Cheeseman Lab Research

We are using a combination of biochemistry, cell biology, proteomics (and any other approach that we can come up with) to understand key questions related to kinetochore function and chromosome segregation.

Phospho-Regulation of Kinetochore Function

The kinetochore is a key site of regulation by the cell as it must respond to cell cycle state, and also monitor and correct any errors in chromosome-microtubule attachments. At least six mitotic kinases appear to function at regulate this structure. However, despite extensive work on these kinases, the key downstream targets for these kinases at kinetochores, and the consequences of specific phosphorylation, remain largely unknown. We have identified multiple phosphorylation sites within the human kinetochore and working to understand the contributions of these sites to chromosome segregation.

Kinetochore-Microtubule Attachments

A key function of the kinetochore is to mediate attachments between chromosomes and microtubule polymers. We are particularly interested in the interface with microtubules to determine how these attachments are generated, regulated, and controlled. Several kinetochore proteins or complexes which interact with microtubules have been identified including the Dam1 ring complex and the Ndc80 complex. We are working to define the specific functions and activities of the kinetochore microtubule binding proteins, and to understand how these proteins are coordinated with each other to generate kinetochore-micrortubule attachments that are able to sustain emense forces, and remain attached to growing or shrinking microtubules.

Kinetochore Assembly

Although a subset of kinetochore proteins are constitutively present at centromeres, the vast majority of proteins are assembled and disassembled over the course of the cell cycle. We are working to understand what controls of the timing of this assembly, and to understand how the mulitple kinetochore proteins interact with each other within the higher order structure. These processes are critical to defining the organization and coordination of this important macromolecular structure.

Kinetochore Proteomics

The identification of kinetochore proteins is a critical challenge for the analysis of chromosome segregation. The first human kinetochore proteins were identified in the mid-80s using autoimmune antibodies from human patients that recognized centromere regions. Additional proteins have been identified in humans and model organisms using a variety of genetic, biochemical, and functional approaches. However, despite extensive work, only a handful of proteins identified by the late 90s. Proteomic approaches have dramatically increased the identification of kinetochore proteins. Chief among these are affinity tagging strategies in which a known kinetochore protein is tagged and used to isolate additional interacting proteins.

We are working to identify the complete compliment of kinetochore proteins in human cells. To do this, we are generating stable clonal human tissue culture cell lines expressing LAP tag fusions to kinetochore components. Purified kinetochore complexes are analyzed by mass spectrometry using Multi-dimensional Protein Identification Technology (MudPIT) by our lab using our inhouse facilitities. This approach identifies novel kinetochore proteins and defines which kinetochore proteins interact most closely within the kinetochore.

Currently, about 80 kinetochore proteins that have been identified in vertebrates. We estimate that there are likely to be more than 100 kinetochore localized proteins.

---