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 a gainst the two centrosome-specific proteins, pericentrin and gamma tub ulin. The inhibition of anti-pericentrin binding was measured by fluor escence imaging. Heat had the greatest effect on intact cells, followe d in sensitivity by centrosomes attached to their companion nucleus, w ith purified centrosomes being least sensitive. The centrosomal conten t of pericentrin was measured by immunoprecipitation followed by weste rn blotting. Heat caused the amount of pericentrin in the centrosomal fraction to increase, suggesting that pericentrin did not leave the ce ntrosome during heat shock. Furthermore, the pericentrin of the centro somal fraction became less soluble after heat shock, and could only be solublized by the most denaturing condition of boiling in 0.1% SDS. I mmunoelectron microscopy revealed a heat-induced increase in the elect ron-dense material comprising the pericentriolar material (PCM), consi stent with protein aggregation. Lastly, in heated cells immunoelectron microscopy demonstrated an increase in the binding of heat shock prot ein 70 (HSP70) to numerous locations throughout the cytoplasm. These d ata suggest that heat shock reduces the solubility of centrosomal and other cytoplasmic proteins, most likely through protein aggregation.