engleski

Isoform Specific Phosphorylation of Dynamin1 in Regulating the Cortical Actin Cytoskeleton in Podocytes

BACKGROUND Dynamin is an essential actin regulatory protein in podocyte, and loss of its function is closely connected to podocyte injury and proteinuria. Recently, our studies have shown that dynamin directly regulates actin cytoskeleton via its oligomerization state. Importantly, dynamin specific small drug (Bis- T-23) that induces its oligomerization ameliorates proteinuria in diverse proteinuric animal models through recovering functional actin structures in injured podocytes. Threrfore, it is important to maintain balance between dynamin assembly and disassembly. This dynamin oligomerization can be regulated through interaction with diverse cellular proteins, and it was reported that dynamin1 can differentially alter the affinity for its protein binding partners via phosphorylation by two different serine/thronine kinases, GSK3β and CDK5, in neurons. Based on these data, we hypothesize that phosphorylation-dependent dynamin1 oligomerization is an important molecular mechanism that regulates actin dynamics in podocytes. METHODS Dynamin1 phosphorylation in podocytes was detected by western blot using phospho-dynamin1 specific antibodies in the presence of GSK3β or CDK5 inhibitor. Actin and paxillin in podocytes were stained to observe actin structures and focal adhesions. Cell migration and spreading assays were performed with podocytes expressing phosphor-dynamin1 mutants. For zebrafish experiments, each phosphor-dynamin1 mutant was expressed in dynamin2KD zebrafish. RESULTS 1. Dynamin1 is phosphorylated by GSK3β and CDK5 in podocytes. 2. Expression of phosphor-dynamin1 mutants alters cortical actin networks during cell spreading. 3. Expression of phosphor-dynamin1 mutants attects cell migration. 4. Expression of phosphor-dynamin1 mutants fails to rescue proteinuria in dynamin2KD zebrafish. CONCLUSION The role of dynamin in actin cytoskeleton in podocytes is essential to maintain the glomerular filtration barrier. Dynamin directly regulates actin structures via its oligomerization state. Our data suggest that dynamin1 phosphorylation is implicated in cortical actin dynamics in podocytes, and its balanced phosphorylation by GSK3β and CDK5 is crucial to podocyte's function in glomerular filtration.

dynamin 1, actin cytoskeleton, podocytes, phosphorylation

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