Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell–cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.     

An ion-transporting ATPase encodes multiple apical localization signals

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.     

Cellular localization and stimulation-associated distribution dynamics of syntaxin-1 and syntaxin-3 in gastric parietal cells

Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin-1 and syntaxin-3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin-1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin-3 was found predominantly in the H,K-ATPase-rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP-syntaxin-1 and CFP-syntaxin-3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin-1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin-1 in a subadjacent cytoplasmic compartment. In resting, non-secreting parietal cells, syntaxin-3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin-3 translocated to the apical membrane vacuoles, there co-localizing with H,K-ATPase, syntaxin-1 and F-actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.     

A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase

The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H, K-ATPase alpha subunit and the highly homologous Na,K-ATPase alpha subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase alpha subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na, K-ATPase alpha subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.     

Polarized distribution of IQGAP proteins in gastric parietal cells and their roles in regulated epithelial cell secretion

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K-ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F-actin-containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP-mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921-930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin-containing cell surface structures.     

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin–Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.     

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.     

α-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium

Apical actomyosin activity in animal epithelial cells influences tissue morphology and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the nonmetazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from nonmetazoans to vertebrates and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in?a simple model organism.     

Use of the H,K-ATPase beta subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase beta subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the beta subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric beta subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase beta subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.     

Agonist-induced changes in cell shape during regulated secretion in rat pancreatic acini

The actin cytoskeleton plays an important role in the mediation of exocytosis and the determination of cell shape. Experimentally induced changes in cell shape have been shown to affect stimulated secretion in pancreatic acini. In this study, we have examined whether physiologic agonists induce changes in acinar cell shape to modulate secretion. Computer-enhanced video microscopy, immunofluorescence confocal microscopy, and quantitative Western blotting were used to study cell shape changes and cytoskeletal dynamics in rat pancreatic acini. Amylase assays were performed to study the effect of the actin-myosin cytoskeletal antagonists latrunculin A, BDM, and ML-9 on secretion. We found that pancreatic acini underwent a prominent and reversible shape change in response to the physiologic secretory agonist cholecystokinin. This was accompanied by an apical activation of myosin II as well as a basolateral redistribution of both actin and myosin II. Cytoskeletal antagonists inhibited this shape change and attenuated stimulated amylase secretion. Therefore, in addition to acting as a barrier at the apex, the actin-myosin cytoskeleton may also function to modulate cell shape to further regulate stimulated secretion.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.     

Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells

Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.     

Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.     

Diphosphorylation of regulatory light chain of myosin IIA is responsible for proper cell spreading

The actomyosin cytoskeleton plays prominent roles in cell spreading and migration. To address the roles of myosin II isoforms and to estimate the region where the myosin IIs are activated in spreading cells, we examined the immunolocalization of myosin II isoforms and phosphorylated RLCs in the spreading MRC-5 cells. We observed the formation of actin ring-like structure at the base of the lamella. Both myosin IIA and IIB were predominantly localized there. Myosin IIA and diphosphorylated RLC were distributed outside of the region where myosin IIB and monophosphoryated RLC were distributed predominantly. Inhibition of Rho-kinase resulted in the disappearance of the diphosphorylation of RLC, moreover, it accelerated the rate of cell spreading and induced an aberrant cell shape at later stage of spreading. These results indicate that diphosphorylation of RLCs of myosin IIA by Rho-kinase in lamella is responsible for the cell to spread properly.     

Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation

Gastric ezrin was initially identified as a phosphoprotein associated with parietal cell activation. To explore the nature of ezrin phosphorylation, proteins from resting and secreting gastric glands were subjected to two-dimensional SDS-PAGE. Histamine triggers acid secretion and a series of acidic isoforms of ezrin on two-dimensional SDS-PAGE. Mass spectrometric analysis of these acidic ezrin spots induced by stimulation suggests that Ser66 is phosphorylated. To determine whether Ser66 is a substrate of protein kinase A (PKA), recombinant proteins of ezrin, both wild type and S66A mutant, were incubated with the catalytic subunit of PKA and [32P]ATP. Incorporation of 32P into wild type but not the mutant ezrin verified that Ser66 is a substrate of PKA. In addition, expression of S66A mutant ezrin in cultured parietal cells attenuates the dilation of apical vacuolar membrane associated with stimulation by histamine, indicating that PKA-mediated phosphorylation of ezrin is necessary for acid secretion. In fact, expression of phosphorylation-like S66D mutant in parietal cells mimics histamine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution revealed a blockade of stimulation-induced proton pump mobilization in S66A but not S66D ezrin-expressing parietal cells. These data suggest that PKA-mediated phosphorylation of ezrin plays an important role in mediating the remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells.     

Dynamin2 Organizes Lamellipodial Actin Networks to Orchestrate Lamellar Actomyosin

Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2''s action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.     

A redistribution of actin and myosin IIA accompanies Ca(2+)-dependent insulin secretion

The study addressed the functional link between remodelling of the actomyosin cytoskeleton in pancreatic beta-cells and the regulation of insulin secretion. Confocal microscopy revealed that myosin heavy chain (MHC) IIA co-localized very well with filamentous (F)-actin in RINm5F cells but MHCIIB did not. Subcellular localization of MHCIIB was not altered by stimulation with 30 mM KCl (which evokes Ca(2+)-dependent insulin secretion). In contrast MHCIIA redistributed in a manner similar to F-actin, especially towards the apical surface, but also away from peripheral regions towards cell contact points on the basal surface. Finally, Ca(2+)-dependent insulin secretion was inhibited by stabilization of actin filaments with jasplakinolide. The results support a role for the MHCIIA/actin cytoskeleton in regulating insulin secretion.     

F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca(2+) signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca(2+) signaling.