Cryo-EM study of the human kinetochore

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 the mitotic spindle. At metaphase, condensed sister chromatid pairs are aligned on the metaphase plate. Each chromatid is attached to microtubules by kinetochores, large protein complexes that specifically assemble onto centromeric chromatin. Once all chromosomes achieve bipolar 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. Kinetochores that mediate and regulate this process consist of over 100 proteins, that also function to detect and signal appropriate microtubule attachment and tension. An error-correction mechanism detects the lack of tension at incorrect kinetochore-microtube attachments (indicating lack of biorientation). This triggers a process mediated by protein (de)phosphorylation to correct mis-attachments and generate bioriented sister chromatid pairs.

We recently determined structures of the inner and outer kinetochore complexes from both yeast and human. We are now interested in understanding the mechanism of outer kinetochore attachment to microtubules in humans and how lack of tension is detected by the error correction mechanism in order to establish biorientation. The specific aim of the PhD project will be to reconstitute end-on outer kinetochore-microtubule complexes and determine their structures by cryo-electron microscopy, and to understand how the lack of tension is sensed by the error correction mechanism to correct mis-orientated kinetochores.

The project will include a variety of techniques including single particle cryo-electron microscopy, optical tweezers to measure rupture forces on KT-MT attachments, and in vitro reconstitution approaches involving cloning and protein expression.