Integrin signaling to the actin cytoskeleton

Integrin engagement stimulates the activity of numerous signaling molecules, including the Rho family of GTPases, tyrosine phosphatases, cAMP-dependent protein kinase and protein kinase C, and stimulates production of PtdIns(4,5)P2. Integrins promote actin assembly via the recruitment of molecules that directly activate the actin polymerization machinery or physically link it to sites of cell adhesion.     

Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion

1. Download : Download high-res image (174KB)
2. Download : Download full-size image

     

Mutual regulation between Hippo signaling and actin cytoskeleton

Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.     

Reorganization of the actin cytoskeleton upon G-protein coupled receptor signaling

The actin cytoskeleton is involved in a multitude of cellular responses besides providing structural support. While the role of the actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, reorganization of the actin cytoskeleton upon signaling by G-protein coupled receptors (GPCRs) represents a relatively unexplored area. The G-protein coupled receptor superfamily is an important protein family in mammals, involved in signal transduction across membranes. G-protein coupled receptors act as major signaling hubs and drug targets. The serotonin(1A) receptor is a representative member of the G-protein coupled receptor superfamily and plays a crucial role in the generation and modulation of various cognitive, developmental and behavioral functions. In order to monitor the changes in the actin cytoskeleton upon serotonin(1A) receptor signaling in a quantitative manner, we developed an approach based on high magnification imaging of F-actin in cells, followed by image reconstruction. Our results suggest that the actin cytoskeleton is reorganized in response to serotonin(1A) receptor signaling. In addition, we show that reorganization of the actin cytoskeleton is strongly dependent on adenosine 3',5'-cyclic monophosphate level, and is mediated by the activation of protein kinase A. Our results are consistent with the possibility of a feedback mechanism involving the actin cytoskeleton, adenosine 3',5'-cyclic monophosphate level and the serotonin(1A) receptor.     

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation. atherosclerosis; cytoskeletal dynamics; endothelial cells; mechanotransduction     

Dynamic remodeling of the actin cytoskeleton: Lessons learned from Listeria locomotion

The bacterial pathogen Listeria monocytogenes displays the remarkable ability to reorganize the actin cytoskeleton within host cells as a means for promoting cell-to-cell transfer of the pathogen, in a manner that evades humoral immunity. In a series of events commencing with the biosynthesis of the bacterial surface protein ActA, host cell actin and many actin-associated protein self-assemble to from rocket-tail structures that continually grow at sites proximal to the bacterium and depolymerize distally. Widespread interest in the underlying molecular mechanism of Listeria locomotion stems from the likelihood that the dynamic remodeling of the host cell actin cytoskeleton at the cell's leading edge involves mechanistically analogous interactions. Recent advances in our understanding of these fundamental cytoskeletal rearrangements have been achieved through a clearer recognition of the central role of oligo-proline sequence repeats present in ActA, and these findings provide a basis for inferring the role of analogous host cell proteins in the force-producing and position-securing steps in pseudopod and lamellipod formation at the peripheral membrane.     

Apactin is involved in remodeling of the actin cytoskeleton during regulated exocytosis

Apactin is an 80-kDa type I membrane glycoprotein derived from pro-Muclin, a precursor that also gives rise to the zymogen granule protein Muclin. Previous work showed that apactin is efficiently removed from the regulated secretory pathway and targeted to the actin-rich apical plasma membrane of the pancreatic acinar cell. The cytosolic tail (C-Tail) of apactin consists of 16 amino acids, has Thr casein kinase II and Ser protein kinase C phosphorylation sites, and a C-terminal PDZ-binding domain. Secretory stimulation of acinar cells causes a decrease in Thr phosphorylation and an increase in Ser phosphorylation of apactin. Fusion peptides of the C-Tail domain pulldown actin, ezrin, and EBP50/NHERF in a phosphorylation-dependent manner. HIV TAT-C-Tail fusion peptides were used as dominant negative constructs on living pancreatic cells to study effects on the actin cytoskeleton. During secretory stimulation, TAT-C-Tail-Thr/Asp phosphomimetic peptide caused an increase in actin-coated zymogen granules at the apical surface, while TAT-C-Tail-S/D phosphomimetic peptide caused a broadening of the actin cytoskeleton. These data indicate that stimulation-mediated Thr dephosphorylation allows decreased association of apactin with EBP50/NHERF and fosters actin remodeling to coat zymogen granules. Stimulation-mediated Ser phosphorylation increases apactin association with the actin cytoskeleton, maintaining tight bundling of actin microfilaments at the apical surface. Thus, apactin is involved in remodeling the apical cytoskeleton during regulated exocytosis in a manner controlled by phosphorylation of the apactin C-Tail.     

Filling gaps in signaling to actin cytoskeletal remodeling

A recent publication in the April 4 issue of Cell advances our understanding of stimulus response coupling leading to actin remodeling. It describes the identification of a novel membrane component Mig-2 that engages filamin A through a new intermediary, migfilin, to stimulate actin assembly and cell spreading on a substrate of extracellular matrix.     

Villin enhances hepatocyte growth factor-induced actin cytoskeleton remodeling in epithelial cells

Villin is an actin-binding protein localized to intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a calcium-dependent manner. Although villin is not necessary for the bundling of F-actin in vivo, it is important for the reorganization of the actin cytoskeleton elicited by stress during both physiological and pathological conditions (Ferrary et al., 1999). These data suggest that villin may be involved in actin cytoskeleton remodeling necessary for many processes requiring cellular plasticity. Here, we study the role of villin in hepatocyte growth factor (HGF)-induced epithelial cell motility and morphogenesis. For this purpose, we used primary cultures of enterocytes derived from wild-type and villin knock-out mice and Madin-Darby canine kidney cells, expressing villin in an inducible manner. In vitro, we show that epithelial cell lysates from villin-expressing cells induced dramatic, calcium-dependent severing of actin filaments. In cell culture, we found that villin-expressing cells exhibit enhanced cell motility and morphogenesis upon HGF stimulation. In addition, we show that the ability of villin to potentiate HGF-induced actin reorganization occurs through the HGF-activated phospholipase Cgamma signaling pathway. Collectively, these data demonstrate that villin acts as a regulator of HGF-induced actin dynamics.     

Cell signaling pathways to alphaB-crystallin following stresses of the cytoskeleton

Small heat shock proteins (sHSPs) act as chaperone, but also in protecting the different cytoskeletal components. Recent results suggest that αB-crystallin, a member of sHSPs family, might regulate actin filament dynamics, stabilize them in a phosphorylation dependent manner, and protect the integrity of intermediate filaments (IF) against extracellular stress. We demonstrate that vinblastin and cytochalasin D, which respectively disorganize microtubules and actin microfilaments, trigger the activation of the p38/MAPKAP2 kinase pathway and lead to the specific αB-crystallin phosphorylation at serine 59. Upstream of p38, we found that RhoK, PKC and PKA are selectively involved in the activation of p38 and phosphorylation of αB-crystallin, depending on the cytoskeletal network disorganized. Moreover, we demonstrate that chronic perturbations of IF network result in the same activation of p38 MAPK and αB-crystallin phosphorylation, as with severe disorganization of other cytoskeletal networks. Finally, we also show that Ser 59 phosphorylated αB-crystallin colocalizes with cytoskeletal components. Thus, disturbance of cytoskeleton leads by converging signaling pathways to the phosphorylation of αB-crystallin, which probably acts as a protective effector of the cytoskeleton.     

Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton

We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.     

Role of the p70 pathway in regulating the actin cytoskeleton and cell migration

We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration.     

On the role of protein kinases in regulating neutrophil actin association with the cytoskeleton

We have investigated the effect of staurosporine-type protein kinase inhibitors, displaying different enzyme specificity, on the association of actin with the neutrophil cytoskeleton. In resting cells, nanomolar concentrations of staurosporine induced a rapid increase in cytoskeleton-associated actin. Other inhibitors, more specific for protein kinase C (PKC) or kinases dependent on cyclic nucleotides, induced a much smaller response, indicating that inhibition of these enzymes is not involved in the staurosporine-dependent rise. Therefore, inhibition of an unknown staurosporine-sensitive enzyme, not identical with PKC or one of the cyclic nucleotide-dependent kinases, can trigger an increase in cytoskeletal actin. It is well known that chemotactic peptide induces a rapid rise in cytoskeletal actin, followed by a decrease at later times after the onset of activation. Preincubation with CGP 41,251, a relatively specific inhibitor for PKC, did not affect these two events at concentrations of the drug which, in separate experiments, inhibited markedly phorbol ester induced protein phosphorylation in intact neutrophils. Thus the chemotactic peptide-induced changes in the level of cytoskeletal actin appear to be independent of PKC activation.     

The role of the actin cytoskeleton in calcium signaling in starfish oocytes

Ca(2+) is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca(2+) to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca(2+) signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca(2+)-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca(2+) signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca(2+)-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.     

Microarray phenotyping places cyclase associated protein CAP at the crossroad of signaling pathways reorganizing the actin cytoskeleton in Dictyostelium

Large-scale gene expression analysis has been applied recently to uncover groups of genes that are co-regulated in particular processes. Here we undertake such an analysis on CAP, a protein that participates in the regulation of the actin cytoskeleton and in cAMP signaling in Dictyostelium. microarray analysis revealed that loss of CAP altered the expression of many cytoskeletal components. One of these, the Rho GDP-dissociation inhibitor RhoGDI1, was analyzed further. RhoGDI1 null cells expressed lower amounts of CAP, which failed to accumulate predominantly at the cell cortex. To further position CAP in the corresponding signal transduction pathways we studied CAP localization and cellular functioning in mutants that have defects in several signaling components. CAP showed correct localization and dynamics in all analyzed strains except in mutants with deficient cAMP dependent protein kinase A activity, where CAP preferentially accumulated in crown shaped structures. Ectopic expression of CAP improved the efficiency of phagocytosis in Gβ-deficient cells and restored the pinocytosis, morphology and actin distribution defects in a PI3 kinase double mutant (pi3k1/2 null). Our results show that CAP acts at multiple crossroads and links signaling pathways to the actin cytoskeleton either by physical interaction with cytoskeletal components or through regulation of their gene expression.     

hhLIM is a novel F-actin binding protein involved in actin cytoskeleton remodeling

Human heart LIM protein (hhLIM) is a newly cloned protein. In vitro analyses showed that green fluorescent protein (GFP)-tagged hhLIM protein accumulated in the cytoplasm of C2C12 cells and colocalized with F-actin, indicating that hhLIM is an actin-binding protein in C2C12 cells. Overexpression of hhLIM-GFP in C2C12 cells significantly stabilized actin filaments and delayed depolymerization of the actin cytoskeleton induced by cytochalasin B treatment. Expression of hhLIM-GFP in C2C12 cells also induced significant changes in the organization of the actin cytoskeleton, specifically, fewer and thicker actin bundles than in control cells, suggesting that hhLIM functions as an actin-bundling protein. This hypothesis was confirmed using low-speed co-sedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of hhLIM. hhLIM has two LIM domains. To identify the essential regions and sites for association, a series of truncated mutants was constructed which showed that LIM domain 2 has the same activity as full-length hhLIM. To further characterize the binding sites, the LIM domain was functionally destructed by replacing cysteine with serine in domain 2, and results showed that the second LIM domain plays a central role in bundling of F-actin. Taken together, these data identify hhLIM as an actin-binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network

Dynamic rearrangements of the actin cytoskeleton, following T-cell antigen receptor (TCR) engagement, provide the structural matrix and flexibility to enable intracellular signal transduction, cellular and subcellular remodeling, and driving effector functions. Recently developed cutting-edge imaging technologies have facilitated the study of TCR signaling and its role in actin-dependent processes. In this review, we describe how TCR signaling cascades induce the activation of actin regulatory proteins and the formation of actin networks, and how actin dynamics is important for T-cell homeostasis, activation, migration, and other effector functions.