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Figure 2. Analysis of the mechanism of nocodazole action. (A–C) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cells extracts supplemented with 5 μM pure tubulin in the presence of: (A) ATP-regenerating system and 5 μM okadaic acid; (B) ATP-regenerating system, 5 μM okadaic acid and 20 μM nocodazole; and (C) ATP-regenerating system, 5 μM okadaic acid, 20 μM nocodazole, and 20 μM tubulin. Bar, 10 μm.
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Figure 3. Persistent poisoning of microtubule assembly by CD-complex in reconstituted permeabilized cells. (A–F) Double immunostaining of permeabilized NIH3T3 cells using Tyr-tubulin antibody YL1/2, and Glu-tubulin antibody. Permeabilized NIH3T3 cells were incubated for 5 min at 34°C with TTL− cell extracts supplemented with ATP-regenerating system, and with 5 μM purified Glu-tubulin after a 15-min preincubation at the same temperature under the following conditions: (A–B) permeabilization buffer alone; (C–D) 5 μM CD complex; (E–F) 5 μM colchicine plus 5 μM BSA. In the absence of cell preincubation with CD complex, the tubulin from extracts formed microtubule extensions (B and F) whose formation is extensively inhibited in cells preincubated with CD complexes (C and D). Note that these extensions are stained by Tyr-tubulin antibody due to the presence of low levels of Tyr-tubulin in TTL− cell extracts (A and E), and that inhibition of tubulin assembly by CD complex induces some background staining, including nonspecific nuclear staining (C and D). Bar, 10 μm.
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Figure 4. Inhibition of nocodazole action in the presence of added CD complex. (A–B) Immunostaining of interphasic permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin, ATP-regenerating system and 5 μM okadaic acid in the absence of CD complex (A), or in the presence of 5 μM CD complex (B). (C–D) Immunofluorescence analysis of the microtubule content of control (C) or CD complex–supplemented (D) cell extracts. Cell extracts supplemented with 10 μM tubulin and ATP-regenerating system were incubated for 30 min at 30°C. Aliquots were then incubated for 30 min in the absence (C) or presence of 5 μM CD complex (D). Microtubules were then cross-linked, spun onto coverslips, and immunostained as described in Saoudi et al. (1995). (E–F) Immunostaining of interphase-permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cells were permeabilized and then incubated for 30 min at 34°C with NIH3T3 cell extracts supplemented with 5 μM pure tubulin, ATP-regenerating system, 5 μM okadaic acid, and 20 μM nocodazole in the absence of CD complex (E) or the presence of 5 μM CD complex (F). Bar, 10 μm.
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Figure 5. Assay of microtubule turnover in permeabilized NIH3T3 cells reconstituted with mammalian interphase cell extracts. Permeabilized NIH 3T3 cells were incubated at 34°C with TTL− cell extracts containing 1 μM purified Glu-tubulin, ATP-regenerating system, and 5 μM okadaic acid. (A–B) Double immunostaining of interphase microtubule arrays in permeabilized cells with Tyr-tubulin monoclonal antibody YL1/2 (green) and Glu-tubulin antibody (red) after a 30-min incubation with cell extracts. (A) The tubulin from the extract forms short tails on preexisting interphase microtubules, but fails to invade the interphase network. (B) 2× enlargement of a peripheral zone of the cell showing the Glu-tubulin tails at the ends of Tyr-microtubules. (C) Immunostaining of interphase permeabilized NIH3T3 cells with tubulin antibody mAb YL1/2. Cell extracts were supplemented with 20 μM nocodazole; this control experiment shows that microtubules were nocodazole-sensitive during microtubule turnover assay. Bars: (A and C) 10 μm; (B) 5 μm. (D) Quantitative analysis of microtubule turnover. Cells were incubated with TTL− cell extracts as described in A. At the indicated time points, cells were fixed and stained with Tyr-tubulin antibody, and the amount of Tyr-tubulin in interphase microtubule networks was quantified as described in Materials and Methods.
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Figure 6. Reconstitution of microtubule turnover in permeabilized cells supplemented with Xenopus egg extracts. Permeabilized TTL− cells were incubated at 34°C with interphase Xenopus egg extracts for the indicated periods of time. Cells were then double-stained using Glu-tubulin antibody (A, C, and E) and Tyr-tubulin mAb YL1/2 (B, D, and F). Within 15 min, most of the original interphase microtubules had depolymerized. At intermediate stages of microtubule turnover, Glu-microtubule fragments, apparently detached from the centrosome, were visible (C). The Tyr-tubulin from the soluble tubulin pool initially formed tails and then invaded the whole interphase network (B, D, and F). Image superposition showed that microtubule tails grew on the ends of preexisting microtubules (data not shown). Bar, 10 μm.
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Figure 7. Reconstitution of microtubule turnover in permeabilized cells involves disassembly of fragmented microtubules. TTL− permeabilized cells were labeled with Glu-tubulin antibody and secondary Cy 3 goat anti–rabbit IgG antibody in warm permeabilization buffer without cell fixation. Cells were washed with the same buffer, and then the antibody-labeled cells were incubated with interphase Xenopus egg extracts (A) or with NIH3T3 cell extracts supplemented with 1 μM purified Tyr-tubulin, ATP-regenerating system, and 5 μM okadaic acid (B). (A; left) Successive images of the same cell were taken at the indicated time points to visualize the pathway of disassembly of the original interphase Glu-microtubules in the presence of Xenopus egg extracts. Images showed microtubule fragmentation and segmental disassembly. Bar, 5 μm. The zero time point corresponds to the beginning of observation and not to the beginning of incubation (∼5-min difference). Therefore, this time point differs from the zero time point in Fig. 6 (fixed cell experiments). (A; right) Videomicroscopic images (4× enlarged) of disassembling original Glu-interphase microtubules. A randomly selected part of a permeabilized TTL− cell labeled with Glu-antibody and reconstituted with Xenopus egg extracts was observed using videomicroscopy over a period of 20 s. Successive images showed apparent microtubule breakage followed by variable extents of microtubule depolymerization. Arrows show microtubule breakage events. Zero time point is as above. Bar, 100 nm. (B) Control experiment showing apparent absence of microtubule severing in permeabilized TTL− cells incubated with soluble NIH3T3 cell extracts. Bar, 5 μm.
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Figure 8. Microtubule turnover in permeabilized cells reconstituted with Xenopus egg extracts involves minus end directed depolymerization. (A) Permeabilized TTL− cells were incubated at 34°C with Xenopus interphase egg extracts supplemented with 5 μM CD complex. At the indicated times, cells were double-labeled using Glu-tubulin antibody (left) and Tyr-tubulin monoclonal antibody YL1/2 (right). Within 15 min, most interphase microtubules had depolymerized. No polymerization of the tubulin from the extracts was observed in the presence of CD complex (right), showing blockage of microtubule assembly by CD complexes. Bar, 10 μm. (B) Videomicroscopy images of disassembling original interphase Glu-microtubules. Interphase microtubules of TTL− permeabilized cells were labeled with Glu-tubulin antibody and secondary Cy3 conjugated goat anti–rabbit IgG antibody in warm permeabilization buffer without cell fixation. Cells were washed with the same buffer, and then the antibody-labeled cells were incubated with interphase Xenopus egg extracts supplemented with 5 μM CD complex. The pattern and kinetics of Glu-microtubule disassembly were similar to the ones observed during microtubule turnover (see Fig. 7 A). This result suggests that disassembly proceeds mainly from microtubule minus ends, during turnover (see text). Bar, 10 μm.
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Figure 9. Microtubule turnover in intact cells. TTL− cells were microinjected with pure TTL. At the indicated time points after microinjection, cells were fixed and double-labeled using Tyr-tubulin monoclonal antibody YL1/2 (A, C, E, and G) and Glu-tubulin antibody (B, D, F, and H). Arrows indicate microinjected cells. During microtubule turnover, Tyr-microtubules formed radial arrays arising from centrosomes (C, E, and G), whereas Glu-microtubules appeared as fragments detached from centrosomes not centered as Tyr-microtubules (D, F, and H). (D, insert) Image of a severed microtubule (5×). Bar, 10 μm.
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Figure 10. Microinjection of CD complex in intact cells induces rapid microtubule depolymerization. NIH3T3 cells were injected with a mixture of nonreactive rabbit IgGs and with 25 μM CD complex. Cells were double-labeled using goat rabbit IgG antibody (A and C) and Tyr-tubulin monoclonal antibody YL1/2 (B and D). After 5 min, background staining of depolymerized free tubulin was evident in microinjected cells (A and B), and microtubule depolymerization was complete after 15 min (C and D). Bar, 10 μm.
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