The role of membranes and membrane trafficking in RNA localization

Eukaryotic cells possess highly sophisticated membrane trafficking pathways that define specific membrane domains and provide a means for moving vesicles between them (Mostov, Su, and ter Beest, 2003, Nat. Cell Biol. 5, 287-293). Here, I review recent data that indicate a role for membrane trafficking in mRNA localization. Specifically, I review evidence that some localized mRNAs are anchored to specific membrane domains and/or transported on membranous organelles or vesicles to specific subcellular sites. This review is not intended as a discussion on indirect influences of membrane trafficking on mRNA localization. I will not, for example, discuss the role of membrane trafficking in the regulation of extracellular signalling events that could indirectly influence mRNA localization through polarization of the actin or microtubule cytoskeleton (for examples, see reviews by Drubin and Nelson, 1996, Cell 84, 335-344; Shulman and St Johnston, 1999, Trends Cell Biol. 9, M60-M64).     

Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.     

Protective effects of lysophosphatidic acid (LPA) on chronic ethanol-induced injuries to the cytoskeleton and on glucose uptake in rat astrocytes

Ethanol induces severe alterations in membrane trafficking in hepatocytes and astrocytes, the molecular basis of which is unclear. One of the main candidates is the cytoskeleton and the molecular components that regulate its organization and dynamics. Here, we examine the effect of chronic exposure to ethanol on the organization and dynamics of actin and microtubule cytoskeletons and glucose uptake in rat astrocytes. Ethanol-treated cells cultured in either the presence or absence of fetal calf serum showed a significant increase in 2-deoxyglucose uptake. Ethanol also caused alterations in actin organization, consisting of the dissolution of stress fibres and the appearance of circular filaments beneath the plasma membrane. When lysophosphatidic acid (LPA), which is a normal constituent of serum and a potent intercellular lipid mediator with growth factor and actin rearrangement activities, was added to ethanol-treated astrocytes cultured without fetal calf serum, it induced the re-appearance of actin stress fibres and the normalization of 2-deoxyglucose uptake. Furthermore, ethanol also perturbed the microtubule dynamics, which delayed the recovery of the normal microtubule organization following removal of the microtubule-disrupting agent nocodazole. Again, pre-treatment with LPA prevented this alteration. Ethanol-treated rodent fibroblast NIH3T3 cells that constitutively express an activated Rho mutant protein (GTP-bound form) were insensitive to ethanol, as they showed no alteration either in actin stress-fibre organization or in 2-deoxyglucose uptake. We discuss the putative signalling targets by which ethanol could alter the cytoskeleton and hexose uptake and the cytoprotective effect of LPA against ethanol-induced damages. The latter opens the possibility that LPA or a similar non-hydrolysable lipid derivative could be used as a cytoprotective agent against the noxious effects of ethanol.     

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.     

Reorganization and translocation of the ectoplasmic cytoskeleton in the leech zygote by condensation of cytasters and interactions of dynamic microtubules and actin filaments

The formation and bipolar translocation of an ectoplasmic cytoskeleton of rings and meridional bands was studied in interphase zygotes of the glossiphoniid leech Theromyzon trizonare. Zygotes consisted of a peripheral organelle-rich ectoplasm and an internal yolk-rich endoplasm. After microinjection of labeled tubulin and/or actin, zygotes were examined by time-lapse video imaging, immunofluorescence and confocal microscopy. The rings and meridional bands were formed by condensation of a network of moving cytasters that represented ectoplasmic secondary centers of microtubule and actin filament nucleation. In some cases the network of cytasters persisted between the rings. The cytoskeleton had an outer actin layer and an inner microtubule layer that merged at the irregularly-shaped boundary zone. Bipolar translocation of the rings, meridional bands, or the network of cytasters led to accumulation of the cytoskeleton at both zygote poles. Translocation of the cytoskeleton was slowed or arrested by microinjected taxol or phalloidin, in a dose-dependent fashion. Results of drug treatment probably indicate differences in the degree and speed at which the cytoskeleton becomes stabilized. Moreover, drugs that selectively stabilized either microtubules or actin filaments stabilized and impaired movement of the entire cytoskeleton. Microtubule poisons and latrunculin-B failed to disrupt the cytoskeleton. It is concluded that the microtubule and actin cytoskeletons are dynamic, presumably cross-linked and resistant to depolymerizing drugs. They probably move along each other by a sliding mechanism that depends on the instability of microtubules and actin filaments.     

APC nuclear membrane association and microtubule polarity.

BACKGROUND INFORMATION: Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. RESULTS: We previously showed, using primary fibroblasts and astrocytes in in vitro scratch-induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus-ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. CONCLUSIONS: We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Organization and function of an actin cytoskeleton in Plasmodium falciparum gametocytes

In preparation for transmission to its mosquito vector, Plasmodium falciparum, the most virulent of the human malaria parasites, adopts an unusual elongated shape. Here we describe a previously unrecognized actin‐based cytoskeleton that is assembled in maturing P. falciparum gametocytes. Differential extraction reveals the presence of a highly stabilized population of F‐actin at all stages of development. Super‐resolution microscopy reveals an F‐actin cytoskeleton that is concentrated at the ends of the elongating gametocyte but extends inward along the microtubule cytoskeleton. Formin‐1 is also concentrated at the gametocyte ends suggesting a role in actin stabilization. Immunoelectron microscopy confirms that the actin cytoskeleton is located under the inner membrane complex rather than in the sub‐alveolar space. In stage V gametocytes, the actin and microtubule cytoskeletons are reorganized in a coordinated fashion. The actin‐depolymerizing agent, cytochalasin D, depletes actin from the end of the gametocytes, whereas the actin‐stabilizing compound, jasplakinolide, induces formation of large bundles and prevents late‐stage disassembly of the actin cytoskeleton. Long‐term treatment with these compounds is associated with disruption of the normal mitochondrial organization and decreased gametocyte viability.     

Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model

The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

DNase-I-dependent dissociation of erythrocyte cytoskeletons

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.     

Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I(CaL), I(Na), I(KATP), I(SAC) by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca(2+) release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the beta-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined.     

Regulation of substrate adhesion dynamics during cell motility

The movement of a metazoan cell entails the regulated creation and turnover of adhesions with the surface on which it moves. Adhesion sites form as a result of signaling between the extracellular matrix on the outside and the actin cytoskeleton on the inside, and they are associated with specific assembles of actin filaments. Two broad categories of adhesion sites can be distinguished: (1) "focal complexes" associated with lamellipodia and filopodia that support protrusion and traction at the cell front; and (2) "focal adhesions" at the termini of stress fibre bundles that serve in longer term anchorage. Focal complexes are signaled via Rac1 or Cdc42 and can either turnover on a minute scale or differentiate, via intervention of the RhoA pathway, into longer-lived focal adhesions. All classes of adhesion sites depend on the stress in the actin cytoskeleton for their formation and maintenance. Different cell types use different adhesion strategies to move, in terms of the relative engagement of filopodia and lamellipodia in focal complex formation and protrusion and the extent of focal adhesion formation. These differences can be attributed to variations in the relative activities of Rho family members. However, the Rho GTPases alone are unable to signal asymmetry in the actin cytoskeleton, necessary for polarisation and movement. Polarisation requires the collaboration of the microtubule cytoskeleton. Changes in the polymerisation state of microtubules influences the activities of both Rac1 and RhoA and microtubules interact directly with adhesion foci and promote their turnover. Possible mechanisms of cross-talk between the microtubule and actin cytoskeletons in determining polarity are discussed.     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi-to-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI-coated and uncoated vesicles contain β/γ-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.     

The ion channels to cytoskeleton connection as potential mechanism of mechanosensitivity

As biological force-sensing systems mechanosensitive (MS) ion channels present the best example of coupling molecular dynamics of membrane proteins to the mechanics of the surrounding cell membrane. In animal cells MS channels have over the past two decades been very much in focus of mechanotransduction research. In recent years this helped to raise awareness of basic and medical researchers about the role that abnormal MS channels may play in the pathophysiology of diseases, such as cardiac hypertrophy, atrial fibrillation, muscular dystrophy or polycystic kidney disease. To date a large number of MS channels from organisms of diverse phylogenetic origins have been identified at the molecular level; however, the structure of only few of them has been determined. Although their function has extensively been studied in a great variety of cells and tissues by different experimental approaches it is, with exception of bacterial MS channels, very little known about how these channels sense mechanical force and which cellular components may contribute to their function. By focusing on MS channels found in animal cells this article discusses the ways in which the connections between cytoskeleton and ion channels may contribute to mechanosensory transduction in these cells. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

APC nuclear membrane association and microtubule polarity

Background information. Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. Results. We previously showed, using primary fibroblasts and astrocytes in in vitro scratch‐induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus‐ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. Conclusions. We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. We have identified a novel role for the actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. We show that Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulator gamma-tubulin. Moreover, this change in gamma-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that gamma-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons.     

Effect of cytoskeleton disruptors on L-type Ca channel distribution in rat ventricular myocytes

The cytoskeleton plays an important role in many aspects of cardiac cell function, including protein trafficking. However, the role of the cytoskeleton in determining Ca channel location in cardiac myocytes is unknown. In the present study we therefore investigated the effect of the cytoskeletal disruptors cytochalasin D, latrunculin, nocadazole and colchicine on the distribution of Ca channels in rat ventricular myocytes during culture for up to 96 h. During culture in the absence of these agents, cell edges became rounded, t-tubule density decreased, and the normal transverse distribution of the alpha1 (pore-forming) subunit of the L-type Ca channel became more punctate and peri-nuclear; these changes were associated with loss of synchronous Ca release in response to electrical stimulation. Disruption of tubulin using nocadazole or colchicine or sequestration of monomeric actin by latrunculin had no effect on these changes. In contrast, cytochalasin D inhibited these changes: cell shape, t-tubule density, transverse Ca channel staining and synchronous Ca release were maintained during culture. The protein synthesis inhibitor cycloheximide had similar effects to cytochalasin. These data suggest that cytochalasin stabilizes actin in adult ventricular myocytes in culture, thus stabilizing cell structure and function, and that actin is important in trafficking L-type Ca channels from the peri-nuclear region to the t-tubules, where they are normally located and provide the trigger for Ca release.     

Quantifying RhoA facilitated trafficking of the epithelial Na+ channel toward the plasma membrane with total internal reflection fluorescence-fluorescence recovery after photobleaching

The epithelial Na(+) channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is set, in part, by its membrane levels. The small G protein RhoA increases ENaC activity by increasing the membrane levels of this channel. We hypothesize that RhoA increases ENaC activity by promoting channel trafficking to the plasma membrane. Few experimental methods are available to directly visualize trafficking of ion channels to the plasma membrane. Here we combine electrophysiology with two complementary imaging methods, total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, to study the mechanistic basis of RhoA actions on ENaC. Patch clamp results demonstrate that RhoA increases ENaC activity in an additive manner with dominant-negative dynamin. This is consistent with a mechanism of increased ENaC trafficking to the membrane. Direct visualization of ENaC movement near the plasma membrane with total internal reflection fluorescence-fluorescence recovery after photobleaching revealed that RhoA accelerates ENaC trafficking toward the membrane. RhoA-facilitated movement of the channel was sensitive to disrupting the endomembrane system. Moreover, facilitating retrieval decreased ENaC activity but not trafficking toward the membrane. ENaC at the plasma membrane clustered and was laterally immobile suggesting that the cytoskeleton tethers or corrals membrane resident channels or membrane-directed vesicles containing ENaC. Disrupting microtubules but not microfilaments led to reorganization of ENaC clusters and slowed trafficking toward the membrane. The cytoskeleton is an established target for RhoA signaling. We conclude that RhoA, likely through effects on the cytoskeleton, promotes ENaC trafficking to the plasma membrane to increase channel membrane levels and activity.     

The filament-forming protein Pil1 assembles linear eisosomes in fission yeast

The cortical cytoskeleton mediates a range of cellular activities such as endocytosis, cell motility, and the maintenance of cell rigidity. Traditional polymers, including actin, microtubules, and septins, contribute to the cortical cytoskeleton, but additional filament systems may also exist. In yeast cells, cortical structures called eisosomes generate specialized domains termed MCCs to cluster specific proteins at sites of membrane invaginations. Here we show that the core eisosome protein Pil1 forms linear cortical filaments in fission yeast cells and that purified Pil1 assembles into filaments in vitro. In cells, Pil1 cortical filaments are excluded from regions of cell growth and are independent of the actin and microtubule cytoskeletons. Pil1 filaments assemble slowly at the cell cortex and appear stable by time-lapse microscopy and fluorescence recovery after photobleaching. This stability does not require the cell wall, but Pil1 and the transmembrane protein Fhn1 colocalize and are interdependent for localization to cortical filaments. Increased Pil1 expression leads to cytoplasmic Pil1 rods that are stable and span the length of cylindrical fission yeast cells. We propose that Pil1 is a novel component of the yeast cytoskeleton, with implications for the role of filament assembly in the spatial organization of cells.     

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.