Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

Abstract

The key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far. In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.