Immunofluorescence localization of the 90-kDa heat-shock protein to cytoskeleton.
The 90-kDa heat-shock protein, hsp90, is an abundant and conserved protein located predominantly in the cytoplasm. Previous reports have localized a portion of the hsp90 to microtubules and to microfilaments. Here, we show that colocalization of hsp90 with microtubules is seen after long-term but not short-term fixation of rat pulmonary endothelial cells with formaldehyde. Under conditions of methanol fixation, a significant portion of both hsp90 and the hsp90-associated protein p23 is present on fibrillar structures extending throughout the cytoplasm of endothelial cells, however, when cells are treated with colcemid under conditions that eliminate microtubules, the fibrils condense into bright rope-like bundles located in the immediate perinuclear area and extending toward the cell periphery. Identical images are observed with an antibody against vimentin and the pattern seen after colcemid treatment is classical for intermediate filaments. Preabsorption of the anti-hsp90 antibody with purified hsp90 prevents the immunofluorescence but preabsorption with purified p23 does not, and the reverse is the case for immunofluorescence produced by the anti-p23 antibody. These results suggest that hsp90 is able, either directly or indirectly via other proteins, to associate with both microtubules and microfilaments.
Czar
M J
MJ
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0632, USA.
Welsh
M J
MJ
Pratt
W B
WB
D000818:Animals; D000911:Antibodies, Monoclonal; D030161:Avian Proteins; D001798:Blood Proteins / Q000032:analysis; D002352:Carrier Proteins / Q000032:analysis; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000737:chemistry* / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000032:analysis; D003703:Demecolcine / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis* / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000032:analysis; D007382:Intermediate Filaments / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000032:analysis; D051381:Rats; D022021:Tacrolimus Binding Proteins; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods; D014746:Vimentin / Q000032:analysis
D000911:Antibodies, Monoclonal; D030161:Avian Proteins; D001798:Blood Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D014746:Vimentin; C093858:p22 calcium binding protein, chicken; D015638:Cytochalasin D; D022021:Tacrolimus Binding Proteins; D003703:Demecolcine