Mobility of kinetochore proteins measured by FRAP analysis in living cells.

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS ACS Applied Bio Materials Pub Date : 2022-03-01 Epub Date: 2022-01-08 DOI:10.1007/s10577-021-09678-x

Reito Watanabe, Yasuhiro Hirano, Masatoshi Hara, Yasushi Hiraoka, Tatsuo Fukagawa

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引用次数: 9

Abstract

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

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FRAP法测定活细胞中着丝点蛋白的迁移率。

在有丝分裂过程中，着丝粒对染色体的忠实分离至关重要，并通过涉及许多着丝粒蛋白的动态过程进行组装。不同的实验策略被用来理解着丝点组装过程。光漂白后荧光恢复(FRAP)分析也是揭示着丝点组装动力学的有用策略。在这项研究中，我们将荧光蛋白标记的着丝点蛋白cdna引入每个内源性位点，并对鸡DT40细胞进行了FRAP分析。着丝粒蛋白(CENP)-C在间期高度移动，但在有丝分裂期间不移动。缺乏cenp - a结合结构域的CENP-C突变体在有丝分裂期间变得可移动。与CENP-C相比，CENP-T和CENP-H在间期和有丝分裂期间都是不动的。Dsn1是Mis12复合体的一个组成部分，直接与CENP-C结合，其迁移依赖于有丝分裂过程中CENP-C的迁移。因此，我们的FRAP分析提供了着丝点如何组装的动态方面。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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