优化的印度麂细胞着丝粒纤维成熟的活细胞分析方案。

Optimized protocol for live-cell analysis of kinetochore fiber maturation in Indian muntjac cells.

机构信息

Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Cell Division Group, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal.

出版信息

STAR Protoc. 2023 Mar 17;4(1):102011. doi: 10.1016/j.xpro.2022.102011. Epub 2023 Jan 12.

DOI:10.1016/j.xpro.2022.102011

PMID:36640366

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9846000/

Abstract

Here, we take advantage of the low chromosome number (2N=6) and distinctively large kinetochores of female Indian muntjac cells to investigate the molecular mechanism underlying k-fiber maturation. We describe steps for monitoring kinetochore-microtubule dynamics over time. Specifically, we detail the combination of live-cell super-resolution CH-STED microscopy of microtubule growth events within individual k-fibers and a laser-mediated k-fiber injury/repair assay. These tools provide a direct assessment of microtubule amplification mechanisms within k-fibers in metazoans. For complete details on the use and execution of this protocol, please refer to Almeida et al. (2022)..

摘要

在这里，我们利用雌性印度麂细胞的低染色体数（2N=6）和明显较大的着丝粒来研究 k 纤维成熟的分子机制。我们描述了监测着丝粒-微管动力学随时间变化的步骤。具体来说，我们详细介绍了在单个 k 纤维内微管生长事件的活细胞超分辨率 CH-STED 显微镜与激光介导的 k 纤维损伤/修复测定相结合的方法。这些工具为后生动物中 k 纤维内微管扩增机制提供了直接评估。有关此方案的使用和执行的完整详细信息，请参阅 Almeida 等人（2022 年）。