Heat shock causes protein aggregation and reduced protein solubility at the centrosome and other cytoplasmic locations.
Heat shock markedly inhibited centrosome staining by antisera raised against the two centrosome-specific proteins, pericentrin and gamma tubulin. The inhibition of anti-pericentrin binding was measured by fluorescence imaging. Heat had the greatest effect on intact cells, followed in sensitivity by centrosomes attached to their companion nucleus, with purified centrosomes being least sensitive. The centrosomal content of pericentrin was measured by immunoprecipitation followed by western blotting. Heat caused the amount of pericentrin in the centrosomal fraction to increase, suggesting that pericentrin did not leave the centrosome during heat shock. Furthermore, the pericentrin of the centrosomal fraction became less soluble after heat shock, and could only be solubilized by the most denaturing condition of boiling in 0.1% SDS. Immunoelectron microscopy revealed a heat-induced increase in the electron-dense material comprising the pericentriolar material (PCM), consistent with protein aggregation. Lastly, in heated cells immunoelectron microscopy demonstrated an increase in the binding of heat shock protein 70 (HSP70) to numerous locations throughout the cytoplasm. These data suggest that heat shock reduces the solubility of centrosomal and other cytoplasmic proteins, most likely through protein aggregation.
Vidair
C A
CA
University of California, San Francisco 94143-0806, USA.
Huang
R N
RN
Doxsey
S J
SJ
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000941:Antigens / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D018385:Centrosome / Q000378:metabolism*; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006358:Hot Temperature; D016253:Microscopy, Immunoelectron; D017830:Octoxynol / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D012995:Solubility; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
D000906:Antibodies; D000941:Antigens; D018840:HSP70 Heat-Shock Proteins; D014404:Tubulin; C086178:pericentrin; D017830:Octoxynol