Fig. 6. Inhibition or mislocalization of katanin reduces the rate of nocodazole-mediated spindle disassembly. CV-1 cells with an integrated YFP--tubulin transgene were transiently transfected with different CFP-fusion constructs and mounted in a perfusion chamber. Transfected cells were identified by CFP fluorescence and spindle disassembly was monitored by shuttered, time-lapse imaging of YFP-fluorescence after perfusion with nocodazole. The ratio of spindle fluorescence/cytoplasmic fluorescence was determined for each frame of each YFP-time-lapse sequence and was normalized to a scale of 100% initial fluorescence ratio to 0% of initial fluorescence ratio as described in Materials and Methods. (A) Individual plots of decreasing YFP-fluorescence ratio over time in individual nocodazole-treated mitotic cells. , CFP; , CFP-P loop K-A p60. To compare the effects of different CFP fusions on spindle disassembly rate, the time required to achieve 80% loss of normalized spindle intensity ratio (20% of initial ratio in A) was determined for each half spindle in each time lapse sequence. The number of half spindles exhibiting different ranges of time to 80% loss of spindle intensity ratio are displayed as histograms in B-F. Note that cells expressing CFP or CFP-N-P loop K-A p60 exhibited disassembly times similar to those of untransfected cells (B,D,F). By contrast, 60-65% of cells expressing CFP-P loop K-A p60 or CFP-con80 exhibited spindle disassembly times slower than ever observed in untransfected or CFP-expressing cells (C,E). Grey bars indicate disassembly times observed in untransfected and CFP-transfected control cells. White bars indicate times slower than ever observed in control cells.

Return to article