Myosin 10 uses its MyTH4 and FERM domains differentially to support two aspects of spindle pole biology required for mitotic spindle bipolarity.
Myosin 10 uses its MyTH4 and FERM domains differentially to support two aspects of spindle pole biology required for mitotic spindle bipolarity.
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肌球蛋白 10 不同地使用其 MyTH4 和 FERM 结构域来支持有丝分裂纺锤体双极性所需的纺锤体极生物学的两个方面。
DOI：
10.1101/2023.06.15.545002
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Hammer,JohnA
中科院分区：
文献类型：
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作者：
Yim,Yang-In;Pedrosa,Antonio;Wu,Xufeng;Chinthalapudi,Krishna;Cheney,RichardE;Hammer,JohnA
肌球蛋白10（Myo 10）具有分别凭借其整合素结合FERM结构域和微管结合MyTH 4结构域将肌动蛋白丝连接到基于整合素的粘附和微管的能力。在这里，我们使用Myo 10敲除细胞来定义Myo 10对维持纺锤体双极性的贡献，并通过互补作用来定量其MyTH 4和FERM结构域的相对贡献。Myo 10基因敲除的HeLa细胞和小鼠胚胎成纤维细胞（MEFs）都表现出多极纺锤体频率的显著增加。非同步化中期细胞的染色显示，缺失额外中心体的敲除MEF和敲除HeLa细胞中纺锤体多极化的主要驱动因素是中心粒周围物质（PCM）片段化，其产生γ-微管蛋白阳性无中心粒灶，作为额外的纺锤体极。对于具有额外中心体的HeLa细胞，Myo 10耗尽通过损害额外纺锤体极的聚集进一步加剧纺锤体多极化。互补实验表明，Myo 10必须与整合素和微管相互作用，以促进PCM/极的完整性。相反，Myo 10促进额外中心体聚集的能力只需要它与整合素相互作用。重要的是，Halo-Myo 10敲入细胞的图像显示，肌球蛋白在有丝分裂期间仅定位于粘附收缩纤维内。基于这些和其他结果，我们得出结论，Myo 10促进PCM/极的完整性在一定的距离，并通过促进收缩纤维为基础的细胞粘附，这可能提供了一个锚驱动极聚焦的微管为基础的力量，它有利于额外的中心体聚类。
Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10’s contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates γ-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10’s ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.