Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.     

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.     

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.     

The formin mDia regulates GSK3beta through novel PKCs to promote microtubule stabilization but not MTOC reorientation in migrating fibroblasts

In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3beta (GSK3beta) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3beta inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3beta in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3beta functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3beta phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3beta through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3beta Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3beta through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.     

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.     

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.     

Feedback Mechanism for Microtubule Length Regulation by Stathmin Gradients

We formulate and analyze a theoretical model for the regulation of microtubule (MT) polymerization dynamics by the signaling proteins Rac1 and stathmin. In cells, the MT growth rate is inhibited by cytosolic stathmin, which, in turn, is inactivated by Rac1. Growing MTs activate Rac1 at the cell edge, which closes a positive feedback loop. We investigate both tubulin sequestering and catastrophe promotion as mechanisms for MT growth inhibition by stathmin. For a homogeneous stathmin concentration in the absence of Rac1, we find a switchlike regulation of the MT mean length by stathmin. For constitutively active Rac1 at the cell edge, stathmin is deactivated locally, which establishes a spatial gradient of active stathmin. In this gradient, we find a stationary bimodal MT-length distribution for both mechanisms of MT growth inhibition by stathmin. One subpopulation of the bimodal length distribution can be identified with fast-growing and long pioneering MTs in the region near the cell edge, which have been observed experimentally. The feedback loop is closed through Rac1 activation by MTs. For tubulin sequestering by stathmin, this establishes a bistable switch with two stable states: one stable state corresponds to upregulated MT mean length and bimodal MT length distributions, i.e., pioneering MTs; the other stable state corresponds to an interrupted feedback with short MTs. Stochastic effects as well as external perturbations can trigger switching events. For catastrophe-promoting stathmin, we do not find bistability.     

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.     

Microtubule network asymmetry in motile cells: Role of Golgi-derived array

Cell migration requires polarization of the cell into the leading edge and the trailing edge. Microtubules (MTs) are indispensable for polarized cell migration in the majority of cell types. To support cell polarity, MT network has to be functionally and structurally asymmetric. How is this asymmetry achieved? In interphase cells, MTs form a dynamic system radiating from a centrosome-based MT-organizing center (MTOC) to the cell edges. Symmetry of this radial array can be broken according to four general principles. Asymmetry occurs due to differential modulation of MT dynamics, relocation of existing MTs within a cell, adding an asymmetric nucleation site, and/or repositioning of a symmetric nucleation site to one side of a cell. Combinations of these asymmetry regulation principles result in a variety of asymmetric MT networks typical for diverse motile cell types. Importantly, an asymmetric MT array is formed at a non-conventional MT nucleation site, the Golgi. Here, we emphasize the contribution of this array to the asymmetry of MT network.     

Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.     

Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling

We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally “pioneering” MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 μm/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione–2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that ~80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.     

Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulins are post-translationally modified species that differ by a single amino acid at their respective C-termini. We have examined the distribution of these two species by immunofluorescence in proliferating and differentiated cells using antisera specifically reactive with each of the forms. In proliferating PtK1 cells, Tyr tubulin was the predominant form in almost every cytoplasmic microtubule (MT); only a few MTs contained detectable Glu tubulin. In contrast, staining of centrioles and primary cilia of PtK1 cells suggested that Glu tubulin was the predominant form in these stable assemblies of MTs. An examination of the distribution (by immunofluorescence) and relative amount (by immunoblot analysis) of the two forms of tubulin in the stable assemblies of MTs present in cultured neuronal cells (neurites), sperm and tracheal cells (axonemes and basal bodies), and platelets and erythrocytes (marginal bands) revealed that, in general, the MTs in these arrays contained substantially elevated levels of Glu tubulin in comparison with the levels in MTs of cultured cells. The one exception, the marginal band of toad erythrocytes, which contained only Tyr tubulin, demonstrates that an elevated level of Glu tubulin is not an obligate feature of a stable array of MTs. Nonetheless, an elevated level of Glu tubulin may be a useful indicator of stable MTs in differentiated cells. It is important to note that commonly used sources of tubulin (e.g., brain or flagella) necessarily yield tubulin that differs strikingly from tubulin of proliferating cells in its content of Glu tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level

Interphase cultured monkey kidney (TC-7) cells contain distinct subsets of cellular microtubules (MTs) enriched in posttranslationally detyrosinated (Glu) or tyrosinated (Tyr) alpha tubulin (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779-789). To determine the relative stability of these subsets of MTs, we subjected TC-7 cells to treatments that slowly depolymerized MTs. We found Glu MTs to be more resistant than Tyr MTs to depolymerization by nocodazole in living cells, and to depolymerization by dilution in detergent-permeabilized cell models. However, in cold-treated cells, Glu and Tyr MTs did not differ significantly in their stability. Digestion of permeabilized cell models with pancreatic carboxypeptidase A, to generate Glu MTs from endogenous Tyr MTs, did not significantly alter the resistance of the endogenous Tyr MTs toward dilution-induced depolymerization. Furthermore, in human fibroblasts that contained no distinct Glu MTs, we observed a population of nocodazole-resistant MTs. These data suggest that Glu MTs possess enhanced stability against end-mediated depolymerization, yet detyrosination alone appears to be insufficient to confer this enhanced stability.     

Actin-capping protein promotes microtubule stability by antagonizing the actin activity of mDia1

In migrating fibroblasts, RhoA and its effector mDia1 regulate the selective stabilization of microtubules (MTs) polarized in the direction of migration. The conserved formin homology 2 domain of mDia1 is involved both in actin polymerization and MT stabilization, and the relationship between these two activities is unknown. We found that latrunculin A (LatA) and jasplakinolide, actin drugs that release mDia1 from actin filament barbed ends, stimulated stable MT formation in serum-starved fibroblasts and caused a redistribution of mDia1 onto MTs. Knockdown of mDia1 by small interfering RNA (siRNA) prevented stable MT induction by LatA, whereas blocking upstream Rho or integrin signaling had no effect. In search of physiological regulators of mDia1, we found that actin-capping protein induced stable MTs in an mDia1-dependent manner and inhibited the translocation of mDia on the ends of growing actin filaments. Knockdown of capping protein by siRNA reduced stable MT levels in proliferating cells and in starved cells stimulated with lysophosphatidic acid. These results show that actin-capping protein is a novel regulator of MT stability that functions by antagonizing mDia1 activity toward actin filaments and suggest a novel form of actin–MT cross-talk in which a single factor acts sequentially on actin and MTs.     

Down-regulation of Stathmin Is Required for the Phenotypic Changes and Classical Activation of Macrophages

Macrophages are important cells of innate immunity with specialized capacity for recognition and elimination of pathogens and presentation of antigens to lymphocytes for adaptive immunity. Macrophages become activated upon exposure to pro-inflammatory cytokines and pathogenic stimuli. Classical activation of macrophages with interferon-γ (IFNγ) and lipopolysaccharide (LPS) triggers a wide range of signaling events and morphological changes to induce the immune response. Our previous microtubule (MT) proteomic work revealed that the stathmin association with MTs is considerably reduced in activated macrophages, which contain significantly more stabilized MTs. Here, we show that there is a global decrease in stathmin levels, an MT catastrophe protein, in activated macrophages using both immunoblotting and immunofluorescent microscopy. This is an LPS-specific response that induces proteasome-mediated degradation of stathmin. We explored the functions of stathmin down-regulation in activated macrophages by generating a stable cell line overexpressing stathmin-GFP. We show that stathmin-GFP overexpression impacts MT stability, impairs cell spreading, and reduces activation-associated phenotypes. Furthermore, overexpressing stathmin reduces complement receptor 3-mediated phagocytosis and cellular activation, implicating a pivotal inhibitory role for stathmin in classically activated macrophages.     

Beating rate of isolated neonatal cardiomyocytes is regulated by the stable microtubule subset

We investigated the roles of microtubule (MT) dynamics (growth and shrinkage), the stable, nongrowing MT subset, the posttranslationally detyrosinated MT subset, and artificially elevated tubulin levels in the negative regulation of heart cell beating rate. We manipulated the MT populations in isolated, neonatal cardiomyocytes obtained from normal animals in several ways and then measured heart cell beating rate directly. We found that the stabilized population of MTs was sufficient to maintain a normal beating rate, whereas MT dynamics and detyrosination made no observable contribution. Furthermore, by directly and acutely increasing the level of tubulin within otherwise normally beating cells, we found that the increased tubulin (and MT) levels further depressed the beating rate. In conclusion, the stabilized MT subset is sufficient to maintain the normal beating rate in these cells, whereas increasing the MT density depresses it.     

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.     

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.     

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.     

Establishment of a stable, acetylated microtubule bundle during neuronal commitment

Two posttranslational modifications of alpha-tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment- and differentiation-related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA-induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine-stable, although no MT-associated proteins (MAPs) are detectable using a battery of anti-MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.