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Katanin inhibition prevents the redistribution of -tubulin at mitosis

Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA

View larger version (128K): [in a new window]   Fig. 5. Inhibition of katanin by CFP-P loop K-A p60 reduces the rate of nocodazole-mediated spindle disassembly. CV-1 cells with an integrated YFP--tubulin transgene were transiently transfected with CFP-P loop K-A p60 and mounted in a perfusion chamber. Transfected cells were identified by CFP fluorescence (A) and spindle disassembly was monitored by shuttered, time-lapse imaging of YFP-fluorescence (B-F) after perfusion with nocodazole. Note that the spindle in the untransfected cell (bottom right) shortened and disappeared rapidly, whereas the spindle in the CFP-P loop K-A p60-expressing cell (top left) shortened and faded much more slowly.

View larger version (38K): [in a new window]   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.

View larger version (111K): [in a new window]   Fig. 1. GFP-P loop K-A p60 inhibits the in vitro microtubule-severing activity of wild-type p60. Fluorescence images were captured after taxol-stabilized, tetra-methyl rhodamine labeled microtubules were perfused with different protein solutions containing Mg2+-ATP, incubated for 4 minutes, then fixed with glutaraldehyde. (A) No katanin. (B) 0.1 µM 6his-wt-p60 pre-bound with 0.1 µM GST-con80. (C) 0.1 µM 6his-wt-p60 pre-bound with 0.1 µM GST-con80 + 0.4 µM GFP-P loop K-A p60. (D) 0.1 µM 6his-wt-p60 pre-bound with 0.1 µM GST-con80 + 0.4 µM heat-inactivated GFP-P loop K-A p60.

View larger version (17K): [in a new window]   Fig. 2. The area occupied by -tubulin at prometaphase spindle poles is dependent on polymerized microtubules. CV-1 cells were treated for 30 minutes with either nocodazole added as a stock solution dissolved in DMSO (20 µM nocodazole, 0.1% DMSO final concentration) or with 0.1% DMSO only. Cells were fixed and stained with an anti--tubulin monoclonal antibody. Prometaphase cells were identified by morphology of DAPI-stained chromosomes and the area of increased -tubulin staining intensity was determined as described in Materials and Methods. The histogram shows the fraction of cells with -tubulin areas falling within each range of values shown.

View larger version (34K): [in a new window]   Fig. 3. Katanin inhibition reduces the area occupied by -tubulin at prometaphase spindle poles. (A-D) CV-1 cells were transfected with plasmids encoding GFP fusions to the dominant katanin inhibitor, P loop K-A p60, the weak katanin inhibitor, N-P loop K-A p60, or GFP alone. Cells were fixed and stained with an anti--tubulin monoclonal antibody within 24 hours of transfection or within 12 hours of release from a thymidine block to ensure that mitotic cells were in their first mitosis after expression of the GFP-fusion. The fluorescence micrographs show anti--tubulin staining of representative prometaphase cells expressing GFP (A) or GFP-P loop K-A p60 (B). The histograms in C-E show the fraction of prometaphase cells with -tubulin areas falling within each range of values shown. P indicates the significance of the difference between control (GFP alone) and experimental populations as determined with the Mann-Whitney test. (E) Affinity-purified anti-p60 katanin antibodies or a control IgG were introduced into CV-1 cells by Chariot mediated protein transfection (see Materials and Methods). Cells were fixed 4 hours after initiation of the antibody treatment and prometaphase cells containing significant cytoplasmic IgG concentrations were identified by DAPI-stained chromosome morphology and anti-rabbit IgG staining. Areas of increased -tubulin staining intensity were analyzed as in C and D.

View larger version (25K): [in a new window]   Fig. 4. Prolonged mislocalization of katanin from spindle poles results in a reduction of the -tubulin focus at prometaphase spindle poles. (A,B). CV-1 cells were transfected with plasmids encoding a GFP fusion to the C-terminal, con80, domain of p80 katanin, which causes endogenous p60 to mislocalize from spindle poles to the cytoplasm, or GFP alone. Cells were fixed and stained with an anti--tubulin monoclonal antibody either 20 hours (A) or 48 hours (B) post-transfection. The histogram shows the fraction of cells with -tubulin areas falling within each range of values shown. P indicates the significance of the difference between control (GFP alone) and experimental populations as determined with the Mann-Whitney test.

View larger version (18K): [in a new window]   Fig. 7. Katanin mislocalization does not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching (FRAP). Mitotic CV-1 cells expressing an integrated YFP-tubulin gene and transiently expressing either dsRed2-con80, which mislocalizes endogenous katanin from spindle poles, or dsRed2 alone were located and subjected to FRAP analysis. One half spindle was completely photobleached with the krypton/argon laser of a laser scanning confocal microscope. Recovery of fluorescence due to incorporation of unbleached YFP-tubulin was monitored by time-lapse confocal microscopy. Examples of recovery by two individual spindles are shown. I(bleached/unbleached) is the ratio of fluorescence intensity of the bleached half spindle to that of the unbleached half spindle. t-p is the time to reach 50% recovery to the apparent plateau of recovery. t is the predicted time to reach 50% recovery to an I(bleached/unbleached) ratio of 1. Averages with standard deviations were determined from 10 (dsRed2) or 13 (dsRed2-con80) spindles.