CELLULAR ATP DEPLETION INDUCES DISRUPTION OF THE SPECTRIN CYTOSKELETAL NETWORK

Ischemia in vivo or ATP depletion in vitro result in disruption and ce llular redistribution of the cortical F-actin cytoskeleton in epitheli al cells. However, little is known regarding the effect of these two m aneuvers on other components of the actin cytoskeleton. Because the sp ectrin (fodrin in epithelial cells)-based network links the actin cyto skeleton to the surface membrane, we have utilized a reversible model of ATP depletion in LLC-PK1 cells to study the effect of ATP depletion on fodrin and ankyrin. Under physiological conditions, both ankyrin a nd fodrin were largely Triton X-100 insoluble and colocalized immunofl uorescently along the lateral membranes of LLC-PK1 cells. After ATP de pletion, there was a rapid and duration-dependent increase in Triton X -100 solubility of both proteins. This was not true for villin and myo sin I, as Triton X-100 solubility was unaffected and reduced by ATP de pletion, respectively. The increase in fodrin and ankyrin detergent so lubility during ATP depletion was associated with cytosolic redistribu tion of the proteins, as determined using immunofluorescent techniques . Sucrose gradient fractionation and Western blot-analysis of the Trit on X-100-soluble fraction following ATP depletion revealed lack of ass ociation between fodrin and ankyrin. Furthermore, dual-label digital c onfocal immunofluorescent studies revealed lack of association of cyto plasmic ankyrin and fodrin following ATP depletion. Taken together, th ese data indicate that ATP depletion in LLC-PK1 cells leads to dissoci ation of both ankyrin and fodrin from the actin cytoskeleton. Furtherm ore, the two proteins dissociate from each other and redistribute thro ughout the cytoplasm.