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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis
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Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis

机译：连续的polo样激酶1活性调节有丝分裂期间的扩散，以维持中心体的自组织

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摘要

Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1-containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, causes defects in centrosome structure, which recover when activity is restored. Our findings imply that continuous PLK1 activity during mitosis maintains centrosome self-organization by a mechanism dependent on its reaction and diffusion, suggesting a model for the formation of stable mitotic structures using dynamic protein kinases.
机译：尚不清楚诸如中心体之类的有丝分裂结构是否可以通过其动态蛋白质成分的调节迁移率而自组织。在这里，我们结合荧光光谱和化学遗传学研究活细胞中polo样激酶1（PLK1）的扩散，这是一种在有丝分裂开始时对中心体成熟至关重要的酶。功能性EGFP-PLK1融合蛋白的细胞质扩散与其在细胞周期中酶活性的已知变化呈负相关。使用化学遗传学对EGFP-PLK1的特异性抑制作用增强了流动性，点突变也使负责底物识别和催化作用的polo-box或激酶结构域失活。 EGFP-PLK1扩散跨活细胞的空间分布图，使用光栅图像相关光谱法和线扫描，可检测中心体中低迁移率区域。这些区域表现出增加的瞬时递归EGFP-PLK1结合的特征，与稳定的含EGFP-PLK1的复合物在细胞质中的扩散不同。即使在中心体成熟后，化学遗传抑制有丝分裂EGFP-PLK1的活性也会导致中心体结构的缺陷，并在恢复活性时恢复。我们的发现暗示有丝分裂期间连续的PLK1活性通过依赖于其反应和扩散的机制维持着中心体的自组织，这提示了使用动态蛋白激酶形成稳定的有丝分裂结构的模型。

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