Inverted Formin 2 Regulates Actin Dynamics by Antagonizing Rho/Diaphanous-related Formin Signaling
ABSTRACT Mutations in inverted formin 2 INF2 are a common cause of familial FSGS. INF2 interacts with diaphanous-related formins (mDia) and antagonizes mDia-mediated actin polymerization in response to active Rho signaling, suggesting that dysregulation of these pathways may mediate the development of INF2-related FSGS. However, the precise mechanisms by which INF2 regulates actin-dependent podocyte behavior remain largely unknown. Here, we investigated the possible role of INF2 in both lamellipodia-associated actin dynamics and actin-dependent slit diaphragm (SD) protein trafficking by manipulating the expression of INF2 and the activity of Rho/mDia signaling in cultured podocytes. Activation of mDia in the absence of INF2 led to defective formation of lamellipodia and abnormal SD trafficking. Effects of mutations disrupting the INF2-mDia interaction suggested the specificity of the mDia-antagonizing effect of INF2 in maintaining the lamellipodium. Furthermore, we found that SD trafficking requires INF2 interaction with lipid raft components. In summary, INF2 regulates lamellipodial actin dynamics and the trafficking of slit diaphragm proteins by opposing Rho/mDia-mediated actin polymerization. Thus, in podocytes, INF2 appears to be an important modulator of actin-dependent behaviors that are under the control of Rho/mDia signaling.
SourceAvailable from: sciencedirect.com[Show abstract] [Hide abstract]
ABSTRACT: Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.12/2014; 1(2-3):107-115. DOI:10.1016/j.ebiom.2014.11.009
[Show abstract] [Hide abstract]
ABSTRACT: Podocytes are specialized epithelial cells that cover the outer surfaces of glomerular capillaries. Unique cell junctions, known as slit diaphragms, which feature nephrin and Neph family proteins in addition to components of adherens, tight, and gap junctions, connect adjacent podocyte foot processes. Single gene disorders affecting the slit diaphragm result in nephrotic syndrome in humans, characterized by massive loss of protein across the capillary wall. In addition to specialized cell junctions, interconnecting podocytes also adhere to the glomerular basement membrane (GBM) of the capillary wall. The GBM is a dense network of secreted, extracellular matrix (ECM) components and contains tissue-restricted isoforms of collagen IV and laminin in addition to other structural proteins and ECM regulators such as proteases and growth factors. The specialized niche of the GBM provides a scaffold for endothelial cells and podocytes to support their unique functions and human genetic mutations in GBM components lead to renal failure, thus highlighting the importance of cell-matrix interactions in the glomerulus. Cells adhere to ECM via adhesion receptors, including integrins, syndecans, and dystroglycan and in particular the integrin heterodimer α3β1 is required to maintain barrier integrity. Therefore, the sophisticated function of glomerular filtration relies on podocyte adhesion both at cell junctions and at the interface with the ECM. In health, the podocyte coordinates signals from cell junctions and cell-matrix interactions, in response to environmental cues in order to regulate filtration and as our understanding of mechanisms that control cell adhesion in the glomerulus develops, then insight into the effects of disease will improve. The ultimate goal will be to develop targeted therapies to prevent or repair defects in the filtration barrier and to restore glomerular function.Frontiers in Endocrinology 10/2014; 5:160. DOI:10.3389/fendo.2014.00160
[Show abstract] [Hide abstract]
ABSTRACT: Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF-FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.Proceedings of the National Academy of Sciences 04/2015; DOI:10.1073/pnas.1505035112 · 9.81 Impact Factor