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.
Highlights
Inverted formin 2 (INF2) is a member of the formin family of proteins that regulate actin dynamics (Chhabra and Higgs, 2006; Korobova et al, 2013; Ramabhadran et al, 2011)
We have found that this prevents the dampening effect of INF2 on Rho/diaphanous related formin proteins (Dias) signaling, leading to imbalanced actin dynamics, perturbation of actin polymerization, and disruption of actin based cellular remodeling processes (e.g. lamellipodia formation and trafficking to the membrane of cultured podocytes (Sun et al, 2013))
Loss of INF2 function leads to a nephrosis-like edematous phenotype with podocyte abnormalities accompanied by both malformation and malfunction of the glomerular filtration barrier
Summary
Inverted formin 2 (INF2) is a member of the formin family of proteins that regulate actin dynamics (Chhabra and Higgs, 2006; Korobova et al, 2013; Ramabhadran et al, 2011). The slit diaphragm, a unique cell–cell junctional complex, is an electron dense structure visible between neighboring foot processes (Kubiak and Niemir, 2006; Asanuma and Mundel, 2003) Both the intricate morphology and unique function of podocytes are dependent on the regulation of the actin cytoskeleton, which is under the spatial and temporal control of the Rho family of small GTPases (Zhu et al, 2011; Scott et al, 2012; Akilesh et al, 2011; Mouawad et al, 2013; Oh et al, 2004; Shono et al, 2009). We demonstrate that the function of INF2 in glomerular development is largely mediated by its effect on Rho-activated Dia signaling, as knockdown of either zebrafish Rhoa or Dia rescues the INF2 morphant phenotype. The relative simplicity of this in vivo model of INF2-associated disease and its rescue by transient transgenesis and gene knockdown support its utility in examining genetic and pharmacologic modulation
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