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 diaphanousrelated 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. Podocytes are terminally differentiated epithelial cells with interdigitating processes that wrap around and support the capillaries of the kidney's glomeruli. The terminal portions of these actin-rich extensions, known as foot processes (FPs), are bridged by cell-cell junctions called slit diaphragms (SD), a complex of proteins anchored at adjacent FPs. 1,2 FPs are polarized cytoplasmic processes with an apical-basal junction demarcated by the SD complex. 3 The SD protein complex participates in regulating the morphology and filter function of podocytes through crosstalk with actin remodeling pathways. 4,5 Ultrastructural studies have shown that the FPs contain a central actin bundle surrounded by a cortical actin network. This cortical actin is essential for maintaining the morphology and function of podocytes. 6 Through direct connections with the plasma membrane of FPs, cortical actin serves as a scaffold for SD proteins and their communication with actin filaments through signaling pathways and actin-binding proteins. 6,7 Actin reorganization and SD protein translocation accompany the foot process effacement and loss of the filtration barrier seen in proteinuric diseases. 8

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo

“…Therefore, direct binding between the INFT-2-DID and the CYK-1-DAD might also occur. Importantly, in the EC we found that CYK-1 inhibits the actinpolymerizing activity of INFT-2; the reciprocal effect to that seen in previous studies (Sun et al, 2013(Sun et al, , 2011. Whether this reciprocal effect can also be observed with human mDia and INF2 will require more precise in vitro and cell culture studies.…”

“…Scale bars: 10 µm (B,E); 5 µm (C,F). (Madrid et al, 2010); and CYK-1, the sole ortholog of mDia (MiMi et al, 2012), because a direct interaction between INF2 and mDia, modulated by Cdc42, had been described and found to regulate F-actin in cell culture (Sun et al, 2013(Sun et al, , 2011. In considering the relationship of the two C. elegans paralogs with INF2, we note that phylogenetic analysis clearly places INFT-2 in the same family as human INF2 (Mi-Mi et al, 2012;Fig.…”

“…Cell culture and in vitro analysis suggest that human INF2 binds to mDia and inhibits its ability to polymerize actin (Sun et al, 2013(Sun et al, , 2011. Our work demonstrates that the reciprocal regulatory interaction occurs in vivo: CYK-1 negatively regulates the actinpolymerizing function of INFT-2, and this plays a role in regulating F-actin levels and EXC-6-promoted EC outgrowth.…”

“…We also show that CYK-1 negatively regulates INFT-2 apical localization, inhibits the ability of INFT-2 to promote F-actin accumulation, and may act via a distinct target to inhibit basolateral outgrowth. As INF2 and mDia physically interact and cross-regulate in cultured cells (Sun et al, 2013(Sun et al, , 2011, we propose that the conserved EXC-5−CDC-42 pathway modulates their physical interaction and that this regulation and interaction are functionally important in vivo during tubulogenesis.…”

“…5B). This model is based on the fact that in vitro and in cell culture, binding of the INF2-DID to the mDia-DAD inhibits mDia actin-polymerizing activity (Sun et al, 2013(Sun et al, , 2011. We note that in the partial INFT-2-DID, the residues important for INF2-DID binding to the mDia-DAD, which are mutated in patients with autosomal dominant In the scatter graph, each point represents a measurement carried out on a single 0.5 µm confocal z-section to prevent confounding additive effects from projections.…”