Modulating α‐actinin‐4 dynamics in podocytes

Abstract

Podocytes are epithelial cells that line the outer aspect of renal blood vessels and provide a platform for the kidney's filtering apparatus, the slit diaphragm. Mutations in alpha-actinin-4, an actin bundling protein highly expressed in podocytes, result in increased affinity for actin and cause a familial form of focal segmental glomerulosclerosis. We hypothesized that such gain-of-affinity mutations would override alpha-actinin-4's sensitivity to regulatory factors such as calcium (acting via two EF-hand motifs), and phosphoinositides. We generated calcium- (mutEF) and phosphoinositide- (mutPI) insensitive variants of alpha-actinin-4, comparing their properties to a disease-associated mutant (K256E) and to the wildtype (wt) protein. alpha-Actinin-4(mutPI) displayed increased affinity for actin, while the affinity of alpha-actinin-4(mutEF) was unchanged. Addition of calcium to actin sedimentation assays caused a decrease in the association of alpha-actinin-4(wt) with filamentous actin, while phosphoinositides generally increased this association. Similar to alpha-actinin-4(K256E), alpha-actinin-4(mutPI) was mislocalized in cultured podocytes, being preferentially associated with filamentous actin and focal adhesions. Fluorescence recovery after photobleaching experiments revealed a rapid turnover of alpha-actinin-4(wt) and alpha-actinin-4(mutEF) along stress fibers and focal adhesions, while the turnover of alpha-actinin-4(K256E) and alpha-actinin-4(mutPI) was dramatically reduced at these subcellular locales. Equibiaxial mechanical stimulation of podocytes, a mimic of intraglomerular forces, reduced podocyte surface area by 50%; this decrease was more severe (70%) in the presence of high-affinity mutants of alpha-actinin-4. These data suggest that dynamic regulation of alpha-actinin-4/actin interactions may be necessary for maintaining podocyte structure in response to glomerular hydrostatic forces.

α-Actinin-4 Is Required for Amoeboid-type Invasiveness of Melanoma Cells

“…This also strongly supports the contention that ACTN4 differs from ACTN1 in integrating the membrane with the actin cytoskeleton possibly by bridging between actin and phosphoinositides on the membrane (13,17,38) rather than simply bundling actin filaments. Further studies are needed to decipher the molecular mechanisms by which the expression of ACTN4 but not ACTN1 is elevated in melanoma and the increase of ACTN4 expression regulates the metastasis and invasiveness.…”

Podocyte Injury Associated with Mutantα-Actinin-4

“…For α -actinin-4, the phosphoinositide-binding site is within the CH2 domain. In contrast to findings for α -actinin-1, our studies showed that phosphoinositides increase the interaction of α -actinin-4 with F-actin [19]. Phosphorylation of α -actinin-1 has been reported in activated platelets [20].…”

Identification of Tpr and α-actinin-4 as two novel SLK-interacting proteins

“…Another consideration is that SLK alters the phosphorylation and function of α-actinin-4, including its actin-binding properties. It has been demonstrated that adoption of an "open" conformation of α-actinin-4 by mutational disruption of the CH2 domain increases its affinity for actin [42]. Since SLK can bind to the N-terminus of α-actinin-4, which contains the CH2 domain, SLK may inhibit formation of the open conformation of α-actinin-4, and negatively regulate actin binding.…”