Saoudi Y, Fotedar R, Abrieu A, Dorée M, Wehland J, Margolis R L, Job D
CEA-Grenoble, Département de Biologie Moléculaire et Structurale, Laboratoire du Cytosquelette, INSERM Unité 366, 38054 Grenoble cedex 9, France.
J Cell Biol. 1998 Sep 21;142(6):1519-32. doi: 10.1083/jcb.142.6.1519.
Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end-directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
通透细胞中的微管缺乏动态活性，并且对诸如诺考达唑等解聚药物不敏感。利用这个模型系统，我们已经建立了在补充各种细胞提取物时，通透的间期细胞中微管动力学逐步重建的条件。当在存在蛋白磷酸酶抑制剂的情况下，向通透细胞补充哺乳动物细胞提取物时，微管对诺考达唑变得敏感。诺考达唑诱导的解聚从微管正端开始，而微管负端保持无活性。这种对诺考达唑敏感的微管不表现出亚基周转。相比之下，当向通透细胞补充非洲爪蟾卵提取物时，微管会积极地周转。这涉及通过微管片段化持续产生游离的微管负端。新产生的负端显然作为微管解聚的位点，而微管正端发生净微管聚合。我们提供证据表明，在完整细胞的全细胞水平上发生了类似的微管片段化和负端定向解聚。这些数据表明，类似于体内观察到的动力学的微管动力学可以在通透细胞中重建。这个模型系统应该为体外测定提供手段，以鉴定在调节微管动力学中重要的分子。此外，我们的数据支持最近的工作，表明微管踏车运动是微管周转的一个重要机制。
