p21(Cip1) regulates cell-substrate adhesion and interphase microtubule dynamics in untransformed human mammary epithelial cells

Despite its frequent inactivation in human breast cancers, the role of p21(Cip1) (p21) in morphological plasticity of normal mammary epithelial cells is still poorly understood. To address this question, we have investigated the consequences of p21 silencing in two-dimensional (2D) morphogenesis of untransformed human mammary epithelial cells. Here we show that p21 inactivation causes a reduction of 2D cell spreading and suppresses focal adhesion. In order to investigate the cytoskeletal modifications associated with this altered morphology, we have analyzed the microtubule dynamics in interphase p21-depleted cells. Our results demonstrate that interphase microtubule dynamic instability is strongly increased by p21 silencing. This alteration correlates with severe microtubule hypoacetylation. Next, we show that these microtubule defects in p21-depleted cells can be reversed by the use of the small molecule tubacin, a specific inhibitor of the α-tubulin deacetylase HDAC6. Tubacin-induced microtubule dynamics decrease also correlates with a partial recovery of cell spreading and focal adhesion in those cells. Collectively, these data indicate that p21 regulates the morphological plasticity of normal mammary epithelial cells by modulating dynamics of key cytoskeletal components.     

HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the epsilon-amino group of alpha-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that beta-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro. We find that HDAC-6 is a micro tubule-associated protein capable of deacetylating alpha-tubulin in vivo and in vitro. HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC-6 function by two independent mechanisms--pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)--leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.     

Microtubule plus-end tracking proteins in differentiated mammalian cells

Differentiated mammalian cells are often characterized by highly specialized and polarized structure. Its formation and maintenance depends on cytoskeletal components, among which microtubules play an important role. The shape and dynamic properties of microtubule networks are controlled by multiple microtubule-associated factors. These include molecular motors and non-motor proteins, some of which accumulate specifically at the growing microtubule plus-ends (the so-called microtubule plus-end tracking proteins). Plus-end tracking proteins can contribute to the regulation of microtubule dynamics, mediate the cross-talk between microtubule ends, the actin cytoskeleton and the cell cortex, and participate in transport and positioning of structural and regulatory factors and membrane organelles. Malfunction of these proteins results in various human diseases including some forms of cancer, neurodevelopmental disorders and mental retardation. In this article we discuss recent data on microtubule dynamics and activities of microtubule plus-end binding proteins important for the physiology and pathology of differentiated mammalian cells such as neurons, polarized epithelia, muscle and sperm cells.     

TPPP/p25 Promotes Tubulin Acetylation by Inhibiting Histone Deacetylase 6

TPPP/p25 (tubulin polymerization-promoting protein/p25) is an unstructured protein that induces microtubule polymerization in vitro and is aligned along the microtubule network in transfected mammalian cells. In normal human brain, TPPP/p25 is expressed predominantly in oligodendrocytes, where its expression is proved to be crucial for their differentiation process. Here we demonstrated that the expression of TPPP/p25 in HeLa cells, in doxycycline-inducible CHO10 cells, and in the oligodendrocyte CG-4 cells promoted the acetylation of α-tubulin at residue Lys-40, whereas its down-regulation by specific small interfering RNA in CG-4 cells or by the withdrawal of doxycycline from CHO10 cells decreased the acetylation level of α-tubulin. Our results indicate that TPPP/p25 binds to HDAC6 (histone deacetylase 6), an enzyme responsible for tubulin deacetylation. Moreover, we demonstrated that the direct interaction of these two proteins resulted in the inhibition of the deacetylase activity of HDAC6. The measurement of HDAC6 activity showed that TPPP/p25 is able to induce almost complete (90%) inhibition at 3 μm concentration. In addition, treatment of the cells with nocodazole, vinblastine, or cold exposure revealed that microtubule acetylation induced by trichostatin A, a well known HDAC6 inhibitor, does not cause microtubule stabilization. In contrast, the microtubule bundling activity of TPPP/p25 was able to protect the microtubules from depolymerization. Finally, we demonstrated that, similarly to other HDAC6 inhibitors, TPPP/p25 influences the microtubule dynamics by decreasing the growth velocity of the microtubule plus ends and also affects cell motility as demonstrated by time lapse video experiments. Thus, we suggest that TPPP/p25 is a multiple effector of the microtubule organization.     

Inter-relationship of Histone Deacetylase-6 with Tau-cytoskeletal organization and remodeling

Cytoskeletal elements are the key players in cellular integrity, structure, signalling and migration. Each cytoskeletal element comprises of properties with respect to its structure and stability, which serve a specific array of functions. These structures are highly dynamic and regulated by modulation via direct interaction or post-translational modifications. HDAC6 is a cytoplasmic deacetylase known to regulate a wide range of cellular functions either through its deacetylase activity or direct interaction via its C-terminal ZnF UBP domain. HDAC6 has been widely studied for its role in aggresome formation, which acts as a protective mechanism upon protein aggregation. HDAC6 is known to play a critical role in the regulation of cytoskeletal elements-microtubules and actin filaments. This review summarizes the regulatory role of HDAC6 in cytoskeletal remodeling and dynamics of neuronal cells and its significance in neurodegenerative diseases.     

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ϵ-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.     

Clostridium difficile Toxin A Decreases Acetylation of Tubulin,Leading to Microtubule Depolymerization through Activation of Histone Deacetylase 6, and This Mediates Acute Inflammation

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2′,3′-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439-4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM-1 and MPM-2, decorate throughout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno-electron microscopy showed that these antibodies labeled the electron-dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM-1 and MPM-2) and the epiplasm (MPM-1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.     

How do Abl family kinases regulate cell shape and movement?

Genetic analysis and studies of normal and leukemia cells in culture have shown that Abl family nonreceptor tyrosine kinases regulate cell morphogenesis and motility. Abl family kinases, which include Drosophila (D-) Abl and the vertebrate Abl and Arg proteins, relay signals from cell surface growth-factor and adhesion receptors to promote cytoskeletal rearrangements. Recent biochemical and crystallographic analyses have clarified the mechanisms by which growth-factor and adhesion receptors might regulate the activity of Abl family kinases. When activated, Abl family kinases can regulate cytoskeletal dynamics by phosphorylating several known cytoskeletal regulatory proteins. In addition, the C-terminal half of Abl family kinases has several domains that bind to cytoskeletal components. Emerging evidence suggests that Abl family kinases can use these domains to directly organize cytoskeletal structure in vivo.     

Association of casein kinase II with microtubules

A magnesium-dependent heparin-inhibited protein kinase activity associated with brain microtubule preparations has been identified as casein kinase II using a monospecific polyclonal antibody. This enzyme appears enriched in cold-stable microtubule fractions. By immunofluorescence microscopy using an antiserum against casein kinase II, the in situ immunolabeling of some microtubule assays has been observed. Thus, mitotic spindles are stained by the anti-casein kinase II antibody in fibroblast cells. In neuroblastoma cells induced to differentiate, the labeling of microtubule arrays inside developing axon-like processes is also seen. These results support the view that casein kinase II can modulate cytoskeletal assembly and dynamics through phosphorylation of microtubule proteins.     

Regulation of cell migration by dynamic microtubules

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.     

c-Cbl and Cbl-b Act Redundantly to Protect Osteoclasts from Apoptosis and to Displace HDAC6 from β-Tubulin,Stabilizing Microtubules and Podosomes

c-Cbl and Cbl-b are highly conserved adaptor proteins that participate in integrin signaling, regulating cytoskeletal organization, motility, and bone resorption. Deletion of both c-Cbl and Cbl-b in mice leads to embryonic lethality, indicating that the two proteins perform essential redundant functions. To examine the redundant actions of c-Cbl and Cbl-b in osteoclasts, we depleted c-Cbl in Cbl-b^−/− osteoclasts by using a short hairpin RNA. Depleting both Cbl proteins disrupted both the podosome belt and the microtubule network and decreased bone-resorbing activity. Stabilizing the microtubules with paclitaxel or inhibiting histone deacetylase 6 (HDAC6), which destabilizes microtubules by deacetylating β-tubulin, protected both the microtubule network and the podosome belt. Examination of the mechanism involved demonstrated that the conserved four-helix bundle of c-Cbl''s tyrosine kinase binding domain bound to β-tubulin, and both c-Cbl and Cbl-b displaced HDAC6. In addition to the effects on microtubules and the podosome belt, depleting both Cbls significantly increased the levels of the proapoptotic protein Bim and apoptosis relative to the levels induced by eliminating either protein alone. Thus, both c-Cbl and Cbl-b promote bone resorption via the stabilization of microtubules, allowing the formation of the podosome belt in osteoclasts, and by promoting osteoclast survival.     

Plant microtubule-associated proteins: the HEAT is off in temperature-sensitive mor1.

Microtubules perform essential functions in plant cells and govern, with other cytoskeletal elements, cell division, formation of cell walls and morphogenesis. For microtubules to perform their roles in the cell their organization and dynamics must be regulated and microtubule-associated proteins bear the main responsibility for these activities. We are just beginning to identify these plant microtubule-regulating proteins. Biochemical, molecular and genetic procedures have identified plant homologues of known microtubule-associated proteins, such as kinesins, katanin and XMAP215, and novel classes of plant microtubule-associated proteins, such as MAP65 and MAP190. Showing how these proteins coordinate the microtubule cytoskeleton in vivo is now the challenge. The recent identification and characterization of the Arabidopsis thaliana microtubule organization mutant, mor1, begins to address this challenge and here we highlight the significance of this work.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

Role and regulation of kinesin-8 motors through the cell cycle

Members of the kinesin-8 motor family play a central role in controlling microtubule length throughout the eukaryotic cell cycle. Inactivation of kinesin-8 causes defects in cell polarity during interphase and astral and mitotic spindle length, metaphase chromosome alignment, timing of anaphase onset and accuracy of chromosome segregation. Although the biophysical mechanism by which kinesin-8 molecules influence microtubule dynamics has been studied extensively in a variety of species, a consensus view has yet to emerge. One reason for this might be that some members of the kinesin-8 family can associate to other microtubule-associated proteins, cell cycle regulatory proteins and other kinesin family members. In this review we consider how cell cycle specific modification and its association to other regulatory proteins may modulate the function of kinesin-8 to enable it to function as a master regulator of microtubule dynamics.     

The kinesin-like proteins,KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens

In plants, light determines chloroplast position;these organelles show avoidance and accumulation re-sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light ...     

Loss of α-Tubulin Acetylation Is Associated with TGF-β-induced Epithelial-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-β-induced EMT. We found that TGF-β increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-β type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.