Kuhn Jonathan, Dumont Sophie
Department of Cell & Tissue Biology, University of California, San Francisco, California, USA; Tetrad Graduate Program, University of California, San Francisco, California, USA.
Department of Cell & Tissue Biology, University of California, San Francisco, California, USA; Tetrad Graduate Program, University of California, San Francisco, California, USA; Department of Cellular & Molecular Pharmacology, University of California, San Francisco, California, USA.
Methods Cell Biol. 2014;123:467-87. doi: 10.1016/B978-0-12-420138-5.00025-2.
The kinetochore mediates chromosome segregation at cell division. It is the macromolecular machine that links chromosomes to spindle microtubules, and is made of more than 100 protein species in mammalian cells. Molecular tools are presently revealing the biochemical interactions and regulatory mechanisms that ensure proper kinetochore function. Here, we discuss two approaches for imaging and physically probing kinetochores despite mitotic cell rounding and rapid kinetochore dynamics. First, we describe how mild spindle compression can improve kinetochore imaging and how stronger compression can mechanically perturb the spindle and kinetochores. Second, we describe how simultaneously imaging two-colored kinetochore reporter probes at subpixel resolution can report on kinetochore structural dynamics under cellular forces. We hope that the experimental details we provide here will make these two approaches broadly accessible and help move forward our understanding of kinetochore function--and make these approaches adaptable to the study of other cellular structures.
动粒在细胞分裂时介导染色体分离。它是将染色体与纺锤体微管相连的大分子机器，在哺乳动物细胞中由100多种蛋白质组成。目前，分子工具正在揭示确保动粒正常功能的生化相互作用和调控机制。在这里，我们讨论两种在有丝分裂细胞变圆和动粒快速动态变化的情况下对动粒进行成像和物理探测的方法。首先，我们描述了轻度纺锤体压缩如何改善动粒成像，以及更强的压缩如何机械扰动纺锤体和动粒。其次，我们描述了如何以亚像素分辨率同时对双色动粒报告探针进行成像，以报告细胞力作用下动粒的结构动态。我们希望这里提供的实验细节能使这两种方法广泛可用，并有助于推动我们对动粒功能的理解——并使这些方法适用于其他细胞结构的研究。
