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Journal of Cell Science, Vol 108, Issue 1 343-356, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
KT Trevor, JG McGuire and EV Leonova
Center for Molecular Biology, Wayne State University, Detroit, Michigan, USA.
SW-13 cells that lack cytoplasmic intermediate filaments (IFs) were stably transfected with a human vimentin cDNA expression vector. Isolated subclones displayed two prevalent patterns of vimentin distribution as observed by indirect immuno-localization: (1) cytoplasmic filaments characteristic of a vimentin IF network; and (2) a distinct, juxtanuclear focus with limited filamentous extensions. Comparative analysis of two subclones that uniquely segregated these patterns of vimentin organization indicated that vimentin accumulated as a perinuclear focus in cells that expressed a 4-fold lower level of the protein. The observed variation in cellular organization was not due to detectable differences in vimentin protein modification, as determined by two-dimensional gel analysis. Increasing the amount of vimentin in a low expressing clone by a secondary transfection with human or mouse vimentin cDNA resulted in well-dispersed, cytoplasmic filaments, suggesting that the distinct juxtanuclear organization of vimentin arose due to lower cellular vimentin levels. Employing anti-gamma-tubulin and anti-vimentin antibodies, dual immunofluorescence together with confocal microscopy revealed that the juxtanuclear focus of vimentin was located in the centrosomal region. Electron microscopy showed a spheroidal, filamentous structure with at least some filaments closely associated with the pericentriolar material (PCM). Because vimentin IF organization is at least partially dependent on microtubules, the effects of nocodazole and taxol on perinuclear vimentin foci were examined. Neither drug affected the juxtanuclear localization of foci, although taxol (10 microM, 5 hours) caused a release of pericentriolar gamma-tubulin from the nuclear region in 50-60% of the cells. These studies indicate that lower, in vivo, levels of vimentin fail to form extended IFs but rather are organized as a perinuclear aggregate. Moreover, the PCM of the centrosome appears to possess attachment sites for vimentin IFs.
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