Altered Actin Dynamics Research Articles

Expression of expanded polyglutamine targets profilin for degradation and alters actin dynamics

Huntington's disease is caused by polyglutamine expansion in the huntingtin protein. Huntingtin directly interacts with profilin, a major actin monomer sequestering protein and a key integrator of signals leading to actin polymerization. We observed a progressive loss of profilin in the cerebral cortex of Huntington's disease patients, and in cell culture and Drosophila models of polyglutamine disease. This loss of profilin is likely due to increased degradation through the ubiquitin proteasome system. Profilin loss reduces the F/G actin ratio, indicating a shift in actin polymerization. Overexpression of profilin abolishes mutant huntingtin toxicity in cells and partially ameliorates the morphological and functional eye phenotype and extends lifespan in a transgenic polyglutamine Drosophila model. These results indicate a link between huntingtin and profilin and implicate profilin in Huntington's disease pathogenesis.

Essential Role of Cofilin‐1 and mDia1 in Regulating Thrombin‐Induced Actin Dynamics and RelA/p65 Nuclear Translocation in Endothelial Cells

Activation of the small GTPase RhoA and associated changes in actin dynamics control thrombin‐induced NF‐κB activation and ICAM‐1 expression in endothelial cells. However, the events acting downstream of RhoA to mediate the changes in actin dynamics required for NF‐κB activation and ICAM‐1 expression remain unclear. Here we show that thrombin engages cofilin 1 and mDia1 downstream of RhoA to regulate these responses in endothelial cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser‐3 phosphorylation/inactivation of cofilin in a ROCK‐dependent manner. RNAi knockdown of cofilin‐1 stabilized the actin filaments and prevented thrombin‐ and RhoA‐induced NF‐κB activity. In parallel experiments, knockdown of mDia1 destabilized the actin filaments and inhibited thrombin‐ and RhoA‐induced NF‐κB activity. Inhibition of NF‐κB activity and ICAM‐1 expression following stabilization and destabilization of actin filaments by these approaches occurred downstream of IκBα degradation and was associated with impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. These findings support the notion thatspecific alterations in actin dynamics induced by thrombin are mediated by concerted actions ofcofilin‐1 and mDia1 and are necessary for RelA/p65 nuclear localization, and thereby ICAM‐1 expression in endothelial cells.This abstract is funded by: ALA, NIH HL067424.

Microtubules Are a Target for Self-Incompatibility Signaling in Papaver Pollen

Perception and integration of signals into responses is of crucial importance to cells. Both the actin and microtubule cytoskeleton are known to play a role in mediating diverse stimulus responses. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization. SI in Papaver rhoeas triggers a Ca(2+)-dependent signaling network to trigger programmed cell death (PCD), providing a neat way to inhibit and destroy incompatible pollen. We previously established that SI stimulates F-actin depolymerization and that altering actin dynamics can push pollen tubes into PCD. Very little is known about the role of microtubules in pollen tubes. Here, we investigated whether the pollen tube microtubule cytoskeleton is a target for the SI signals. We show that SI triggers very rapid apparent depolymerization of cortical microtubules, which, unlike actin, does not reorganize later. Actin depolymerization can trigger microtubule depolymerization but not vice versa. Moreover, although disruption of microtubule dynamics alone does not trigger PCD, alleviation of SI-induced PCD by taxol implicates a role for microtubule depolymerization in mediating PCD. Together, our data provide good evidence that SI signals target the microtubule cytoskeleton and suggest that signal integration between microfilaments and microtubules is required for triggering of PCD.

A Critical Role for HSP90 in Cancer Cell Invasion Involves Interaction with the Extracellular Domain of HER-2

HSP90 is a ubiquitously expressed molecular chaperone that controls the folding, assembly, intracellular disposition, and proteolytic turnover of many proteins, most of which are involved in signal transduction processes. Recently, a surface form of HSP90 has been identified and associated with cell migration events. In this paper, we explore the interaction of surface HSP90 with HER-2, a receptor-like glycoprotein and member of the ErbB family of receptor tyrosine kinases that play central roles in cellular proliferation, differentiation, and migration as well as in cancer progress. The involvement of HSP90 in the regulation of HER-2 has been attributed so far to receptor stabilization via interaction with its cytoplasmic kinase domain. Here we present evidence, using glutathione S-transferase pull-down and transfection assays, for a novel interaction between surface HSP90 and the extracellular domain of HER-2. Specific disruption of this interaction using mAb 4C5, a function-blocking monoclonal antibody against HSP90, inhibits cell invasion accompanied by altered actin dynamics in human breast cancer cells under ligand stimulation conditions with heregulin. Additionally, disruption of surface HSP90/HER-2 interaction leads to inhibition of heregulin-induced HER-2-HER-3 heterodimer formation, reduced HER-2 phosphorylation, and impaired downstream kinase signaling. Interestingly, this disruption does not affect HER-2 internalization. Our data suggest that surface HSP90 is involved in heregulin-induced HER-2 activation and signaling, leading to cytoskeletal rearrangement, essential for cell invasion.

Loading alters actin dynamics and up-regulates cofilin gene expression in chondrocytes

Chondrocyte mechanotransduction in response to mechanical loading is essential for the health and homeostasis of articular cartilage. The actin cytoskeleton has been implicated in cell mechanics and mechanotransduction. This study tests the hypothesis that loading modulates actin dynamics and organisation with subsequent changes in gene expression for actin associated proteins. Chondrocytes were transfected with eGFP-actin, seeded in agarose and subjected to cyclic compression (10 cycles, 1Hz, 0–15% strain) on the stage of a confocal microscope. Compression resulted in a subsequent reduction in cortical eGFP-actin intensity and a reduction in fluorescence recovery after photobleaching (FRAP), suggesting net cortical actin de-polymerisation, compared to unloaded controls. Cyclic compression for 10min up-regulated gene expression for the actin depolymerising proteins, cofilin and destrin. Thus mechanical loading alters cortical actin dynamics, providing a potential mechanism through which chondrocytes can adapt their mechanical properties and mechanosensitivity to the local mechanical environment.

Actin Cytoskeleton Dynamics Promotes Leptin-Induced Vascular Smooth Muscle Hypertrophy via RhoA/ROCK- and Phosphatidylinositol 3-Kinase/Protein Kinase B-Dependent Pathways

Obesity is associated with increased leptin production that may contribute to cardiovascular pathology through a multiplicity of effects. Leptin has been shown to contribute to vascular remodeling through various mechanisms, including production of vascular smooth muscle (VSMC) hypertrophy; however, the mechanisms underlying the vascular hypertrophic effect of leptin remain unknown. In the present study, we investigated the contributions of the RhoA/Rho kinase (ROCK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways, actin dynamics, and the expression of serum-response factor (SRF) in the hypertrophic effects of leptin on vascular tissue. Strips of rat portal vein (RPV) were cultured with or without leptin at 3.1 nM for 1 to 3 days. Leptin significantly increased RhoA activity by 163 +/- 20%, whereas phosphorylation of downstream factors, including LIM kinase 1 and cofilin-2, was increased by 160 +/- 25 and 290 +/- 25%, respectively. Leptin also significantly phosphorylated Akt by 130 +/- 30%, which was inhibited by the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). RhoA/ROCK and PI3K/Akt activation was associated with a significant increase in RPV wet weight (11 +/- 1%), protein synthesis (45 +/- 7%), SRF expression (136 +/- 11%), and polymerization of actin, as reflected by an increase in the F-/G-actin ratio, effects that were significantly attenuated by a leptin receptor (leptin obese receptor) antibody, the ROCK inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y-27632) as well as the PI3K inhibitor LY294002. Our results indicate that the activation of RhoA/ROCK and PI3K/Akt plays a pivotal role in leptin signaling, leading to the development of VSMC hypertrophy through a mechanism involving altered actin dynamics.

MAPK‐dependent actin cytoskeletal reorganization underlies BK channel activation by insulin

Numerous brain regions are enriched with insulin and insulin receptors, and several lines of evidence indicate that insulin is an important modulator of neuronal function. Indeed, recent studies have demonstrated that insulin inhibits hippocampal epileptiform-like activity, in part by activating large-conductance Ca2+-activated potassium (BK) channels. Moreover, the mitogen-activated protein kinase (MAPK) signalling cascade has been found to couple insulin to BK channel activation. However, the cellular events downstream of MAPK that underlie this action of insulin are unknown. Here we demonstrate that in hippocampal neurons, BK channel activation by insulin is blocked by actin filament stabilization, suggesting that this process is dependent on the actin cytoskeleton. Stabilizing actin filaments also markedly attenuated the ability of insulin to inhibit the aberrant hippocampal synaptic activity evoked following Mg2+ removal. Insulin also promoted rapid reorganization of fluorescently labelled polymerized actin filaments; an action that was prevented by inhibitors of MAPK activation. Moreover, in parallel studies, insulin increased the level of phospho-MAPK immunostaining in hippocampal neurons. These data are consistent with BK channel activation by insulin involving MAPK-dependent alterations in actin dynamics. This process may have important implications for the role of insulin in regulating hippocampal excitability.

The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE(2) release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE(2) release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE(2) release. In fact, PGE(2) release was increased threefold in the polymerization inhibited cells and this PGE(2) release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE(2) release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE(2) release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton.

Redox Regulation of Ephrin/Integrin Cross-Talk

Interactions linking the Eph receptor tyrosine kinase and ephrin ligands transduce short-range repulsive signals regulating several motile biological processes including axon pathfinding, angiogenesis and tumor growth. These ephrin-induced effects are believed to be mediated by alterations in actin dynamics and cytoskeleton reorganization. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. In particular, some ephrin ligands lead to a decrease in integrin-mediated cell adhesion and spread. Here we show that the ability of ephrinA1 to inhibit cell adhesion and spreading in prostatic carcinoma cells is strictly dependent on the decrease in the activity of the small GTPase Rac1. Given the recognized role of Rac-driven redox signaling for integrin function, reported to play an essential role in focal adhesion formation and in the overall organization of actin cytoskeleton, we investigated the possible involvement of oxidants in ephrinA1/EphA2 signaling. We now provide evidence that Reactive Oxygen Species are an integration point of the ephrinA1/integrin interplay. We identify redox circuitry in which the ephrinA1-mediated inhibition of Rac1 leads to a negative regulation of integrin redox signaling affecting the activity of the tyrosine phosphatase LMW-PTP. The enzyme in turn actively dephosphorylates its substrate p190RhoGAP, finally leading to RhoA activation. Altogether our data suggest a redox-based Rac-dependent upregulation of Rho activity, concurring with the inhibitory effect elicited by ephrinA1 on integrin-mediated adhesion strength.

Gelsolin overexpression alters actin dynamics and tyrosine phosphorylation of lipid raft-associated proteins in Jurkat T cells

Upon T cell receptor engagement, both the actin cytoskeleton and substrates of tyrosine phosphorylation are remodeled to create a signaling complex at the interface of the antigen-presenting cell and responding T cell. While T cell signaling has been shown to regulate actin reorganization, the mechanisms by which changes in actin dynamics affect early T cell signaling have not been fully explored. Using gelsolin, an actin-binding protein with capping and severing activities, and latrunculin, an actin-depolymerizing agent, we have further investigated the interplay between actin dynamics and the regulation of T cell signaling. Overexpression of gelsolin altered actin dynamics in Jurkat T cells, and alteration of actin dynamics correlated with dysregulation of tyrosine phosphorylation of raft-associated substrates. This perturbation of tyrosine phosphorylation was correlated with inhibition of activation-dependent signaling pathways regulating Erk-1/2 phosphorylation, NF-AT transcriptional activation and IL-2 production. Modification of actin by the depolymerizing agent latrunculin also altered the tyrosine phosphorylation patterns of proteins associated with lipid rafts, and pre-treatment with latrunculin inhibited anti-CD3 mAb-mediated NF-AT activation. Thus, our data indicate that actin cytoskeletal dynamics modulate the tyrosine phosphorylation of raft-associated proteins and subsequent downstream signal transduction.

Actin Dynamics and Ethanol Sensitivity

The risk of alcoholism has been associated with resistance to the acute intoxicating effects of alcohol, leading researchers to explore model systems that might give insight into the molecular factors that modulate ethanol sensitivity. Two studies suggest that ability to regulate the actin cytoskeleton could influence sensitivity to alcohol. Rothenfluh et al . found that mutations of the Drosophila RhoGAP18B locus, which was predicted to encode three RhoGAP18 proteins that shared only the guanosine triphosphatase (GTPase) activating protein (GAP) domain, led to resistance to ethanol-induced sedation (assessed by the loss-of-righting test and locomotor tracking) that was specifically associated with loss of the RhoGAP18B-RC transcript. Experiments with dominant-negative or constitutively active Rho GTPases and loss-of-function alleles suggested that the effects of RhoGAP18B-RC on ethanol-induced sedation were mediated though Rho1 or the Rac GTPases (or both) but not through Cdc42. In contrast to RhoGAP18B-RC , the RhoGAP18B-RA transcript appeared to be involved in mediating ethanol’s stimulant effects. As Sordella and Van Aelst note, the Rho GTPases have been implicated in regulation of actin dynamics; the second study, by Offenhäuser et al ., investigated ethanol resistance in mice of Eps8, which is also involved in regulating actin dynamics. Mice lacking Eps8 ( Eps8 -KO mice) showed increased ethanol consumption and were less sensitive to the sedative and motor-incoordinating effects of ethanol but not to its stimulation of locomotor activity. Eps8 was observed in dendritic articulations of cerebellar granule neurons (CGNs); it was present in cerebellar postsynaptic density fractions, and it coimmunoprecipitated with the N -methyl-𝒟-aspartate (NMDA)-type glutamate receptor (NMDAR). Electrophysiological recordings from CGNs in cerebellar slices indicated that the NMDA component of the composite excitatory postsynaptic current was increased in Eps8 -KO compared with that of wild-type mice and was less sensitive to inhibition by 400 mM ethanol. Introduction of Eps8 into Eps8 -KO neurons reduced NMDAR-mediated currents, as did latrunculin (which disrupts actin filaments). Eps8 was present in F-actin-rich clusters in the neurites of cultured CGNs; glutamate or NMDA treatment led to a decrease in cofilin phosphorylation and a loss of F-actin from these structures, effects that were attenuated in Eps8 -KO neurons. Ethanol treatment also elicited a loss of F-actin from these structures and in cerebellar slices, effects to which Eps8 -KO neurons were resistant. Thus, both Drosophila and mouse studies suggest that disruption of the mechanisms involved in regulating actin dynamics affects sensitivity to ethanol. A. Rothenfluh, R. J. Threlkeld, R. J. Bainton, L. T.-Y. Tsai, A. W. Lasek, U. Heberlein, Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms. Cell 127 , 199-211 (2006). [PubMed] N. Offenhäuser, D. Castelletti, L. Mapelli, B. E. Soppo, M. C. Regondi, P. Rossi, E. D'Angelo, C. Frassoni, A. Amadeo, A. Tocchetti, B. Pozzi, A. Disanza, D. Guarnieri, C. Betsholtz, G. Scita, U. Heberlein, P. P. Di Fiore, Increased ethanol resistance and consumption in Eps8 knockout mice correlates with altered actin dynamics. Cell 127 , 213-226 (2006). [PubMed] R. Sordella, L. Van Aelst, Driving actin dynamics under the influence of alcohol. Cell 127 , 37-39 (2006). [PubMed]

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

A role for actin in aging and apoptosis.

The actin cytoskeleton is central to many cell processes including membrane trafficking and generation of cell polarity. We have identified a role for actin in cell death and in promoting longevity of the budding yeast, Saccharomyces cerevisiae. Aging in yeast appears to occur via an apoptotic-like pathway with changes including DNA fragmentation, loss of mitochondrial membrane permeability, increase in levels of ROS (reactive oxygen species) and exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This pathway can be induced by alterations in actin dynamics, such that reduced dynamics correlates with increased levels of ROS and decreased viability. Conversely, increased actin dynamics correlates with low ROS levels and increased survival. Our current studies have focused on identifying pathways which couple changes in actin dynamics to cell death.

A role for actin in aging and apoptosis

The actin cytoskeleton is central to many cell processes including membrane trafficking and generation of cell polarity. We have identified a role for actin in cell death and in promoting longevity of the budding yeast, Saccharomyces cerevisiae. Aging in yeast appears to occur via an apoptotic-like pathway with changes including DNA fragmentation, loss of mitochondrial membrane permeability, increase in levels of ROS (reactive oxygen species) and exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This pathway can be induced by alterations in actin dynamics, such that reduced dynamics correlates with increased levels of ROS and decreased viability. Conversely, increased actin dynamics correlates with low ROS levels and increased survival. Our current studies have focused on identifying pathways which couple changes in actin dynamics to cell death.

DictyosteliumLIS1 Is a Centrosomal Protein Required for Microtubule/Cell Cortex Interactions, Nucleus/Centrosome Linkage, and Actin Dynamics

The widespread LIS1-proteins were originally identified as the target for sporadic mutations causing lissencephaly in humans. Dictyostelium LIS1 (DdLIS1) is a microtubule-associated protein exhibiting 53% identity to human LIS1. It colocalizes with dynein at isolated, microtubule-free centrosomes, suggesting that both are integral centrosomal components. Replacement of the DdLIS1 gene by the hypomorphic D327H allele or overexpression of an MBP-DdLIS1 fusion disrupted various dynein-associated functions. Microtubules lost contact with the cell cortex and were dragged behind an unusually motile centrosome. Previously, this phenotype was observed in cells overexpressing fragments of dynein or the XMAP215-homologue DdCP224. DdLIS1 was coprecipitated with DdCP224, suggesting that both act together in dynein-mediated cortical attachment of microtubules. Furthermore, DdLIS1-D327H mutants showed Golgi dispersal and reduced centrosome/nucleus association. Defects in DdLIS1 function also altered actin dynamics characterized by traveling waves of actin polymerization correlated with a reduced F-actin content. DdLIS1 could be involved in actin dynamics through Rho-GTPases, because DdLIS1 interacted directly with Rac1A in vitro. Our results show that DdLIS1 is required for maintenance of the microtubule cytoskeleton, Golgi apparatus and nucleus/centrosome association, and they suggest that LIS1-dependent alterations of actin dynamics could also contribute to defects in neuronal migration in lissencephaly patients.

New Partners for Ena/VASP

The Ena/VASP family proteins are important regulators of the cytoskeleton that are recruited to sites of actin remodeling during cell adhesion, polarization, and migration. Within locations such as focal adhesions and lamellipodia, Ena/VASP proteins alter local actin dynamics through their interactions with actin and profilin, as well as through their associations with proteins that bind to their proline-rich protein interaction domain, EVH1. Less well understood is how Ena/VASP proteins locally alter actin dynamics in response to specific cell signals. Two related adaptor proteins have now been identified as Ena/VASP binding partners that could provide the link between localized membrane-based signaling events and cytoskeletal remodeling. Krause et al. have determined that mammalian Lamellipodin (Lpd), whose C. elegans ortholog, Mig-10, controls neuronal migration, binds to Ena/VASP proteins. Lpd and Ena/VASP proteins colocalized at the tips of lamellipodia in a variety of mammalian cell types and were isolated in a protein complex from primary cortical neurons. However, Lpd localization did not depend on the presence of Ena/VASP. Rather, Lpd directed Ena/VASP protein localization, a recruitment dependent on the EVH1-binding motifs within Lpd. A Ras-association (RA) and lipid-binding PH domain in Lpd were required for targeting to the leading edge of lamellipodia. The PH domain specifically associated with PI(3,4)P2, a phosphoinositide involved in cell polarization. Overexpression of Lpd also increased lamellipodia protrusion velocity in fibroblasts, an effect that required Ena/VASP expression. Knockdown of Lpd by RNA interference reduced actin polymerization in lamellipodia and impaired lamellipodia formation. Similar in Lpd, Rap1-interacting adaptor molecule (RIAM) was identified by Lafuente et al. as another Ena/VASP interacting protein. Both RIAM and Lpd exhibit similar protein domain structure, comprising a new family of adaptor proteins called MRL (Mig-10/RIAM/Lpd). In activated T cells, RIAM associated with activated Rap1, a Ras superfamily member that localizes to the plasma membrane to control cell adhesion and spreading. RIAM was also required for activated Rap1 to localize to the plasma membrane and stimulate integrin-mediated adhesion in T cells. RIAM was distributed throughout the leading edge of lamellipodia, and its overexpression induced cell adhesion. Knockdown of RIAM expression by RNA interference reduced the cellular content of polymerized F actin and adhesion. Although RIAM and Lpd are both found in the leading edge of lamellipodia, Lpd did not bind to activated Rap1 and did not localize to focal adhesions, which suggests that the two adaptors may couple Ena/VASP to distinct signaling pathways within similar subcellular locations. The two adaptor proteins may integrate signals from both phosphoinositides and Ras superfamily proteins and direct actin through association with Ena/VASP. M. Krause, J. D. Leslie, M. Stewart, E. M. Lafuente, F. Valderrama, R. Jagannathan, G. A. Strasser, D. A. Rubinson, H. Liu, M. Way, M. B. Yaffe, V. A. Boussiotis, F. B. Gertler, Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Devel. Cell 7 , 571-583 (2004). [Online Journal] E. M. Lafuente, A. A. F. L. van Puijenbroek, M. Krause, C. V. Carman, G. J. Freeman, A. Berezovskaya, E. Constantine, T. A. Springer, F. B. Gertler, V. A. Boussiotis, RIAM, an Ena/VASP and profilin ligand, interacts with RAP1-GTP and mediates Rap1-induced adhesion. Devel. Cell 7 , 585-595 (2004). [Online Journal]

Migration on rafts

The plasma membrane polarizes in moving cells by reorganizing lipid rafts, according to Gomez-Mouton et al. on page 759. This polarization puts migration tools at the correct end of the cell. Figure PI3K (red) is recruited (left to right) to membrane rafts (green) at the leading edge. The polarization of signaling molecules such as PI3K allows migrating cells to respond directionally to chemotactic signals by forming lamellipodia at the front and uropods at the rear. Gomez-Mouton et al. show that this localized signaling is a result of redistribution of plasma membrane lipid compartments that puts chemokine receptors at the front of cells. Using live migrating white blood cells, the group watched in real time as some rafts moved to the cell's leading edge, while other rafts moved to the rear. Rafts that moved to the front contained the CCR5 chemotactic receptor. Wherever CCR5 was found, the authors also saw activated PI3K—a key determinant in the orientation of migration. Disruption of raft polarization, by removing cholesterol, prevented this localized PI3K recruitment and thus stopped directed migration. So far it is unclear how different rafts choose between the front and rear of the cell. Perhaps activation of a few receptors on one side of the cell initiates the clustering of several receptor- containing rafts. As the actin cytoskeleton is needed for raft polarization, the authors speculate that the PI3K brought to these rafts may activate proteins that alter actin dynamics to recruit more of the appropriate rafts. One of these proteins could be the GTPase Rac1, whose activation at the leading edge is required for cell migration. Rac1 was recently shown to bind to and be activated specifically at lipid rafts (del Pozo, M.A., et al. 2004. Science. 303:839–842). Other proteins in different rafts might initiate raft movement to the rear. Further insight awaits the characterization of the lipid and protein components of the different rafts. ▪

Regulation of growth cone actin dynamics by ADF/cofilin.

Nervous system development is reliant on neuronal pathfinding, the process in which axons are guided to their target cells by specific extracellular cues. The ability of neurons to extend over long distances in response to environmental guidance signals is made possible by the growth cone, a highly motile structure found at the end of neuronal processes. Growth cones detect directional cues and respond with either attractive or repulsive movements. The motility of growth cones is dependent on rapid reorganization of the actin cytoskeleton, presumably mediated by actin-associated proteins under the control of incoming guidance signals. This article reviews how one such family of proteins, the ADF/cofilins, are emerging as key regulators of growth cone actin dynamics. These proteins are essential for rapid actin turnover in a variety of different cell types. ADF/cofilins are heavily co-localized with actin in growth cones and are necessary for neurite outgrowth. ADF/cofilin activities are regulated through reversible phosphorylation by LIM kinases and slingshot phosphatases. LIM kinases are downstream effectors of the Rho GTPases Rho, Rac, and Cdc42. Growing evidence suggests that extracellular guidance cues may locally alter actin dynamics by regulating the activity of LIM kinase and ADF/cofilin phosphatases via the Rho GTPases. In this way, ADF/cofilins and their upstream effectors may be pivotal to our understanding of how guidance information is translated into physical alterations of the growth cone actin cytoskeleton.

EphrinA1 inactivates integrin-mediated vascular smooth muscle cell spreading via the Rac/PAK pathway.

Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA1 is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA1 in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA1-induced integrin inactivation. EphrinA1 did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA1 effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA1-mediated inhibition of spreading in VSMCs. The ephrinA1-mediated morphological alterations correlated with inhibition of Rac1 and p21-activated kinase 1 (PAK1) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover, repression of Rac1 synthesis with siRNA amplifies the ephrinA1-induced inhibition of spreading. Finally, sphingosine-1-phosphate (S1P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA1 effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA1-mediated inhibition of spreading. In this way, ephrinA1, alone or in synergy with S1P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.

Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor.

Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics. SRF activity can be inhibited by ectopic expression of beta-actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations. To distinguish between these possibilities, we studied actin mutants. Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies. These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio. Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor. SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B. Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals. These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding. Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF.