Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology , Marburg, Germany.
Department of Physics, Carnegie Mellon University , Pittsburgh, PA, USA.
J Cell Biol. 2023 Apr 3;222(4). doi: 10.1083/jcb.202209096. Epub 2023 Jan 27.
The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.
确保有丝分裂过程中染色体正确分离的关键是动粒（kinetochore，KT），这是一个紧密调控的多蛋白复合物，它将着丝粒染色质与纺锤体微管连接起来，从而引导分离过程。因此，了解其结构、功能和调控机制至关重要。然而，由于其复杂性和动态性，迄今为止，只能对其单个亚复合物进行结构细节研究。在这项研究中，我们使用多色单分子定位显微镜构建了一个纳米精度的人类样局部动粒的原位图谱。我们结合两个参考物（cenp1CENP-A 在着丝粒处和 sad1 在纺锤极处）来测量每个感兴趣的蛋白质（protein of interest，POI）。这使我们能够确定细胞周期和有丝分裂平面，并分别可视化单个着丝粒区域。我们使用贝叶斯推断确定复合物内的蛋白质距离，确定每个 POI 的化学计量比，并因此以前所未有的精度构建原位 KT 模型，为其结构提供新的见解。
Zotero 插件
