Funder: Boehringer Ingelheim FondsFunder: Bonn UniversityFunder: Max Planck SocietyFunder: Carnegie Mellon UniversityThe 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
