Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body.
Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body.
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DOI：
10.1091/mbc.e12-03-0223
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发表时间：
2012-06
影响因子：
3.3
通讯作者：
Huffaker TC
中科院分区：
文献类型：
--
作者：
Bergman ZJ;Xia X;Amaro IA;Huffaker TC
动力蛋白抑制剂She 1的缺失导致微管从酵母纺锤体极体脱离的速率增加。这些脱离事件的分子性质的特点，它表明，它们的频率取决于微管被锚定到SPB的方式。研究了She 1的作用机理。在大多数细胞中，微管的组织由微管组织中心决定，微管组织中心使微管组装成核并锚定它们的负端。在缺乏She 1的酿酒酵母细胞中，细胞质微管以高速率从纺锤体极体分离。增加的脱离率取决于动力蛋白的活性，支持以前的证据表明，She 1抑制动力蛋白。在G1期的脱落率高于中期细胞，我们发现，这主要是由于在这些阶段的细胞周期中的微管附着到纺锤体极体的强度的差异。分离的微管的负端通过γ-微管蛋白和Spc 72的存在而稳定，Spc 72是一种将γ-微管蛋白复合物拴在纺锤体极体上的蛋白质。Spc 72-Kar 1融合蛋白抑制G1细胞的分离，表明这两种蛋白之间的相互作用对微管锚定至关重要。She 1的过表达抑制了动力蛋白的加载，但不是动力蛋白，微管加端。此外，She 1在体外直接与微管结合，因此它可能与dynactin竞争进入微管。总体而言，这些结果表明，抑制动力蛋白活性的She 1是重要的，以防止过度分离的细胞质微管，特别是在G1细胞。
Loss of the dynein inhibitor She1 causes increased rates of microtubule detachment from the yeast spindle pole body. The molecular nature of these detachment events is characterized, and it is shown that their frequency depends on the way in which microtubules are anchored to the SPB. The mechanism of She1 action is investigated. The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72–Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.