BCL2 interaction with actin in vitro may inhibit cell motility by enhancing actin polymerization

In addition to its well-defined role as an antagonist in apoptosis, we propose that BCL2 may act as an intracellular suppressor of cell motility and adhesion under certain conditions. Our evidence shows that, when overexpressed in both cancer and non-cancer cells, BCL2 can form a complex with actin and gelsolin that functions to decrease gelsolin-severing activity to increase actin polymerization and, thus, suppress cell adhesive processes. The linkage between increased BCL2 and increased actin polymerization on the one hand and suppression of cell adhesion, spreading and motility on the other hand, is a novel observation that may provide a plausible explanation for why BCL2 overexpression in some tumors is correlated with improved patient survival. In addition, we have identified conditions in vitro in which F-actin polymerization can be increased while cell motility is reduced. These findings underscore the possibility that BCL2 may be involved in modulating cytoskeleton reorganization and may provide an opportunity to explore signal transduction pathways important for cell adhesion and migration and to develop small molecule therapies for suppression of cancer metastasis.Key words: BCL2, actin polymerization, cell motility, adhesion     

Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.     

Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

Cyclooxygenase and cAMP-dependent protein kinase reorganize the actin cytoskeleton for motility in HeLa cells

The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility.     

Lysyl oxidase regulates actin filament formation through the p130(Cas)/Crk/DOCK180 signaling complex

We have previously demonstrated that lysyl oxidase (LOX) is expressed in invasive breast cancer cells compared to poorly invasive cells. Additionally, we have recently shown that LOX regulates cell migration, a key step in the invasion process, through a hydrogen peroxide-dependent mechanism involving the focal adhesion kinase (FAK)/Src signaling complex. Here we further elucidate the role of LOX in cell motility/migration by examining the role of LOX in actin filament polymerization. We demonstrate that inhibition of LOX leads to an increase in phalloidin staining, directly associated with an increase in actin stress fiber formation. This increase in staining was confirmed by activity assays showing an increase in Rho activity with decreased LOX activity. Additionally, Rac and Cdc42 activity decreased with the reduction in LOX activity. Taken together, these data demonstrate a loss of a motogenic phenotype with decreased LOX activity. Finally, in order to elucidate the mechanism by which LOX regulates actin polymerization, we have demonstrated that LOX facilitates p130(Cas) phosphorylation, which allows for the binding to CAS related kinase (Crk) and formation of the p130(Cas)/Crk/DOCK180 signaling complex. Formation of this complex leads to an increase in Rac-GTP, which decreases actin stress fiber formation and increases formation of lamellipodium. These data demonstrate that LOX regulates cell motility/migration through changes in actin filament polymerization, which involve the regulation of the p130(Cas)/Crk/DOCK180 signaling pathway. Elucidating the role of LOX in the regulation of cell motility will allow the development of more effective therapeutic strategies to treat invasive/metastatic breast cancer.     

Antiepileptic teratogen valproic acid (VPA) modulates organisation and dynamics of the actin cytoskeleton

The antiepileptic drug valproic acid (VPA) and teratogenic VPA analogues have been demonstrated to inhibit cell motility and affect cell morphology. We here show that disruption of microtubules or of microfilaments by exposure to nocodazole or cytochalasin D had different effects on morphology of control cells and cells treated with VPA, indicating that VPA affected the cytoskeletal determinants of cell morphology. Furthermore, VPA treatment induced an increase of F-actin, and of FAK, paxillin, vinculin, and phosphotyrosine in focal adhesion complexes. These changes were accompanied by increased adhesion of VPA-treated cells to the extracellular matrix. Treatment with an RGD-containing peptide reducing integrin binding to components of the extracellular matrix partially reverted the motility inhibition induced by VPA, indicating that altered adhesion contributed to, but was not the sole reason for the VPA mediated inhibition of motility. In addition it is shown that the actomyosin cytoskeleton of VPA-treated cells was capable of contraction upon exposure to ATP, indicating that the reduced motility of VPA-treated cells was not caused by an inhibition of actomyosin contraction. On the other hand, VPA caused a redistribution of the actin severing protein gelsolin, and left the cells unable to respond to treatment with a gelsolin-peptide known to reduce the amount of gelsolin bound to phosphatidylinositol bisphosphate (PIP2), leaving a larger amount of the protein in a potential actin binding state. These findings indicate that VPA affects cell morphology and motility through interference with the dynamics of the actin cytoskeleton.     

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.     

From Abl to actin: Abl tyrosine kinase and associated proteins in growth cone motility

The Abl tyrosine kinase plays an important role in axonogenesis. Recent reports indicate that this role involves interaction with several different protein families, including LAR phosphatases, catenin/cadherin cell adhesion complexes, Trio family GEFs, and Ena/VASP family actin regulatory proteins. These findings suggest that Abl and its associated proteins may regulate cell adhesion and actin polymerization, thereby regulating growth cone motility during axonogenesis.     

SIRT2 reduces actin polymerization and cell migration through deacetylation and degradation of HSP90

SIRT2, a member of the class III histone deacetylase family, has been identified as a tumor suppressor, which is associated with various cellular processes including metabolism and proliferation. However, the effects of SIRT2 on cancer cell migration caused by cytoskeletal rearrangement remain uncertain. Here we show that SIRT2 inhibits cell motility by suppressing actin polymerization. SIRT2 regulates actin dynamics through HSP90 destabilization and subsequent repression of LIM kinase (LIMK) 1/cofilin pathway. SIRT2 directly interacts with HSP90 and regulates its acetylation and ubiquitination. In addition, the deacetylase activity of SIRT2 is required for the regulation of actin polymerization and the ubiquitin-mediated proteasomal degradation of HSP90 induced by SIRT2.     

Deformations in actin comets from rocketing beads

The mechanical and dynamical properties of the actin network are essential for many cellular processes like motility or division, and there is a growing body of evidence that they are also important for adhesion and trafficking. The leading edge of migrating cells is pushed out by the polymerization of actin networks, a process orchestrated by cross-linkers and other actin-binding proteins. In vitro physical characterizations show that these same proteins control the elastic properties of actin gels. Here we use a biomimetic system of Listeria monocytogenes, beads coated with an activator of actin polymerization, to assess the role of various actin-binding proteins in propulsion. We find that the properties of actin-based movement are clearly affected by the presence of cross-linkers. By monitoring the evolution of marked parts of the comet, we provide direct experimental evidence that the actin gel continuously undergoes deformations during the growth of the comet. Depending on the protein composition in the motility medium, deformations arise from either gel elasticity or monomer diffusion through the actin comet. Our findings demonstrate that actin-based movement is governed by the mechanical properties of the actin network, which are fine-tuned by proteins involved in actin dynamics and assembly.     

Mammalian CAP (Cyclase-associated protein) in the world of cell migration

Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.     

The coordination between actin filaments and adhesion in mesenchymal migration

Mesenchymal cell motility is characterized by a polarized distribution of actin filaments, with a network of short branched actin filaments at the leading edge, and polymers of actin filaments arranged into distinct classes of actin stress fibres behind the leading edge. Importantly, the distinct actin filaments are characteristically associated with discrete adhesion structures and both the adhesions and the actin filaments are co-ordinately regulated during cell migration. While it has long been known that these macromolecular structures are intimately linked in cells, precisely how they are co-ordinately regulated is presently unknown. Live imaging data now suggests that the focal adhesions may act as sites of actin polymerization resulting in the generation of tension-bearing actin bundles of actin filaments (stress fibres). Moreover, a picture is emerging to suggest that the tropomyosin family of proteins that can determine actin filament dynamics may also play a key role in determining the transition between adhesion states. Molecules such as the tropomyosins are therefore tantalizing candidates to orchestrate the coordination of actin and adhesion dynamics during mesenchymal cell migration.     

VASP governs actin dynamics by modulating filament anchoring

Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.

Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.     

The CEACAM1-L glycoprotein associates with the actin cytoskeleton and localizes to cell-cell contact through activation of Rho-like GTPases

Associations between plasma membrane-linked proteins and the actin cytoskeleton play a crucial role in defining cell shape and determination, ensuring cell motility and facilitating cell-cell or cell-substratum adhesion. Here, we present evidence that CEACAM1-L, a cell adhesion molecule of the carcinoembryonic antigen family, is associated with the actin cytoskeleton. We have delineated the regions involved in actin cytoskeleton association to the distal end of the CEACAM1-L long cytoplasmic domain. We have demonstrated that CEACAM1-S, an isoform of CEACAM1 with a truncated cytoplasmic domain, does not interact with the actin cytoskeleton. In addition, a major difference in subcellular localization of the two CEACAM1 isoforms was observed. Furthermore, we have established that the localization of CEACAM1-L at cell-cell boundaries is regulated by the Rho family of GTPases. The retention of the protein at the sites of intercellular contacts critically depends on homophilic CEACAM1-CEACAM1 interactions and association with the actin cytoskeleton. Our results provide new evidence on how the Rho family of GTPases can control cell adhesion: by directing an adhesion molecule to its proper cellular destination. In addition, these results provide an insight into the mechanisms of why CEACAM1-L, but not CEACAM1-S, functions as a tumor cell growth inhibitor.     

Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe

Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci. Here we investigate the mechanism of actin-patch motility in S. pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p. Actin patches are associated with cables and move with rates of 0.32 microm s(-1) primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips. Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants. In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner. Patches are sites of Arp3-dependent F-actin polymerization in vitro. Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization. Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

Interaction of annexin VI with actin

Actin polymerization was investigated using fluorescence probe N-(1-pyrenyl)iodoacetamide, which was bound covalently to reactive sulfhydryl group, Cys-373. Labeled actin in the bulk was 0.5 to 1% of total actin concentration. Actin polymerization at concentration 12 mM was started by addition of 20 mM KCl and 2 mM MgCl2. The label fluorescence was excited at 365 nm and registered at 386 nm. Under actin polymerization the label fluorescence increased almost 10 times. Two main phases may be distinguished in the process of actin polymerization: 1) monomer activation and nucleus (trimer) formation, 2) growth of actin filaments on the nuclei. In our experimental conditions, both for pure actin and for that with added annexin VI, the 1st phase continued for about 3 min and after that the 2nd phase was perfectly approximated by exponential dependence. An analysis of the exponential curves showed that actin monomer lifetime increased from 327 s, at annexin absence, to about 373 s at 0.7 microM annexin and more. Calculation of rate constants at two ends of growing actin filament suggests that annexin VI binds with pointed ("slow") end so that at sufficient annexin concentration the filament grows only on barbed ("fast") end. Our results, together with data of other researchers showing that annexin VI binds with the inner membrane surface of smooth muscle cell through Ca2+, may indicate that, at Ca2+ entering the cell, this annexin binds actin filament pointed ends to cell surface making it ready for the act of contraction.