Role Of α-actinins In Podocyte Motility

Objectives To investigate the effect of α-actinins ablation and mutation on podocyte cytoskeleton changes, cell motility and traction force.Methods Conditionally immortalized wild type, α-actinin-4K256E heterozygous and homozygous mutant podocytes were cultured in vitro. α-Actinin-1and α-actinin-4were ablated in wild type podocytes using lentiviral RNA interference. Western blot was used to detect the efficiency of transfection. Immunostaining was performed to characterize cell morphology, α-actinins and filamentous actin distribution in podocytes. Wound healing assay was used to measure cell motility. Traction force microscopy was used to measure cell traction.Results Highly efficient ablation of α-actinin-1and α-actinin-4were achieved at protein level using the lentiviral system. α-Actinin-1and α-actinin-4ablation were associated with disorganization of actin stress fibers, increased cell traction force and motility. In contrast, α-actinin-4mutant cells showed decreased cell tration force and motility.Conclusions α-Actinins participate in cytoskeletal organization and traction force generation, tending to regulate podocyte contractility and restrict cell motility. Mutant α-actinin-4may gain some functions to decrease cell contractility and motility. These findings suggest that altered α-actinin function could cause glomerular damage by altering podocyte contractility and motility. Objectives To explore the effects of a-actinins ablation or mutation on mRNA and protein expressions of focal adhesion markers vinculin and (33integrin in vitro and in vivo.Methods Indirect immunostaining was performed to characterize vinculin and filamentous actin distribution in podocytes and active β3integrin in wild type, a-actinin-4knockout, a-actinin-4K256E heterozygous and homozygous mutant knockin mice. Real-time PCR and western blot were used to detect vinculin mRNA and protein expressions in a-actinins ablated and mutant cells. Real-time PCR was also used to detect β3integrin mRNA expression in above cells.Results The mRNA and protein expressions of vinculin were increased in a-actinin-4ablated cells, but not in a-actinin-1ablated and a-actinin-4mutant cells. The mRNA expression of β3integrin was markedly decreased in both a-actinin-4mutant cells. In addition, active (33integrin was increased in a-actinin-4knockout mice and decreased in a-actinin-4K256E heterozygous and homozygous mutant mice.Conclusions a-Actinin-4ablation can affect expression of vinculin. a-Actinin-4knockout and mutation can dramatically affect active β3integrin expression. These data suggested that a-actinins can affect expressions of focal adhesions. Objectives To investigate the effect of a-actinins ablation and mutation on activities of small GTPases and observe changes of small GTPases activities in wild type, a-actinin-4K256E heterozygous and homozygous mutant mice.Methods Small GTPases activities in a-actinins ablated and mutant cells were measured by G-LISA RhoA, Racl and Cdc42activity assays. The glomeruli lysates of wild type, a-actinin-4K256E heterozygous and homozygous mutant mice were also measured by above G-LISA activity assays.Results RhoA was significantly decreased, Cdc42was markedly increased but Racl activity was unchanged in a-actinin-4ablated cells. RhoA was also increased but not significantly changed in a-actinin-1ablated cells. Racl and Cdc42activities were not affected by a-actinin-1ablation. In a-actinin-4mutant cells, RhoA was increased in both a-actinin-4mutant cells. This mutation can not affect Racl and Cdc42activities.Conclusions a-Actinins ablation or mutation can affect small GTPases activities in different degree. These changes are related to cytoskeleton reorganization, adhesion and migration in podocytes.