Life and Medical Sciences
Cell & Developmental Biology
Wiley InterScience Backfile Collection 1832-2000
Fluorescent derivatives of cellular proteins that retain their native characteristics have become useful probes to investigate the dynamics of specific cytoskeletal proteins. In the experiments reported here, a previously characterized fluorescent derivative of tubulin, bimane-tubulin [Wadsworth and Sloboda, 1982a], was used to investigate microtubule assembly in vitro. The results demonstrate that bimanetubulin was competent to assemble onto a variety of organizing centers in vitro, including microtubule organizing centers (MTOCs) present in homogenates of sea urchin eggs, isolated mitotic apparatuses (MAs), and lysed mitotic cells. When homogenates of fertilized sea urchin eggs containing MTOCs were incubated with bimane-tubulin at 37°C, discrete areas of linear fluorescence were observed. Only diffuse fluorescence was observed when calcium or colchicine was added to the homogenate or if the temperature was maintained at 0°C. Negative-stain electron microscopy of the fluorescent arrays revealed morphologically normal microtubules radiating from electron dense regions. When mitotic spindles, isolated in glycerol containing buffers and therefore cold stable, were incubated with bimane-tubulin, linear fluorescence was observed emanating from the spindle poles but not from the region occupied by the kinetochores. MAs incubated with bimane-labeled bovine serum albumin or bimane-labeled microtubule-associated proteins showed only diffuse fluorescence. However, when mitotic cells which were hypotonically lysed in the absence of detergents or microtubule stabilizing solvents, were perfused with bimane-tubulin intense fluorescence was observed in the asters and throughout the spindle. Two experiments suggested that the fluorescence observed in the results outlined above was due to the assembly of normal microtubules from the fluorescent subunits. First, the observed fluorescence was sensitive to cold temperataure, which is known to disassemble microtubules. Second, when the isolated, fluorescent MAs were examined by thin section electron microscopy, microtubules of normal diameter were seen. No aggregated material appeared associated with the walls of the microtubules, which might have been expected if the fluorescent protein was nonspecifically adsorbed to the microtubules. The results of these experiments demonstrate that isolated, stabilized MAs support the growth of new microtubules from the spindle poles while labile spindles, present in lysed cells, incorporate fluorescent tubulin throughout the spindle and asters. The significance of these results for hypotheses concerning microtubule assembly and disassembly during mitosis is discussed.
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