Quantitative measurements of kinetochore protein copy numbers via FCS-calibrated molecular counting.

Abstract

The kinetochore is a protein complex for faithful chromosome segregation. While its architecture has been characterized in vitro based on structural studies, the molecular organization of the kinetochore in living cells remains unclear. To understand the molecular organization of kinetochore proteins in cells, we applied fluorescence correlation spectroscopy (FCS), which enables absolute molecular counting based on its theoretical framework. This approach confirmed that CENP-C is present at roughly double the copy number compared with other components of the constitutive centromere-associated network such as CENP-T. We also analyzed the KMN network, specifically the Mis12 complex (Mis12C) and the Ndc80 complex (Ndc80C), and asked whether the recruitment of these outer kinetochore components relies on CENP-T and/or CENP-C. When either CENP-T or CENP-C lacks the Mis12C-binding domain, cells utilize the remaining domain as a redundant pathway to sustain Ndc80C recruitment. This study provides molecular abundance at the kinetochore with theory-grounded accuracy and offers insights into its structural organization in cells.

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