Research Projects

The fidelity of error-free chromosome segregation is ensured primarily by kinetochore, a multiprotein complex formed at each centromere of every chromosome, which connects the chromosomes to the microtubules during cell division. The mitotic and meiotic kinetochores differ in their behavior in microtubule connection which provides the basis of achieving equational and reductional chromosome segregation in mitosis and meiosis, respectively. Any error in the kinetochore-microtubule attachment leads gain or loss of chromosomes by a cell generating a condition called aneuploidy which is the hallmark of several aneuploidy-associated diseases including cancers, developmental disorders, spontaneous abortion and still birth. Therefore, it is not surprising that several kinetochore proteins have been found to be mutated and/or dysregulated in various cancers and developmental disorders patients. Earlier cell biological and biochemical studies hinted an organizational difference in the ensemble between mitotic and meiotic kinetochores; however, no ultrastructure of endogenous kinetochores is available to pinpoint the structural differences. Notably, a few studies revealing ultrastructure of partial kinetochores reconstituted from a handful of recombinantly purified kinetochore proteins obviously failed to capture the endogenous complexity and the mitotic- or meiotic-specific differences. This study, for the first time, will attempt to elucidate the high-resolution ultrastructure of the endogenous kinetochore complex purified from the mitotic and meiotic budding yeast cells using cryo-EM and cryo-ET techniques. We have following objectives to address this - 1. Construction of yeast strains where cells can be arrested in the mitotic and meiotic metaphase stage as maximum difference in the kinetochore ensemble between mitosis and meiosis is expected at this stage and verification of kinetochore integrity in those arrested cells, 2. Purification of the kinetochores from the constructed strains using affinity pull down methods, 3. Elucidation of the high-resolution ultrastructure of mitotic and meiotic kinetochore complexes using single particle cryo-EM and cryo-ET approaches. The rationale of using budding yeast for the work is i) the budding yeast kinetochore being small in size is comparatively easy to purify and has been purified before for biochemical analysis, ii) the pattern of kinetochore organization and the constituent proteins are conserved from yeast to humans and in fact it is believed that the human kinetochore is made of multiple repeating units like yeast kinetochore; therefore the obtained knowledge from yeast can be extrapolated to understand human kinetochores and iii) advancement in cryo-EM/ET methodologies can reveal the high-resolution structure of the supramolecular complexes from lesser amount of protein complexes than before enabling usage of smaller kinetochores like yeast. The major methods that would be employed are i) affinity-based purification of the endogenous kinetochore complexes from cells arrested at metaphase (mitosis) or metaphase I (meiosis) and ii) cryo-EM, sub-tomogram averaging and cryo-ET analysis of the purified kinetochore complexes as well as their analysis within the intact cells from metaphase and metaphase I. This study would be a culmination of multiple fields viz., molecular biology, cell biology, biochemistry, structural biology and bioinformatics. The outcome of this work will reveal the organizational difference between mitotic and meiotic kinetochore ensembles in high resolution which will have immense implication to understand the fundamental difference and modus operandi of mitotic and meiotic kinetochores. The study will also provide a structural insight of various functional modules, present as sub-complexes within the kinetochore complex, which can be harnessed in future as physiological targets for drug discovery and development against aneuploidy-associated diseases.