Project Summary Accurate segregation of chromosomes during cell division is one of the most fundamental requirements in biology. Without proper chromosomal segregation, genetic information cannot be faithfully transmitted across cell and organismal generations, leading to severe consequences including cell death, developmental defects, or progression of cancer. Furthermore, improper chromosome segregation in cancer cells has been shown to lead to anti-tumor inflammatory responses. Central to the process of chromosome segregation is the centromere, the chromosomal locus at which spindle microtubules bind. The centromere is defined epigenetically by the presence of nucleosomes containing the histone variant CENP-A. Centromeric chromatin serves as the foundation of the kinetochore, a large protein complex which assembles on CENP-A nucleosomes and mediates microtubule binding. Research into the centromere is necessary to better understand the processes that underlie chromosome segregation in both health and disease, but our understanding of the human centromere remains largely incomplete. This proposal aims to answer fundamental questions about the structure and function of the centromere and its associated proteins. Recent advances in reconstitution of large centromeric protein complexes have increased our understanding of the structure of the human kinetochore, but reconstituted complexes can only approximate in vivo structures, and currently there are multiple competing models for the structure and organization of the centromere and kinetochore. The emerging technology of cryo-electron tomography (cryo-ET) provides the opportunity to interrogate the structure of the centromere and kinetochore in their native context within vitreous hydrated cells. To this end, in Aim 1 cryo-ET will be used to obtain the first in situ structures of the human centromere and kinetochore in the interphase and mitosis stages of the cell cycle. The second focus of this proposal is to elucidate the interactions among centromeric proteins that are required for the essential functions of the centromere, including formation of microtubule attachments and maintenance of centromeric identity. Two kinetochore proteins, Ndc80 and CENP-Q, have both been shown to contribute to microtubule binding in vitro and in vivo, but their respective roles in microtubule binding in vivo have not been fully characterized. Multiple proteins within the constitutive centromere-associated network (CCAN) have similarly been shown to contribute to maintenance and deposition of CENP-A at the centromere, but the CCAN contains multiple interconnected subcomplexes whose contributions have never been systematically tested. In Aim 2 mutagenesis of the respective endogenous gene loci (and rapid depletion of the respective wild type gene products) will be used to elucidate in vivo and with temporal accuracy the interactions that underlie spindle attachment and maintenance of centromeric identity. These exp...