Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells
Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells
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DOI：
10.1101/gad.1886810
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发表时间：
2010-05-01
影响因子：
10.5
通讯作者：
Desai, Arshad
中科院分区：
文献类型：
--
作者：
Gassmann, Reto;Holland, Andrew J.;Desai, Arshad
纺锤体检查点在未连接的动粒处产生“等待后期”信号以防止后期过早开始。定位于动粒的动力蛋白被认为通过将检查点蛋白从微管附着的动粒转运到纺锤体极来沉默检查点。在整个后生动物中，动力蛋白向动粒的募集需要蛋白质Spindly。在这里，我们确定了一个保守的基序纺锤体是必不可少的动粒靶向动力蛋白。纺锤体基序突变体，内源性纺锤体耗尽后表达，通常靶向动粒，但防止动力蛋白募集。纺锤体缺失和纺锤体基序突变体，尽管它们对动粒动力蛋白的影响相似，但对染色体对齐和检查点沉默具有相反的后果。Spindly耗尽延迟染色体对齐，但Spindly基序突变体改善了这一缺陷，表明Spindly在对齐中具有动力蛋白募集独立的作用。在纺锤体耗竭中，检查点在通过动粒动力蛋白独立机制延迟对齐后沉默。相反，纺锤体基序突变体与检查点蛋白一起保留在微管连接的动粒上，导致持续的检查点信号传导。因此，动力蛋白介导的去除纺锤体从微管连接的动粒，而不是向极运输本身，是在检查点沉默的关键反应。在缺乏纺锤体的情况下，第二种机制使检查点沉默;这种机制可能是进化上古老的，因为真菌和高等植物缺乏动粒动力蛋白。
The spindle checkpoint generates a "wait anaphase'' signal at unattached kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached kinetochores to spindle poles. Throughout metazoans, dynein recruitment to kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubule-attached kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein.