CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta

CLASPs are mammalian microtubule-stabilizing proteins that can mediate the interaction between distal microtubule ends and the cell cortex. Using mass spectrometry-based assays, we have identified two CLASP partners, LL5beta and ELKS. LL5beta and ELKS form a complex that colocalizes with CLASPs at the cortex of HeLa cells as well as at the leading edge of motile fibroblasts. LL5beta is required for cortical CLASP accumulation and microtubule stabilization in HeLa cells, while ELKS plays an accessory role in these processes. LL5beta is a phosphatidylinositol-3,4,5-triphosphate (PIP3) binding protein, and its recruitment to the cell cortex is influenced by PI3 kinase activity but does not require intact microtubules. Cortical clusters of LL5beta and ELKS do not overlap with focal adhesions but often form in their vicinity and can affect their size. We propose that LL5beta and ELKS can form a PIP3-regulated cortical platform to which CLASPs attach distal microtubule ends.     

“CLASPing” tungsten's effects on microtubules with “PINs”

Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed.     

Epiblast integrity requires CLASP and Dystroglycan-mediated microtubule anchoring to the basal cortex

Amniote epiblast cells differentiate into mesoderm and endoderm lineages during gastrulation through a process called epithelial-to-mesenchymal transition (EMT). Molecular regulation of gastrulation EMT is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules to the basal cortex via CLIP-associated protein (CLASP), a microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation epiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediated through the CLASP-binding partner LL5. Live-imaging using EB1–enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilized basal microtubules. We further show that Dystroglycan is localized to basolateral membrane in epiblast cells. Basal but not lateral localization of Dystroglycan was regulated by CLASP. We propose that epiblast–BM interaction requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which initiates gastrulation EMT.     

A Highlights from MBoC Selection: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gα_i-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gα_i-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gα_i/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gα_i/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.     

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.     

MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis

Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein-dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These findings have wide biological importance, because spindle positioning is essential during embryogenesis and stem-cell homeostasis.     

Identification of a Role for CLASP2 in Insulin Action

Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. We present evidence that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin stimulation. Using mass spectrometry-based protein identification combined with phosphoantibody immunoprecipitation in L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the anti-phosphoserine immunoprecipitates upon insulin stimulation. Western blotting of CLASP2 immunoprecipitates with the phosphoantibody confirmed the finding that CLASP2 undergoes insulin-stimulated phosphorylation, and a number of novel phosphorylation sites were identified. Confocal imaging of L6 myotubes revealed that CLASP2 colocalizes with GLUT4 at the plasma membrane within areas of insulin-mediated cortical actin remodeling. CLASP2 is responsible for directing the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the concept that CLASP2 plays a role in the trafficking of GLUT4 at the cell periphery, CLASP2 knockdown by siRNA in L6 myotubes interfered with insulin-stimulated GLUT4 localization to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport. We therefore propose a new model for CLASP2 in insulin action, where CLASP2 directs the delivery of GLUT4 to cell cortex landing zones important for insulin action.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.     

Rho of Plant GTPase Signaling Regulates the Behavior of Arabidopsis Kinesin-13A to Establish Secondary Cell Wall Patterns

Plant cortical microtubule arrays determine the cell wall deposition pattern and proper cell shape and function. Although various microtubule-associated proteins regulate the cortical microtubule array, the mechanisms underlying marked rearrangement of cortical microtubules during xylem differentiation are not fully understood. Here, we show that local Rho of Plant (ROP) GTPase signaling targets an Arabidopsis thaliana kinesin-13 protein, Kinesin-13A, to cortical microtubules to establish distinct patterns of secondary cell wall formation in xylem cells. Kinesin-13A was preferentially localized with cortical microtubules in secondary cell wall pits, areas where cortical microtubules are depolymerized to prevent cell wall deposition. This localization of Kinesin-13A required the presence of the activated ROP GTPase, MICROTUBULE DEPLETION DOMAIN1 (MIDD1) protein, and cortical microtubules. Knockdown of Kinesin-13A resulted in the formation of smaller secondary wall pits, while overexpression of Kinesin-13A enlarged their surface area. Kinesin-13A alone could depolymerize microtubules in vitro; however, both MIDD1 and Kinesin-13A were required for the depolymerization of cortical microtubules in vivo. These results indicate that Kinesin-13A regulates the formation of secondary wall pits by promoting cortical microtubule depolymerization via the ROP-MIDD1 pathway.     

A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase), which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host–pathogen interactions.     

Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration

Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.     

Dynamics of myosin,microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells

Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.     

RanGTP and CLASP1 cooperate to position the mitotic spindle

Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-β function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.     

Cortical microtubules are responsible for gravity resistance in plants

Mechanical resistance to the gravitational force is a principal gravity response in plants distinct from gravitropism. In the final step of gravity resistance, plants increase the rigidity of their cell walls. Here we discuss the role of cortical microtubules, which sustain the function of the cell wall, in gravity resistance. Hypocotyls of Arabidopsis tubulin mutants were shorter and thicker than the wild-type, and showed either left-handed or right-handed helical growth at 1 g. The degree of twisting phenotype was intensified under hypergravity conditions. Hypergravity also induces reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells. In tubulin mutants, the percentage of cells with longitudinal microtubules was high even at 1 g, and it was further increased by hypergravity. The left-handed helical growth mutants had right-handed microtubule arrays, whereas the right-handed mutant had left-handed arrays. Moreover, blockers of mechanoreceptors suppressed both the twisting phenotype and reorientation of microtubules in tubulin mutants. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in signal perception in gravity resistance. Space experiments will confirm whether this view is applicable to plant resistance to 1 g gravity, as to the resistance to hypergravity.Key words: cortical microtubules, gravity, gravity resistance, hypergravity, mechanoreceptor, microgravity, tubulin mutants     

A Mechanochemical Model Explains Interactions between Cortical Microtubules in Plants

Microtubules anchored to the two-dimensional cortex of plant cells collide through plus-end polymerization. Collisions can result in rapid depolymerization, directional plus-end entrainment, or crossover. These interactions are believed to give rise to cellwide self-organization of plant cortical microtubules arrays, which is required for proper cell wall growth. Although the cell-wide self-organization has been well studied, less emphasis has been placed on explaining the interactions mechanistically from the molecular scale. Here we present a model for microtubule-cortex anchoring and collision-based interactions between microtubules, based on a competition between cross-linker bonding, microtubule bending, and microtubule polymerization. Our model predicts a higher probability of entrainment at smaller collision angles and at longer unanchored lengths of plus-ends. This model addresses observed differences between collision resolutions in various cell types, including Arabidopsis cells and Tobacco cells.     

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

Organization of cortical microtubules at the plasma membrane in Arabidopsis

 To understand the role of microtubules in the regulation of cell elongation, we characterized microtubule patterns in fass, a cell shape mutant of Arabidopsis thaliana (L.) Heynh. Examining microtubule patterns via immunocytochemistry, we found that fass cells were able to organize their microtubules into mitotic spindles and phragmoplasts. During interphase or preprophase, fass cells had cortical microtubules, verified by transmission electron microscopy, but these microtubules were not organized into the cortical array or preprophase band. Using chromatin condensation and tubulin localization on the nuclear envelope as preprophase stage markers, we found that although fass cells lacked the preprophase band and cortical array, their cell division cycle appeared normal. To pinpoint the defect in fass cells, we delineated the sequential events leading to cortical array formation in Arabidopsis cells and found that fass cells initiated and recolonized cortical microtubules in the same manner as wild-type cells, but failed to order them into the cortical array. Taken together, these results suggest fass cells are impaired in a component of the microtubule organizing center(s) required for the proper ordering of cortical microtubules at the plasma membrane. Received: 23 August 1996 / Accepted: 25 September 1996