A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics.
A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics.
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DOI：
10.7554/elife.10113
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发表时间：
2015-10-06
期刊：
影响因子：
7.7
通讯作者：
Rice LM
中科院分区：
文献类型：
--
作者：
Geyer EA;Burns A;Lalonde BA;Ye X;Piedra FA;Huffaker TC;Rice LM
微管动态不稳定性取决于聚合αβ-微管蛋白亚基的GTdR活性，当它们移入和移出微管时，所述聚合αβ-微管蛋白亚基通过至少三种不同的构象循环。这种构象循环如何促进微管的生长、收缩和转换仍然是未知的。在这里，我们报告了αβ-微管蛋白中的埋藏突变产生了具有显著降低的收缩率和灾难频率的微管。该突变通过抑制晶格中通常响应GTP水解而发生的构象变化而引起这些效应，而不会可检测地改变未聚合的αβ-微管蛋白的构象。因此，该突变减弱了αβ-微管蛋白的构象循环和GTP酶循环之间的耦合。通过显示突变主要影响后GTdR的微管的构象和动力学性质，我们的数据显示，在晶格中对GDP的变构反应的强度决定了灾难的频率和快速收缩的严重程度。被称为微管的蛋白质细丝帮助细胞内的货物移动。http://dx.doi.org/10.7554/eLife.10113.001染色体包含着细胞的遗传蓝图，是微管最珍贵的货物。在细胞分裂之前，微管从分裂细胞的末端向中间生长，在那里它们附着在沿着中心线排列的染色体上。然后微管收缩并将染色体拖回细胞的两端。这允许每个新细胞获得每个染色体的一个拷贝。当微管生长时，一种叫做三磷酸鸟苷（或GTP）的分子附着在微管末端的蛋白质上。这就像一个帽子，保护微管收缩。后来，一个化学反应将GTP转化为GDP（鸟苷二磷酸的简称）。如果没有保护性的GTP帽，微管会迅速收缩。与此同时，构成微管的蛋白质也会改变形状。在微管中，当GTP附着时，蛋白质采取直的形状。当GDP附着时，蛋白质在微管中倾向于不同的形状。然而，目前还不清楚这些形状变化是否或如何有助于微管的生长或收缩。盖耶等人现在展示了这种形状变化如何影响微管收缩，首先确定了酵母微管蛋白中的突变，该突变导致蛋白质即使在GDP附着时也保持直的。接下来，强大的显微镜被用来制作突变微管的延时视频。这使得盖耶等人能够观察突变微管的行为，并将其与正常微管的行为进行比较。实验表明，突变的微管比典型的微管更不可能开始收缩。突变的微管也收缩得更慢。这些发现表明，形状的变化控制着收缩的速度和进入收缩阶段的频率。这些关于微管生长和收缩控制的新细节可能有助于科学家研究健康细胞和癌细胞中细胞分裂的发生方式。DOI：http://dx.doi.org/10.7554/eLife.10113.002网站
Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking. DOI: http://dx.doi.org/10.7554/eLife.10113.001 Protein filaments called microtubules help move cargo around inside cells. Chromosomes, which contain the cell’s genetic blueprints, are the microtubule’s most precious cargo. Before a cell divides, microtubules grow from the ends of the dividing cell towards the middle, where they attach to the chromosomes that are lined up along the centerline. Then the microtubules shrink and drag the chromosomes back to the opposite ends of the cell. This allows each of the new cells to get one copy of each chromosome. When the microtubules are growing, a molecule called guanosine triphosphate (or GTP) is attached to the proteins at the end of the filament. This acts like a cap and protects the microtubule from shrinking. Later a chemical reaction converts GTP into GDP (short for guanosine diphosphate). Without the protective GTP cap, the microtubule quickly shrinks. At the same time, the proteins that make up the microtubule also change shape. In the microtubule, the proteins adopt a straight shape when GTP is attached. The proteins favor a different shape in the microtubule when GDP is attached. However, it is unclear if or how these shape changes contribute to how a microtubule grows or shrinks. Geyer et al. now show how this shape shifting can influence microtubule shrinking, by first identifying a mutation in yeast microtubule proteins that cause the proteins to remain straight even when GDP is attached. Next, powerful microscopes were used to make time-lapse videos of the mutated microtubules. This allowed Geyer et al. to observe how the mutated microtubules behaved and compare this to the behavior of normal microtubules. The experiments revealed that the mutated microtubules were less likely to begin shrinking than typical microtubules. The mutated microtubules also shrunk more slowly. These findings indicate that the shape changes control the speed of shrinking and frequency of entering the shrinking phase. These new details about the control of microtubule growth and shrinkage may help scientists studying how cell division happens in both healthy and cancerous cells. DOI: http://dx.doi.org/10.7554/eLife.10113.002