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Palladin, a cytoskeletal protein with essential functions for stress fiber formation, is found in developing and mature tissues, including the kidney. To define its role in the kidney, we measured its expression in mouse kidney and found it co-localized with F-actin in smooth muscle cells of renal arterial vessels, mesangial cells, and podocytes but not in tubular epithelium. Using immunoelectron microscopy, we confirmed that palladin was present in podocytes. In cultured mouse podocytes, palladin co-localized with F-actin in dense regions of stress fibers, focal adhesions, cell-cell contacts and motile cell margins. Transfection with the N-terminal half of palladin targeted it to F-actin-containing structures in podocytes while the C-terminal half accumulated in the nucleus, a result also found for endogenous palladin in cultured cells after leptomycin B was used to block nuclear export. Green fluorescent protein (GFP)-tagged palladin was found in dynamic ring-like F-actin structures and ruffles in cultured podocytes after stimulation with epidermal growth factor. Inhibition of palladin expression by transfection of an antisense construct reduced the formation of ring-like structures. Photo-bleaching analysis showed that GFP-palladin turned over with a half-time of 10 s in focal adhesions and dense regions of stress fibers, suggesting that palladin is a dynamic scaffolding protein. Our study shows that palladin is expressed in podocytes and plays an important role in actin dynamics.

Mutations in the ACTN4 gene, encoding the actin crosslinking protein alpha-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E alpha-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E alpha-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E alpha-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on alpha-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type alpha-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E alpha-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E alpha-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E alpha-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E alpha-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E alpha-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS.