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Journal of Cell Science, Vol 113, Issue 4 597-609, Copyright © 2000 by Company of Biologists
JOURNAL ARTICLES |
RV Silverman-Gavrila and A Forer
Biology Department, York University, Toronto, Ontario M3J 1P3, Canada.
We studied the effects of various drugs on the poleward flux of tubulin in kinetochore microtubules in metaphase-I crane-fly spermatocytes. We used as a measure of tubulin flux a 'gap' in acetylation of kinetochore microtubules immediately poleward from the kinetochore; the 'gap' is caused by a time lag between incorporation of new tubulin subunits at the kinetochore and subsequent acetylation of those subunits as they flux to the pole. We confirmed that the 'gap' is due to flux by showing that the 'gap' disappeared when cells were treated briefly with the anti-tubulin drug nocodazole, which decreases microtubule dynamics. The 'gap' disappeared when cells were treated for 10 minutes with anti-actin drugs (cytochalasin D, latrunculin B, swinholide A), or with the anti-myosin drug 2,3-butanedione 2-monoxime. The 'gap' did not disappear when cells were treated with the actin stabilizing drug jasplakinolide. We studied whether these drugs altered spindle actin. We used fluorescent phalloidin to visualize spermatocyte F-actin, which was associated with kinetochore spindle fibers as well as the cell cortex, the contractile ring and finger-like protrusions at the poles. Spindle F-actin was no longer seen after cells were treated with cytochalasin D, swinholide A or a high concentration of latrunculin B, whereas a low concentration of latrunculin B, which did not completely remove the 'gap', caused reduced staining of spindle actin. Neither 2,3-butanedione 2-monoxime nor jasplakinolide altered spindle actin. These data suggest that an actomyosin mechanism drives the metaphase poleward tubulin flux.
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