Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.
Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.
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DOI：
10.1083/jcb.109.2.637
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发表时间：
1989-08
期刊：
影响因子：
--
通讯作者：
Mitchison TJ
中科院分区：
文献类型：
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作者：
Mitchison TJ
我已经合成了一种新的羧基荧光素衍生物，它不是荧光的，但可以通过暴露在365纳米的光下转化为荧光形式。这种可光激活的荧光探针共价附着在微管蛋白上，并微注射到有丝分裂组织培养细胞中，在那里它被纳入功能纺锤体。为了在有丝分裂纺锤体上产生荧光条，用狭缝微束照射中期细胞。该条在第一分钟内强度下降，可能是由于非着丝点微管的周转。剩下的荧光区，现在可能仅限于着丝点微管，以0.3-0.7微米/分钟的速度向两极移动。这一结果为着丝点微管的极向通量提供了强有力的证据。结合早期的生物素-微管蛋白结合实验（Mitchison, t.j.， L. Evans， E. Schulze, and M. Kirschner. 1986）。细胞。45:515-527)，我的结论是微管在着丝点聚合，并在中期在两极附近解聚合。讨论了这一观察结果对纺锤体结构和功能的意义。局部光激活荧光应该是跟踪活细胞内分子动力学的一种普遍有用的方法。
I have synthesized a novel derivative of carboxyfluorescein that is nonfluorescent, but can be converted to a fluorescent form by exposure to 365-nm light. This photoactivable, fluorescent probe was covalently attached to tubulin and microinjected into mitotic tissue culture cells, where it incorporated into functional spindles. To generate a fluorescent bar across the mitotic spindle, metaphase cells were irradiated with a slit microbeam. This bar decreased in intensity over the first minute, presumably due to turnover of nonkinetochore microtubules. The remaining fluorescent zones, now presumably restricted to kinetochore microtubules, moved polewards at 0.3-0.7 microns/min. This result provides strong evidence for polewards flux in kinetochore microtubules. In conjunction with earlier biotin-tubulin incorporation experiments (Mitchison, T. J., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527), I conclude that microtubules polymerize at kinetochores and depolymerize near the poles throughout metaphase. The significance of this observation for spindle structure and function is discussed. Local photoactivation of fluorescence should be a generally useful method for following molecular dynamics inside living cells.