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分裂GFP技术的AlphaFold2指导工程使得跨物种内源性管素的标记能够实现,同时保持功能

Kaiming Xu¹, Zhiyuan Li², Linfan Mao³

¹Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, McGovern Institute for Brain Research, State Key Laboratory of Membrane Biology, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China.

|August 19, 2024

概括

研究人员开发了一种新的方法,在体内以光标记素同型. 这种技术允许详细研究微管子动力学和管病变,而不会破坏细胞功能.

科学领域:

细胞生物学细胞生物学
分子生物学分子生物学
生物物理学的生物物理.

背景情况:

微管 (MT) 动态对于细胞功能至关重要.
现有的体内MT成像方法在区分素同型时存在局限性.
图布林同型变异有助于各种MT功能.

研究的目的:

开发一种最少侵入性的方法,用于体内光标记内源性突类异型的体内光.
为了实现MT及其特定异型的高保真可视化.
为了研究突突变和动态的功能后果.

主要方法:

利用AlphaFold2管道进行蛋白质结构预测.
设计了一种新的16-氨基酸链接器,使用sfGFP11进行分裂-sfGFP标签.
整合了标签系统到H1-S2循环的tubulin.
在Caenorhabditis elegans,人类细胞和小鼠卵细胞中应用了该技术.

主要成果:

在体内成功实现了微侵袭性,同型特异性管素标记.
证明标签策略不会影响MT动态,胚胎发育或纤毛发育.
可视化了内源性突同型和具有高保真度和最小背景光度的致病性突同型.
验证了该技术在不同物种和生物环境中的实用性.

结论:

开发的光标签策略为研究蛋白动力学和同型特异性功能提供了一个强大的新工具.
这种方法为调查管病变和相关疾病提供了范式的转变.
该技术的广泛适用性增强了我们对微管体生理学的理解.

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