Augmin-dependent microtubule nucleation at microtubule walls in the spindle

The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.     

Intermediate filament-associated cytolinker plectin 1c destabilizes microtubules in keratinocytes

The transition of microtubules (MTs) from an assembled to a disassembled state plays an essential role in several cellular functions. While MT dynamics are often linked to those of actin filaments, little is known about whether intermediate filaments (IFs) have an influence on MT dynamics. We show here that plectin 1c (P1c), one of the multiple isoforms of the IF-associated cytolinker protein plectin, acts as an MT destabilizer. We found that MTs in P1c-deficient (P1c^−/−) keratinocytes are more resistant toward nocodazole-induced disassembly and display increased acetylation. In addition, live imaging of MTs in P1c^−/−, as well as in plectin-null, cells revealed decreased MT dynamics. Increased MT stability due to P1c deficiency led to changes in cell shape, increased velocity but loss of directionality of migration, smaller-sized focal adhesions, higher glucose uptake, and mitotic spindle aberrations combined with reduced growth rates of cells. On the basis of ex vivo and in vitro experimental approaches, we suggest a mechanism for MT destabilization in which isoform-specific binding of P1c to MTs antagonizes the MT-stabilizing and assembly-promoting function of MT-associated proteins through an inhibitory function exerted by plectin''s SH3 domain. Our results open new perspectives on cytolinker-coordinated IF-MT interaction and its physiological significance.     

Hsp90 binds microtubules and is involved in the reorganization of the microtubular network in angiosperms

Microtubules (MTs) are essential for many processes in plant cells. MT-associated proteins (MAPs) influence MT polymerization dynamics and enable them to perform their functions. The molecular chaperone Hsp90 has been shown to associate with MTs in animal and plant cells. However, the role of Hsp90-MT binding in plants has not yet been investigated. Here, we show that Hsp90 associates with cortical MTs in tobacco cells and decorates MTs in the phragmoplast. Further, we show that tobacco Hsp90_MT binds directly to polymerized MTs in vitro. The inhibition of Hsp90 by geldanamycin (GDA) severely impairs MT re-assembly after cold-induced de-polymerization. Our results indicate that the plant Hsp90 interaction with MTs plays a key role in cellular events, where MT re-organization is needed.     

Tobacco microtubule-associated protein,MAP65-1c,bundles and stabilizes microtubules

Three genes that encode MAP65-1 family proteins have been identified in the Nicotiana tabacum genome. In this study, NtMAP65-1c fusion protein was shown to bind and bundle microtubules (MTs). Further in vitro investigations demonstrated that NtMAP65-1c not only alters MT assembly and nucleation, but also exhibits high MT stabilizing activity against cold or katanin-induced destabilization. Analysis of NtMAP65-1c-GFP expressing BY-2 cells clearly demonstrated that NtMAP65-1c was able to bind to MTs during specific stages of the cell cycle. Furthermore, in vivo, NtMAP65-1c-GFP-bound cortical MTs displayed an increase in resistance against the MT-disrupting drug, propyzamide, as well as against cold temperatures. Taken together, these results strongly suggest that NtMAP65-1c stabilizes MTs and is involved in the regulation of MT organization and cellular dynamics.     

γ‐Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ‐Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ‐Tubulin Ring Complexes (γ‐TuRCs). While the subunits that constitute γ‐Tubulin Small Complexes (γ‐TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ‐TuRC‐specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ‐TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ‐TuRCs on astral MTs. γ‐TuRCs locate along the length of astral MTs, and depletion of γ‐TuRC‐specific proteins increases MT dynamics and causes the plus‐end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down‐regulation rescues spindle orientation defects induced by γ‐TuRC depletion. Therefore, we propose a role for γ‐TuRCs in regulating spindle positioning by controlling the stability of astral MTs.     

SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.     

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid ( t _1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.     

Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast

Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.     

Regulation of leading edge microtubule and actin dynamics downstream of Rac1

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, "pioneer" MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.     

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length‐dependent increase in the catastrophe frequency that is mediated by kinesin‐8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin‐8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.     

Arabidopsis microtubule-associated protein AtMAP65-2 acts as a microtubule stabilizer

Nine genes that encode proteins of the MAP65 family have been identified in the Arabidopsis thaliana genome. In this study, we reported that AtMAP65-2, a member of the AtMAP65 family, could strongly stabilize microtubules (MTs). Bacterially-expressed AtMAP65-2 fusion proteins induced the formation of large MT bundles in vitro. Although AtMAP65-2 showed little effect on MT assembly or nucleation, AtMAP65-2 greatly stabilized MTs that were subjected to low-temperature treatment in vitro. Analyses of truncated versions of AtMAP65-2 indicated that the region that encompassed amino acids 495–578, which formed a flexible extended loop, played a crucial role in the stabilization of MTs. Analysis of suspension-cultured Arabidopsis cells that expressed the AtMAP65-2-GFP fusion protein showed that AtMAP65-2 co-localized with MTs throughout the cell cycle. Cortical MTs that were decorated with AtMAP65-2-GFP were more resistant to the MT-disrupting drug propyzamide and to ice treatment in vivo. The results of this study demonstrate that AtMAP65-2 strongly stabilizes MTs and is involved in the regulation of MT organization and dynamics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Li and X. Zeng have contributed equally to this paper and are considered as joint first authors.     

Straighten up and fly right: microtubule dynamics and organization of non-centrosomal arrays in higher plants

Live cell imaging and genetic studies are demonstrating that cortical microtubule arrays in plant cells are dynamic structures in which microtubule (MT) bundles play a key role in creating array organization and function. Steps important for creating and organizing these arrays include recruitment of nucleation complexes to the cell cortex and to the lattices of previously established MTs, association of newly created MTs to the cell cortex, release of MTs from sites of nucleation, transport of released MTs by polymer treadmilling, and subsequent interactions between treadmilling MTs. The results of MT interactions include induced catastrophe, severing, and the capture and reorientation of growing polymer ends by bundling interactions. Together, these properties predict a capacity for self-ordering that is likely to play an important role in establishing the parallel organization of the arrays.     

JNK1 is required for maintenance of neuronal microtubules and controls phosphorylation of microtubule-associated proteins

Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.     

Randomness in microtubule dynamics: an error that requires correction or an inherent plasticity required for normal cellular function?

Microtubules (MTs) play roles in regulating the mechanical structure and dynamics of cells. While MTs appear to be highly ordered structures, recent data suggest some randomness in their structure and dynamics. Part of this inherent randomness is attributed to errors and correction mechanisms are being investigated to overcome these ‘mistakes.’ However, this randomness may also be part of the normal intracellular function of MTs. It is possible that random events in MT structure and dynamics may contribute to their normal function and may even be part of an improved efficacy mechanism. An alternative view, wherein MT and kinetochore errors are part of required cell plasticity, is also discussed. These data may further support the concept of randomness in biological pathways as part of self‐organization or accurate and enhanced function.     

The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery

The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.     

Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis

Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Rho Guanosine Triphosphatase Mediates the Selective Stabilization of Microtubules Induced by Lysophosphatidic Acid

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.     

Feedback interactions between cell-cell adherens junctions and cytoskeletal dynamics in newt lung epithelial cells

To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.     

Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

Summary Post-mitotic epidermal cells of barley leaves were found to contain, in addition to cortical microtubules (CMTs), distinct arrays of endoplasmic microtubules (EMTs). These encircle nuclei and continuously merge into the CMT arrays that underly the plasmalemma. Detailed three-dimensional reconstruction of both types of MTs during fungal infection showed that profound and very rapid MT rearrangements occurred especially in the case of incompatible (resistant) barley-powdery mildew genotype combination. The most early MT responses, followed by their subsequent complete disintegration, were recorded around nuclei. These events might be relevant for the induction of such nuclear processes as onset of DNA synthesis and nuclear chromatin condensation. Observed pattern of early infection events, as well as less prominent responses in the case of compatible (susceptible) barley-powdery mildew genotype combination, both findings suggest that rapid reorganization of the MT cytoskeleton could be involved in recognition of the fungus by host cells and in the initiation of resistance responses in barley leaves. We hypothesize that the integrity and dynamics of the MT cytoskeleton, especially of its perinuclear part, might participate in control mechanisms involved in activation of resistance genes.Abbreviations CMTs cortical microtubules - EMTs endoplasmic microtubules - MT microtubules - PI propidium iodide - SC sensitive combination - RC resistant combination