Cellular ATP depletion induces disruption of the spectrin cytoskeletal network.
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. Because 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 physiological conditions, both ankyrin and fodrin were largely Triton X-100 insoluble and colocalized immunofluorescently along the lateral membranes of LLC-PK1 cells. After 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 1, 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.
Department of Medicine, Indiana University School of Medicine, Indianapolis, USA.
B A
BA
Molitoris
R
R
Dahl
M
M
Hosford
D000199:Actins / Q000502:physiology; D000255:Adenosine Triphosphate / Q000172:deficiency*; D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D003902:Detergents; D005455:Fluorescent Antibody Technique; D018374:LLC-PK1 Cells; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D017830:Octoxynol; D012995:Solubility; D013049:Spectrin / Q000502:physiology*; D013552:Swine
D000199:Actins; D000255:Adenosine Triphosphate; D017487:Ankyrins; D002352:Carrier Proteins; D003902:Detergents; D008840:Microfilament Proteins; D009218:Myosins; D017830:Octoxynol; D013049:Spectrin; C026615:villin