotubule encounters that are favorable for subsequent bundle formation. Consistent with this, cortical microtubules in clasp-1 leaf epidermal cells are hyper-parallel. On the basis of these data, we identify a novel mechanism where modulation of CLASP activity governs microtubule-cortex attachment, thereby contributing to self-organization of cortical microtubules.     

Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP

The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends.     

MAPs: cellular navigators for microtubule array orientations in Arabidopsis

Microtubules are subcellular nanotubes composed of α- and β-tubulin that arise from microtubule nucleation sites and are mainly composed of γ-tubulin complexes. Cell wall encased plant cells have evolved four distinct microtubule arrays that regulate cell division and expansion. Microtubule-associated proteins, the so called MAPs, construct, destruct and reorganize microtubule arrays thus regulating their spatiotemporal transitions during the cell cycle. By physically binding to microtubules and/or modulating their functions, MAPs control microtubule dynamic instability and/or interfilament cross talk. We survey the recent analyses of Arabidopsis MAPs such as MAP65, MOR1, CLASP, katanin, TON1, FASS, TRM, TAN1 and kinesins in terms of their effects on microtubule array organizations and plant development.     

TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.     

Spline and flagellar microtubules are resistant to mitotic disrupter herbicides

Summary Microtubule arrays in developing spermatogenous cells of pteridophytes have unique microtubule organizing centers and post-translation modifications of tubulin. Sensitivity of these arrays to the microtubule-destabilizing effects of the mitotic disrupter herbicides was examined by immunofluorescence, transmission and immunogold electron microscopy. Acetylated, stabilized arrays, such as the spline, and microtubules of the basal bodies and flagella are formed after the final mitotic division and are resistant to these herbicides. Non-acetylated, dynamic arrays that exist prior to the final mitosis, such as interphase and mitotic arrays, are eliminated by all of these herbicides, with symptomology (arrested prometaphase, lobed nuclei, irregular cell plate formation) similar to that observed in other land plants. The only exception to the instability of these mitotic microtubule arrays are the few microtubules that are collected by kinetochores into short tufts. The presence of structurally-distinguishable MTOCs, such as the blepharoplast, did not confer resistance, despite the anchoring of the minus ends of the microtubules. Simultaneous treatment with herbicide and 5-bromodeoxyuridine (BrdU), with subsequent detection with anti-BrdU of cells that had gone through S-phase during the BrdU incubation, reveals that only acetylated arrays formed prior to herbicide treatment are resistant. These data indicate that only actively polymerizing, dynamic microtubule arrays are sensitive to the destabilizing effects of the mitotic disrupter herbicides.Abbreviations MTOC microtubule organizing center - BrdU 5-bromodeoxyuridine     

Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.     

CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo

Microtubule dynamics are thought to play an important role in regulating microtubule interactions with cortical force generating motor proteins that position the spindle during asymmetric cell division. CLASPs are microtubule-associated proteins that have a conserved role in regulating microtubule dynamics in diverse cell types. Caenorhabditis elegans has three CLASP homologs in its genome. CLS-2 is known to localize to kinetochores and is needed for chromosome segregation at meiosis and mitosis; however CLS-1 and CLS-3 have not been reported to have any role in embryonic development. Here, we show that depletion of CLS-2 in combination with either CLS-1 or CLS-3 results in defects in nuclear rotation, maintenance of spindle length, and spindle displacement in the one-cell embryo. Polarity is normal in these embryos, but reduced numbers of astral microtubules reach all regions of the cortex at the time of spindle positioning. Analysis of the microtubule plus-end tracker EB1 also revealed a reduced number of growing microtubules reaching the cortex in CLASP depleted embryos, but the polymerization rate of astral microtubules was not slower than in wild type. These results indicate that C. elegans CLASPs act partially redundantly to regulate astral microtubules and position the spindle during asymmetric cell division. Further, we show that these spindle pole-positioning roles are independent of the CLS-2 binding proteins HCP-1 and HCP-2.     

Essential role of tubulin-folding cofactor D in microtubule assembly and its association with microtubules in fission yeast.

The main structural components of microtubules are alpha- and beta-tubulins. A group of proteins called cofactors are crucial in the formation of assembly-competent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature-sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild-type cells, resulting in altered cell shape, but is rescued by co-overexpression of beta-tubulin. Alp1 co-localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co-sediments with taxol (paclitaxel)-stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of beta-tubulin, cofactor D may play a vital role in microtubule-dependent processes as a microtubule-associated protein.     

Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles

Proteins that in cells specifically bind to growing microtubule plus ends (+TIPs) are thought to play important roles in polarization of the cytoskeleton. However, most +TIPs do not show a bias of their microtubule-binding behavior toward different subcellular regions. Here, we examine the dynamics of the +TIP CLASP in migrating PtK1 epithelial cells. We find that, although CLASPs track microtubule plus ends in the cell body, they dynamically decorate the entire microtubule lattice in the leading edge lamella and lamellipodium. Microtubule lattice binding is mediated by the COOH-terminal region of the CLASP microtubule-binding domain and is regulated downstream of Rac1. Phosphorylation of sites in the NH2-terminal part of the microtubule-binding domain by glycogen synthase kinase 3beta likely regulates the affinity of CLASPs for microtubule lattices. These results demonstrate the striking difference of the microtubule cytoskeleton in the lamella as compared with the cell body and provide the first direct observation of subcellular regulation of a microtubule-associated protein in migrating cells.     

Stu2p,the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer

The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.     

Analysis of cortical arrays from Tradescantia virginiana at high resolution reveals discrete microtubule subpopulations and demonstrates that confocal images of arrays can be misleading

Cortical microtubule arrays are highly organized networks involved in directing cellulose microfibril deposition within the cell wall. Their organization results from complex interactions between individual microtubules and microtubule-associated proteins. The precise details of these interactions are often not evident using optical microscopy. Using high-resolution scanning electron microscopy, we analyzed extensive regions of cortical arrays and identified two spatially discrete microtubule subpopulations that exhibited different stabilities. Microtubules that lay adjacent to the plasma membrane were often bundled and more stable than the randomly aligned, discordant microtubules that lay deeper in the cytoplasm. Immunolabeling revealed katanin at microtubule ends, on curves, or at sites along microtubules in line with neighboring microtubule ends. End binding 1 protein also localized along microtubules, at microtubule ends or junctions between microtubules, and on the plasma membrane in direct line with microtubule ends. We show fine bands in vivo that traverse and may encircle microtubules. Comparing confocal and electron microscope images of fluorescently tagged arrays, we demonstrate that optical images are misleading, highlighting the fundamental importance of studying cortical microtubule arrays at high resolution.     

Mechanisms of Self-Organization of Cortical Microtubules in Plants Revealed by Computational Simulations

Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel yet dispersed array transverse to the elongation axis for proper cell wall expansion. Some of these microtubules exhibit free minus-ends, leading to migration at the cortex by hybrid treadmilling. Collisions between microtubules can result in plus-end entrainment (“zippering”) or rapid depolymerization. Here, we present a computational model of cortical microtubule organization. We find that plus-end entrainment leads to self-organization of microtubules into parallel arrays, whereas catastrophe-inducing collisions do not. Catastrophe-inducing boundaries (e.g., upper and lower cross-walls) can tune the orientation of an ordered array to a direction transverse to elongation. We also find that changes in dynamic instability parameters, such as in mor1-1 mutants, can impede self-organization, in agreement with experimental data. Increased entrainment, as seen in clasp-1 mutants, conserves self-organization, but delays its onset and fails to demonstrate increased ordering. We find that branched nucleation at acute angles off existing microtubules results in distinctive sparse arrays and infer either that microtubule-independent or coparallel nucleation must dominate. Our simulations lead to several testable predictions, including the effects of reduced microtubule severing in katanin mutants.     

The quadripolar microtubule system in lower land plants

The quadripolar microtubule system (QMS) is a complex array that is associated with predivision establishment of quadripolarity in sporocytes of lower plants (bryophytes and lycopsids). The QMS unerringly predicts the polarity of the two meiotic divisions and plays a central role in development of both the mitotic apparatus (MA) and cytokinetic apparatus (CA) which together accomplish quadripartitioning of the sporocyte into four haploid spores. The QMS is typically, but not exclusively, associated with monoplastidy and precocious quadrilobing of the cytoplasm. In early meiotic prophase the single plastid divides and the resultant plastids migrate so that either the tips of two plastids or the four plastids resulting from a second division are located in the future spore domains. Microtubules that emanate from the plastid tips or from individual plastids in the spore domains interact in the future planes of cytokinesis and give rise to the QMS. The QMS, which encages the prophase nucleus, consists of at least four and usually six (when spore domains are in tetrahedral arrangement) bipolar spindle-like arrays of microtubules presumably with minus ends at plastids in spore domains and plus ends interacting in the future plane of cytokinesis. Each of the six arrays is essentially like the single axial microtubule system (AMS) that intersects the division site and is transformed into the spindle in monoplastidic mitosis in hornworts. As comparative data accumulate, it appears that the AMS is not unique to monoplastidic cell division but instead represents a basic microtubule arrangement that survives as spindle and phragmoplast in cell division of higher plants.     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.