Cellular atp depletion induces disruption of the spectrin
cytoskeletal network.
Molitoris, Bruce A., Rolf Dahl, and Melanie Hosford.
Renal Epithelial Biology Experimental Laboratories, Department of
Medicine, Indiana University School of Medicine and the Veterans
Administration Medical Center, ndianapolis, IN, Department of
Medicine, University of Colorado School of Medicine, Denver, CO
APStracts 3:0119F, 1996.
Ischemia in vivo or ATP depletion in vitro result in disruption and
cellular redistribution of the cortical F-actin cytoskeleton in
epithelial cells. However, little is known regarding the effect of
these two maneuvers on other components of the actin cytoskeleton.
Since the spectrin (fodrin in epithelial cells) based network links
the actin cytoskeleton 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 physiologic
conditions, both ankyrin and fodrin were largely Triton-X-100
insoluble and colocalized immunofluorescently along the lateral
membranes of LLC-PK1 cells. Following ATP depletion, there was a
rapid and duration-dependent increase in Triton-X-100 solubility of
both proteins. This was not true for villin and myosin I as Triton-X
-100 solubility was unaffected and reduced by ATP depletion,
respectively. The increase in fodrin and ankyrin detergent solubility
during ATP depletion was associated with cytosolic redistribution of
the proteins, as determined using immunofluorescent techniques.
Sucrose gradient fractionation and Western blot analysis of the
Triton-X-100 soluble fraction following ATP depletion revealed lack
of association between fodrin and ankyrin. Furthermore, dual-label
digital confocal immunofluorescent studies revealed lack of
association of cytoplasmic ankyrin and fodrin following ATP
depletion. Taken together, these data indicate that ATP depletion in
LLC-PK1 cells leads to dissociation of both ankyrin and fodrin from
the actin cytoskeleton. Furthermore, the two proteins dissociate from
each other and redistribute throughout the cytoplasm.
Received 13 July 1995; accepted in final form 25 June 1996.
APS Manuscript Number F229-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 25 July 1996