Cheeseman Lab Research
The goal of our laboratory is to define the molecular mechanisms by which accurate cell division occurs. To grow from a single cell to the 30 trillion cells present in the human body, cells duplicate through cell division. During each cell division, the entire complement of genetic material must be accurately partitioned to the daughter cells. Even a single chromosome mis-segregation event can be catastrophic, resulting in the loss or gain of hundreds of genes, with severe consequences for development and disease.
Our research focuses on the kinetochore, the central player in directing chromosome segregation. The kinetochore is a macromolecular structure composed of more than 100 different proteins that act to connect chromosomes to the microtubule polymers that power their movement, and integrate regulatory signals to ensure the proper timing and fidelity of chromosome segregation. Although the central importance of the kinetochore has long been appreciated, the molecular basis for its many activities remains poorly understood. We use parallel biochemical and cell biological approaches to analyze kinetochore composition, structure, organization, regulation, and how kinetochore proteins function to achieve proper chromosome segregation.
A platform for kinetochore assembly
A subset of kinetochore components stably associates with centromeric DNA to provide a platform to assemble the entire kinetochore structure. Our laboratory is analyzing the nature of the kinetochore-DNA interface and how these DNA binding proteins direct kinetochore assembly. In vertebrates, centromeres are not defined by specific sequences and are instead controlled by epigenetic mechanisms that involve specialized nucleosomes containing the histone H3 variant CENP-A. Although prior work demonstrated that CENP-A is necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. As part of an ongoing collaboration with Tatsuo Fukagawaâ€™s lab, we identified the histone fold-containing CENP-T-W-S-X complex as a key, conserved DNA binding protein complex at centromeres. CENP-T plays an essential role in kinetochore assembly. In fact, we demonstrated that artificial targeting of CENP-T to an ectopic locus is sufficient to direct assembly of a functional kinetochore-like structure in the absence of CENP-A. Interestingly, although the CENP-T-W-S-X complex does not display strong sequence similarity to histones, crystallographic analyses revealed that this complex has structural similarity to canonical nuclesomes. This suggests that the CENP-T-W-S-X complex could provide a chromatin foundation at centromeres in parallel to CENP-A. Our current focus is to determine how CENP-A and the CENP-T-W-S-X complex are targeted to centromeres.
Dynamic kinetochore-microtubule attachments
A key goal for our lab is to define how kinetochores harness the force generated by microtubule depolymerization to direct chromosome alignment and segregation. Our work implicated the KNL1/Mis12 Complex/Ndc80 Complex (KMN) network as an essential player in mediating attachments between kinetochores and microtubules. However, it was unknown how kinetochores remain associated with dynamic microtubule polymers to generate force and direct chromosome movement. We found that the Ndc80 complex lacks the intrinsic ability to remain associated with microtubules as they depolymerize. Importantly, we demonstrated that the human Ska1 complex remains processively associated with depolymerizing microtubules and can impart this tip-tracking activity to the Ndc80 complex. Together, this allows the Ndc80 and Ska1 complexes to form a robust and processive kinetochore-microtubule binding interface. We are currently analyzing the structural and mechanistic basis for these activities. We are also reconstituting larger assemblies of kinetochore components to analyze the ability of the higher order kinetochore structure to harness microtubule-generated force.
Regulating kinetochore function
Chromosome segregation must occur with high fidelity, with even minor defects leading to cell inviability or disease. We have defined specific phosphorylation events in kinetochore proteins that are critical for ensuring accurate chromosome segregation. First, in the presence of inappropriate microtubule attachments, such as both sister kinetochores attaching to the same spindle pole, incorrect kinetochore-microtubule attachments must be eliminated to allow new, proper attachments to be formed. We demonstrated Aurora B kinase phosphorylation inhibits the microtubule binding activities of multiple kinetochore components thereby inactivating the kinetochore-microtubule interface. Importantly, these substrates are differentially phosphorylated in response to changes in kinetochore tension and attachment status, providing a flexible mechanism to correct improper kinetochore-microtubule interactions. Second, during the course of mitosis, kinetochore function must be altered to facilitate the changing requirements to capture microtubules, stabilize attachments, and segregate chromosomes. We found that phosphorylation plays a key role in controlling the kinetochore assembly state during mitosis, changing kinetochore function by altering its components rather than regulating their activities. Third, most kinetochore proteins localize to centromeres only during mitosis, but the mechanisms that control mitotic kinetochore assembly and disassembly were unknown. We demonstrated that cyclin-dependent kinase (CDK) controls kinetochore assembly by directly phosphorylating CENP-T and other kinetochore proteins to promote interactions between the constitutively localized inner kinetochore proteins and mitosis-specific outer kinetochore components. We are currently analyzing kinetochore phospho-regulation downstream of additional mitotic kinases to define the paradigms that control kinetochore assembly and activity.