Ischemia in vivo and cellular ATP depletion in vitro are known to result in actin polymerization in several cell types including renal epithelial cells, fibroblasts and endothelial cells. This seemingly universal event is associated with rapidly occurring, duration-dependent decreases in G- actin, yet the underlying cellular mechanisms and effects remain unknown. We hypothesize ischemia-induced actin polymerization occurs in an unregulated fashion secondary to direct effects of cellular ATP depletion resulting in the accumulation of ADP-G actin. This, in turn, results in quantifiable alterations in actin sequestering protein affinity, accumulation of free G-actin and un-regulated actin assembly in cellular locations where the critical concentration of actin assembly is exceeded. To evaluate this hypothesis we will utilize an in vitro cell culture model of reversible deletion in LLCPK1 cells and complimentary studies in freshly isolated proximal tubules. Biochemical analysis of G-actin associated nucleotides (ATP, ADP), quantitation of total, bound and free actin sequestering proteins including thymosin beta4, profilin and actin depolymerizing factor (ADF) will be performed. Pyrene actin and etheno- ATP techniques will be utilized to quantify the direct effects of altering ATP and ADP levels on actin polymerization in the presence of varying sequestering proteins. We also postulated ATP depletion results in dissociation of capping protein from barbed ends of actin filaments, and enhanced nucleation of actin polymerization. This, in turn, enhances actin polymerization of existing uncapped filaments and unregulated cytosolic polymerization of F-actin by nucleation. Fluorescent analogue techniques using both dynamic wide field and confocal techniques image dynamics, cellular location and effects of ischemia on capping protein and ADF. Finally, the role of ADF in microfilament severing during cellular ATP depletion and repletion will be investigated using similar biochemical and microscopic techniques. Specifically, we will investigate the effect of ischemia on dephosphorylation (activation) of ADF, and recruitment of the surface membrane. Western blotting techniques, immunofluorescent, immunogold TEM and fluorescent analogue techniques using gfp-ADP constructs and 3-D reconstruction techniques will be used.. We believe the resulting data will provide mechanistic information regarding unregulated actin assembly during ATP depletion and actin severing during ATP repletion. This may have direct clinical applicability in ischemic acute renal failure.
Showing the most recent 10 out of 37 publications
