Imaging and physically probing kinetochores in live dividing cells.

Abstract 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.