微丝束结合蛋白对胞质分裂调控分子机理的研究

Cytokinesis is the last and important step of cell division. However, the molecular mechanism regulating cytokinesis is still unclear. Understanding the cytokinetic mechanism can provide new insights and new target sites for efficient maintaining normal human cell division as well as for tumorigenesis prevention. By utilizing a new fluorescent tag and microscopic time-lapse imaging, the applicant was the first to report that during cytokinesis, actin cables/bundles were directly involved in the cytokinetic ring assembly, disassembly and its regulation. This original and preliminary finding suggested that, the key for understanding the molecular mechanism of cytokinesis could be to investigate the functions of actin cables/bundles-localizing proteins. In this proposal, which is based on the previous original technologies and findings, the applicant would like to combine different technologies such as another new bright fluorescent tag, time-lapse imaging, quantitative biological analysis, overexpression, gene deletion and mutations etc. to further understand: 1) key proteins localizing on both the actin cables/bundles and the cytokinetic ring 2) the functions and mechanism of the key proteins during cytokinesis. By these methods, it is hope that the molecular mechanism of cytokinesis can be further revealed.

胞质分裂是细胞分裂的最后一个关键环节，目前对胞质分裂调控的分子机理所知甚少。阐明其调控的分子机理，能为有效维持人类细胞正常分裂与增殖，及有效防控肿瘤发生提供新的分子机理及作用靶点。使用新的荧光标记技术和显微镜延时拍摄技术，申请人率先在国际上报道：微丝束是胞质分裂期间产生的，直接参与胞质分裂环的形成、分解和调控胞质分裂的重要调控结构。该原创的前期研究结果提示：进一步阐明定位于微丝束上的功能蛋白，及其对胞质分裂的调控功能，是揭示胞质分裂分子机理的关键。本申请项目将在上述前期原创技术与发现的基础上，结合使用新的强荧光蛋白标记、延时拍摄、量化生物学分析、过表达、基因敲除和突变体技术，进一步：1）筛选共定位于微丝束和胞质分裂环的关键功能蛋白，并证明其结构的必须性；2）对经筛选的关键蛋白在胞质分裂期间的功能及机制进行研究，以阐明相应的作用机制，从而为阐释胞质分裂的分子机理带来新的突破。

围绕微丝和微丝相关蛋白，课题组完成了研究计划里的：split mNeonGreen11相关新荧光标记技术相关载体构建、药物CK-666处理、激光共聚焦显微镜成像、延时拍摄成像、量化生物学分析、双色成像、过表达、敲降、敲除微丝结合蛋白基因（规律间隔成簇短回文重复序列）等技术，在多种微丝/肌动蛋白相关的方向进行了尝试与探索，在细胞分裂反向的细胞衰老领域及一种新型的程序性细胞死亡领域取得了阶段性进展。课题组以微丝、微丝相关蛋白和显微成像为基础，取得了以下成果：发表1篇关于微丝和微丝相关蛋白的综述文章（第1标注基金号，doi: 10.3389/fcell.2020.634849），参与1篇文章正式发表（已标注基金号），2篇文章正在整理数据准备投稿，获批1项实用新型专利，培养研究生和本科生共11名。