Permselective dysfunction of podocyte-podocyte contact upon angiotensin II unravels the molecular target for renoprotective intervention

Ameliorating the function of the glomerular barrier to circulating proteins by blocking angiotensin II (Ang II) translates into less risk of progression toward end-stage renal failure in diabetic and nondiabetic nephropathies. However, the mechanisms underlying this barrier protection are not clear. Specialized contacts between adjacent podocytes are major candidate targets, and the actin cytoskeleton is emerging as a regulatory element. Here, we present data demonstrating that Ang II induced reorganization of F-actin fibers and redistribution of zonula occludens-1 (ZO-1) that is physically associated with actin in murine podocytes. These effects were paralleled by increased albumin permeability across podocyte monolayers. The F-actin stabilizer jasplakinolide prevented both ZO-1 redistribution and albumin leakage, suggesting that actin cytoskeleton rearrangement is instrumental to podocyte permselective dysfunction induced by Ang II. Changes in both F-actin and ZO-1 patterns were confirmed in glomeruli of rat isolated perfused kidneys on short infusion of Ang II, leading to increased protein excretion. Podocyte dysfunction was mediated by Ang II type 1 receptor and was partly dependent on Src kinase-phospholipase C activation. These data demonstrate that strategies aimed at stabilizing podocyte-podocyte contacts and targeting the relevant intracellular signal transduction are crucial to renoprotection.     

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin–angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of α actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of α actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nephrin稳定血管紧张素Ⅱ诱导的足细胞细胞骨架改变的机制研究

 目的：研究足细胞裂孔膜分子nephrin调节血管紧张素Ⅱ（AngⅡ）诱导的足细胞骨架分布改变的分子机制。方法：用AngⅡ及AngⅡ受体拮抗剂氯沙坦或Akt抑制剂LY294002刺激足细胞,FITC-phalloidin染色标记F-actin,分析足细胞骨架运动。Real-time RT-PCR、RT-PCR和Western blotting检测nephrin mRNA和蛋白表达。转染nephrin全长表达质粒（pcDNA3.1-mNPHS1）,建立稳定转染足细胞系,Western blotting检测转染细胞的Akt磷酸化水平,FITC-phalloidin染色分析高表达nephrin对F-actin分布影响。结果：AngⅡ和LY294002刺激后,足细胞的F-actin重组,应力纤维减少,形成F-actin外周环。氯沙坦显著抑制F-actin重排。AngⅡ刺激后nephrin mRNA和蛋白表达显著降低,Akt磷酸化水平降低。pcDNA3.1-mNPHS1转染显著上调足细胞Akt磷酸化水平,促进足细胞短线状足突形成,部分抑制AngⅡ诱导的骨架重排。结论：PI3K/Akt是nephrin和AngⅡ的共同下游通路。Nephrin能通过PI3K/Akt途径部分稳定AngⅡ诱导的足细胞细胞骨架改变。     

Nef interaction with actin compromises human podocyte actin cytoskeletal integrity

The HIV-1 accessory protein Nef is considered to play an important role in the development of a podocyte phenotype in HIV-1 associated nephropathy. We hypothesized that Nef may be altering the podocyte phenotype both structurally and functionally. To elucidate the involved mechanisms, podocyte proteins interacting with Nef were identified using GST pull down assay and yeast two hybrid assay. The GST pull down assay on protein extracts made from stable colonies of conditionally immortalized human podocytes expressing Nef (Nef/CIHP) displayed a band at 45 kD, which was identified as actin by mass spectrometry. Yeast two hybrid assay identified the following Nef-interacting proteins: syntrophin, filamin B, syntaxin, translational elongation factor 1, and zyxin. The Nef–actin and Nef–zyxin interactions were confirmed by co-localization studies on Nef/CIHP stable cell lines. The co-localization studies also showed that Nef/CIHP stable cell lines had a decreased number of actin filaments (stress fibers), displayed formation of lamellipodia, and increased number of podocyte projections (filopodia). Nef/CIHP displayed an enhanced cortical F-actin score index (P < 0.001) and thus indicated a reorganization of F-actin in the cortical regions. Microarray analysis showed that Nef enhanced the expression of Rac1, syndecan-4, Rif, and CDC42 and attenuated the expression of syndecan-3 and syntenin. In addition, Nef/CIHPs displayed a diminished sphingomyelinase (ASMase) activity. Functionally, Nef/CIHPs displayed diminished attachment and enhanced detachment to their substrate. These findings indicate that Nef interaction with actin compromises the podocyte cytoskeleton integrity.     

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils.

Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils.     

Loss of PTEN promotes podocyte cytoskeletal rearrangement,aggravating diabetic nephropathy

In diabetic nephropathy (DN), podocyte cytoskeletal rearrangement occurs followed by podocyte effacement and the development of proteinuria. PTEN (phosphatase and tensin homologue) is a ubiquitously expressed phosphatase that plays a critical role in cell proliferation, cytoskeletal rearrangement, and motility. In mouse models of diabetes mellitus, PTEN expression is reportedly decreased in mesangial cells, contributing to expansion of the mesangial matrix, but how PTEN in the podocyte influences the development of DN is unknown. We observed that PTEN expression is down‐regulated in the podocytes of diabetic db/db mice and patients with DN. In cultured podocytes, PTEN inhibition caused actin cytoskeletal rearrangement and this response was associated with unbalanced activation of the small GTPases Rac1/Cdc42 and RhoA. In mice treated with PTEN inhibitor, actin cytoskeletal rearrangement occurred in podocytes and was accompanied by increased albumin excretion. We also created mice with an inducible deletion of PTEN selectively in podocytes. These mice exhibited increased albumin excretion and moderate foot process effacement. When the mice were challenged with a high fat diet, podocyte‐specific knockout of PTEN resulted in substantially increased proteinuria and glomeruloclerosis compared to control mice fed a high fat diet or mice with PTEN deletion fed a normal diet. These results indicate that PTEN is involved in the regulation of cytoskeletal rearrangement in podocytes and that loss of PTEN predisposes to the development of proteinuria and DN. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Multiple downstream signalling pathways from ROCK, a target molecule of Rho small G protein, in reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells

BACKGROUND: In Madin-Darby canine kidney cells, Rho small G protein regulates formation of stress fibres, focal adhesions, and peripheral bundles through reorganization of the actin cytoskeleton. There are two morphologically distinguishable types of Rho-regulated stress fibres: parallel and stellate. Of these, effects of Rho small G protein, mDia1 regulates the formation of parallel stress fibres, whereas ROCK regulates the formation of stellate stress fibres, peripheral bundles and focal adhesions. Both mDia1 and ROCK are direct downstream targets of Rho small G protein. RESULTS: The ROCK-induced formation of stellate stress fibres is regulated mainly through the myosin light chain kinase-dependent phosphorylation of myosin light chain and the LIM-kinase-dependent phosphorylation of cofilin. The ROCK-induced formation of focal adhesions is mainly regulated through a downstream pathway of ROCK other than myosin light chain and cofilin. The ROCK-induced formation of peripheral bundles is regulated at least through ERM proteins, but not through the myosin light chain or cofilin. CONCLUSION: Our present and previous findings suggest the presence of multiple downstream signalling pathways from ROCK to reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells.     

Cytoskeletal changes in podocytes associated with foot process effacement in Masugi nephritis.

Foot process effacement represents the most characteristic change in podocyte phenotype under a great variety of experimental as well as human glomerulopathies. It consists in simplification up to a total disappearance of an interdigitating foot process pattern. Finally, podocytes affix to the glomerular basement membrane by outspread epithelial sheets. Structural and immunocytochemical techniques were applied to analyze the cytoskeletal changes associated with foot process effacement in Masugi nephritis. Three days after injection of the anti-glomerular-basement-membrane serum an interdigitating foot process pattern was almost fully lost; more than 90 percent of the outer glomerular capillary surface were covered by expanded sheets of podocyte epithelium that contain a highly organized cytoskeleton adhering to the basal cell membrane. Structurally, this cytoskeleton consists of an interwoven network of microfilaments with regularly distributed dense bodies, which obviously serve as cross-linkers within this network. Immunocytochemically, the expression of actin, alpha-actinin, and pp44 (a specific podocyte protein normally associated with the cytoskeleton of foot processes) were increased in this structure; alpha-actinin was especially prominent in the dense bodies. The results are consistent with the view that foot process effacement represents an adaptive change in cell shape including hypertrophy of the contractile apparatus, reinforcing the supportive role of podocytes. Several factors associated with increased distending forces to podocytes may underlie this phenotype change including loss of mesangial support, elevated glomerular pressures, and impairment of GBM substructure as well as of podocyte-GBM-contacts. Twenty-eight days after serum injection a remodeling of the foot process pattern was seen. It appears that this restitution depends on a preceding repair of mesangial support function to glomerular capillaries.     

Synaptopodin protects against proteinuria by disrupting Cdc42:IRSp53:Mena signaling complexes in kidney podocytes

The actin-based foot processes of kidney podocytes and the interposed slit diaphragm form the final barrier to proteinuria. Mutations affecting several podocyte proteins cause disruption of the filtration barrier and rearrangement of the highly dynamic podocyte actin cytoskeleton. Proteins regulating the plasticity of the podocyte actin cytoskeleton are therefore of critical importance for sustained kidney barrier function. Synaptopodin is an actin-associated protein essential for the integrity of the podocyte actin cytoskeleton because synaptopodin-deficient mice display impaired recovery from protamine sulfate-induced foot process effacement and lipopolysaccharide-induced nephrotic syndrome. Moreover, bigenic heterozygosity for synaptopodin and CD2AP is sufficient to induce spontaneous proteinuria and focal segmental glomerulosclerosis-like glomerular damage in mice. Mechanistically, synaptopodin induces stress fibers by blocking the proteasomal degradation of RhoA. Here we show that synaptopodin directly binds to IRSp53 and suppresses Cdc42:IRSp53:Mena-initiated filopodia formation by blocking the binding of Cdc42 and Mena to IRSp53. The Mena inhibitor FP(4)-Mito suppresses aberrant filopodia formation in synaptopodin knockdown podocytes, and when delivered into mice protects against lipopolysaccharide-induced proteinuria. The identification of synaptopodin as an inhibitor of Cdc42:IRSp53:Mena signaling defines a novel antiproteinuric signaling pathway and offers new targets for the development of antiproteinuric therapeutic modalities.     

The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.     

Junctional Rab13‐binding protein (JRAB) regulates cell spreading via filamins

We previously showed that Rab13 and its effector protein, junctional Rab13‐binding protein (JRAB)/molecules interacting with CasL‐like 2 (MICAL‐L2), regulate junctional development by modulating cell adhesion molecule transport and actin cytoskeletal reorganization in epithelial cells. Here, we investigated how JRAB regulates reorganization of the actin cytoskeleton in NIH3T3 fibroblasts, in an attempt to obtain novel insights into the mechanism of JRAB action. To this end, we expressed mutant proteins that adopt a constitutively open or closed state and then examined effect on cellular morphology of the resulting actin cytoskeletal reorganization. Expression of the JRABΔCT mutant (constitutively ‘closed’ state) induced stress fibers, whereas expression of the JRABΔCC mutant (constitutively ‘open’ state) caused cell spreading with membrane ruffles. Next, we identified the proteins involved in JRAB‐induced rearrangement of actin cytoskeleton leading to morphological changes. In NIH3T3 cells expressing HA‐JRABΔCC, filamin, an actin cross‐linking protein, coimmunoprecipitated with HA‐JRABΔCC. Expression of ASB2 induced degradation of all three filamin isoforms and inhibited the JRABΔCC‐induced cell spreading. Consistent with our previous results, actinin‐1/‐4 were also immunoprecipitated with HA‐JRABΔCC. However, actinin‐1/‐4 have no effect on the cell spreading regulated by JRABΔCC. These data suggest that JRAB contributes to the rearrangement of the actin cytoskeleton during cell spreading via filamins.     

Rho GTPase在信号转导和细胞骨架中的作用

Rho GTPases参与多种重要的细胞生命活动,如肌动蛋白细胞骨架的重构、细胞黏附、细胞运动、囊泡运输、转录激活、基因表达和细胞周期的调控等。当Rho GTPase蛋白水平改变,活性状态改变,效应蛋白丰度改变后,出现异常的Rho 信号,从而影响细胞骨架重组使细胞迁移。调节这些生物信号的转导通路非常复杂, 因此,Rho GTPases已成为近年来的研究热点。     

Rho modulates hepatic sinusoidal endothelial fenestrae via regulation of the actin cytoskeleton in rat endothelial cells

The presence of actin-like microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) indicates that the cytoskeleton of sinusoidal endothelial cells (SEC) plays an important role in the modulation of SEF. Rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The present study aimed to examine how a Rho stimulator; lysophosphatidic acid (LPA), and a Rho inhibitor; bacterial toxin C3 transferase (C3-transferase), affect the morphology of SEF. Monolayers of SEC culture were established by infusing a rat liver with collagenase for 30 min and then culturing in RMPI medium for 24 h. The cells were separated into three groups; control, LPA-treated (15 microM), and C3-transferase-treated (15 microg/ml) groups. SEF morphology was observed by scanning electron microscopy. Formation of F-actin stress fibers was observed by confocal microscopy. Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. Active Rho was measured by Ren's modification. Treatment of SECs with LPA contracted the SEF, concomitant with increases in F-actin stress fiber and actin microfilament, and high expression of phosphorylated myosin light-chain kinase. Following treatment with C3-transferase, SEF dilated and fused, concomitant with a loss of F-actin and microfilament, and low expression of phosphorylated myosin light chain. Rho A expression does not change by both treatments. In conclusion, these results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.     

Modulation of cytoskeletal organization of podocytes during the course of aminonucleoside nephrosis in rats

By immunoelectron microscopy the modulation of cytoskeletal organization of podocytes during the course of puromycin aminonucleoside-induced nephrosis was examined. In control rats, tubulin and vimentin were present, limited to the podocyte cell body and the major processes. Their distribution in the foot processes was virtually negative. Myosin exhibited the same distribution pattern, albeit much more scattered, with no relation to any podocyte organelles or cell structures. Actin was scattered over the fibrillar zones of the cell body and its processes, including the foot processes. In proteinuric rats, loss of foot processes occurred and the glomerular basement membrane was covered by broad cytoplasmic sheets of podocytes, which contained these four subunits of cytoplasmic filaments. Accompanied by the disappearance of proteinuria, the structural organization of the foot processes was completely restored, in which tubulin, vimentin, and myosin were scarcely observed. Our results confirmed that the loss of foot processes is caused by their retraction, and indicated that the specific localization of the podocytic cytoskeleton contributes to the maintenance of the particular cell shape. Its reorganization may account for the structural modification of podocytes.     

Annexin 2 regulates intestinal epithelial cell spreading and wound closure through Rho-related signaling

Epithelial cell migration is a critical event in gastrointestinal mucosal wound healing and is dependent on actin cytoskeletal reorganization. We observed increased expression of an actin regulatory protein, annexin 2, in migrating intestinal epithelial cells. Small interfering RNA (siRNA)-mediated knockdown of annexin 2 expression in Caco-2 epithelial cells resulted in significant reductions in cell spreading and wound closure associated with decreased formation of filamentous actin bundles along the base of migrating cells. Because annexin 2 has been shown to influences actin cytoskeletal remodeling through targeting signaling molecules to membrane domains, we examined the membrane association and activation status of Rho GTPases after annexin 2 knockdown. We observed Rho dissociation from membranes and decreased Rho activity following annexin 2 siRNA transfection. Inhibition of cell spreading and wound closure in annexin 2 siRNA-transfected cells was prevented by expression of constitutively active RhoA. Rho colocalized with annexin 2 in lamellipodia and along the cytoplasmic face of the plasma membrane. In addition, annexin 2 was observed to co-immunoprecipitate with endogenous Rho and constitutively active RhoA. These findings suggest that annexin 2 plays a role in targeting Rho to cellular membranes, thereby modulating Rho-related signaling events regulating cytoskeletal reorganization during epithelial cell migration.     

Ankycorbin: a novel actin cytoskeleton-associated protein

BACKGROUND: Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity and cytokinesis. Many actin filament (F-actin)-binding proteins have been isolated and implicated in the maintenance and reorganization of actin cytoskeleton structures. RESULTS: We purified here a novel protein with a molecular mass of about 125 kDa (p125) from rat liver. We cloned its cDNA from a mouse kidney cDNA library and determined its nucleotide and deduced amino acid sequences. p125 was a protein of 979 amino acids with a calculated Mr of 108 847. p125 contained six ankyrin repeats in the N-terminal region and a domain predicted to form a coiled-coil structure in the C-terminal region. We named p125 ankycorbin (ankyrin repeat- and coiled-coil structure-containing protein). Northern blot analysis indicated that ankycorbin was ubiquitously expressed in all the tissues examined. Immunofluorescence and immunoelectron microscope analyses revealed that ankycorbin was associated with the cortical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibres. However, ankycorbin did not directly bind to F-actin as estimated by the F-actin co-sedimentation assay. CONCLUSIONS: These results indicate that ankycorbin is indirectly associated with the actin cytoskeleton structures, presumably through an unidentified factor and suggest that it is involved in their maintenance and/or reorganization.     

Rho kinase inhibition initiates apoptosis in human airway epithelial cells

Disruption of the actin cytoskeleton elicits profound changes in cell survival and function. The actin cytoskeleton is regulated in a hierarchical manner by Rho GTPases. Rho kinase, a downstream effector of RhoA, regulates the formation of stress fibers and focal adhesions. Disruption of the actin cytoskeleton causes apoptosis in airway epithelial cells. To examine further the relation of cytoskeletal integrity and apoptosis, we tested whether inhibition of Rho kinase would elicit apoptosis in airway epithelial cells. Inhibition with either Y-27632 or HA1077 induced membrane ruffling and loss of actin stress fibers, and apoptosis in airway epithelial cells that was blocked by inhibiting caspase function or by inhibiting protein synthesis. Cells overexpressing constitutively active Rho kinase, but not native Rho kinase, were resistant to Rho kinase inhibitor-induced stress fiber disruption and apoptosis. Inhibition of Rho kinase disrupted actin stress fibers but did not induce apoptosis in 3T3 cells. We demonstrate that Rho kinase inhibition induces airway epithelial cell apoptosis associated with changes in actin filament integrity. Our data suggest that Rho kinase may be a regulator of early initiation of apoptosis.     

Ezrin/radixin/moesin proteins and Rho GTPase signalling in leucocytes

The ezrin/radixin/moesin (ERM) family of actin-binding proteins act both as linkers between the actin cytoskeleton and plasma membrane proteins and as signal transducers in responses involving cytoskeletal remodelling. The Rho family of GTPases also regulate cytoskeletal organisation, and several molecular pathways linking ERM proteins and Rho GTPases have been described. This review discusses recent findings on ERM protein function in leucocytes and how these may be integrated with Rho GTPase signalling.