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Fig. 4. FCPT inhibited poleward microtubule movement and spindle-pole polymerization. (A) FCPT inhibition of poleward microtubule movement was titratable. Localizations of speckle level Alexa-Fluor-647-conjugated tubulin on Xenopus egg-extract spindles with 0, 40 and 200 µM FCPT. The kymographs (measured from colored broken lines) of image sequences obtained over the course of approximately 3.5 minutes (with one image every 3 seconds) show that poleward movement is only inhibited upon addition of 200 µM FCPT. Intermediate concentrations of FCPT had intermediate microtubule poleward rates (see B). Untreated control spindles had a microtubule poleward movement of approximately 1.9 ± 0.2 µm/minute (± s.e.m.). The spindle is outlined with a white line. Scale bar: 5 µm. (B) The reduction of spindle-pole density (as measured by the ratio of spindle-pole:mid-spindle fluorescence) only occurred at FCPT concentrations sufficient to completely inhibit poleward microtubule movement (200 µM). Error bars, s.e.m.; n=3 for each point. (C) Alexa-Fluor-488-EB1 localized on control and FCPT-treated spindles. FCPT removed spindle-pole localization of EB1 compared with controls (imaged approximately 4-5 minutes after FCPT addition; The spindle is outlined with a white line). See supplementary material Movies 1, 2. Scale bar: 10 µm.
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