Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera

Formation of a novel structure, the aggresome, has been proposed to represe nt a general cellular response to the presence of misfolded proteins (Johns ton, J.A,, C.L, Ward, and R,R. Kopito, 1998. J, Cell Biol. 143:1883-1898; W igley, W. C., R.P. Fabunmi, M.G, Lee, C,R, Marine, S, Muallem, G.N. DeMarti no, and P.J, Thomas, 1999. J. Cell Biol. 145:481-490). To test the generali ty of this finding and characterize aspects of aggresome composition and it s formation, we investigated the effects of overexpressing a cytosolic prot ein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP- 250 within the aggresome appeared not to be ubiquitinated. In vivo time-lap se analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region whe re they remain as distinct but closely apposed particulate structures. Over expression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting th at the minus-end-directed motor activities of cytoplasmic dynein are requir ed for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtub ules, However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by sol uble, nonubiquitinated proteins as well as by integral transmembrane ubiqui tinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.