David Barford

Our research is focused on understanding the mechanisms and regulation of chromosome segregation in mitosis. During the cell cycle, accurate chromosome segregation ensures that both daughter cells inherit the correct complement of chromosomes. Errors in this process cause aneuploidy leading to cancer and developmental defects. Duplicated sister chromatids are segregated in mitosis by a large cellular apparatus, the bi-polar mitotic spindle. The spindle is organized from centrosomes, located at opposite poles of the cell. At metaphase, condensed sister chromatid pairs are aligned on the metaphase plate at the centre of the mitotic spindle. Each chromatid is attached to microtubules by kinetochores, large protein complex that specifically assembles onto centromeric chromatin. Once all chromosomes achieve bi-polar orientation on the mitotic spindle, and tension is exerted at the kinetochore-microtubule attachment site, anaphase is triggered. This results in the loss of sister chromatid cohesion and the segregation of each sister chromatid to opposite poles of the cell, a process powered by microtubule depolymerization.

Anaphase onset is triggered by the anaphase-promoting complex (APC/C), a large multi-subunit complex (~1.2 MDa) that functions as an E3 ubiquitin ligase to regulate different stages of the cell cycle. APC/C activates the protease separase to cleave the kleisin subunit of cohesin, the multi-subunit complex responsible for sister chromatid cohesion. The APC/C is regulated by the spindle assembly checkpoint (SAC) that ensures that chromosome segregation is coordinated with the correct attachment of chromosomes to the mitotic spindle. Unattached kinetochores trigger the SAC by catalysing assembly of the mitotic checkpoint complex (MCC) that inhibits the APC/C. Using a combination of cryo-electron microscopy, protein crystallography and biochemistry we have determined the high-resolution structures of different functional states of the APC/C, and other protein complexes involved in mediating and controlling chromosome segregation. We have determined how the APC/C recognizes and ubiquitinates its substrates, is controlled by protein phosphorylation, how the MCC inhibits the APC/C, and how the APC/C is spontaneously activated once the last unattached kinetochore attaches to the mitotic spindle.

Our current research is directed to reconstituting mitosis in vitro to understand the structure and mechanism of the kinetochore and spindle pole body in budding yeast, how the SAC is activated, and how microtubules mediate chromosome movement in anaphase. We use a variety of techniques including single particle cryo-electron microscopy, cryo-electron tomography, super-resolution fluorescence microscopy and in vitro reconstitution approaches.