Ultrastructural localization of alpha A-crystallin to the bovine lens fiber cell cytoskeleton.
Monoclonal antibodies to the bovine alpha A-crystallin were developed and used to probe the relationship between alpha A-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble", and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KCl. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the alpha A-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subfraction of the total alpha A-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.
FitzGerald
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PG
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis 95616.
Graham
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D
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003459:Crystallins / Q000378:metabolism / Q000648:ultrastructure*; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007150:Immunohistochemistry; D007904:Lens Cortex, Crystalline / Q000378:metabolism / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D003459:Crystallins; D003598:Cytoskeletal Proteins