Structural biology of cell division
Cell division is an essential biological process that ensures genome integrity by equally and identically distributing chromosomes to the daughter cells. Errors in cell division often result in daughter cells with inappropriate chromosome numbers, a condition associated with cancers and birth defects. Key events that determine the accuracy of cell division include centromere specification, kinetochore assembly, physical attachment of kinetochores to spindle microtubules and successful completion of cytokinesis. These cellular events are regulated by a number of mitotic molecular assemblies (including the Chromosomal Passenger Complex (CPC), KMN (Knl1-Mis12-Ndc80) network, the Ska complex, Spindle Assembly Checkpoint and the Anaphase Promoting Complex) involving an extensive network of protein-protein interactions.
Although much is known about the basic mechanisms of cell division, structural level mechanistic details of pathways regulating error free chromosome segregation are still emerging. In particular, a high-resolution understanding of centromere inheritance and how kinetochores employ dynamic protein interaction to harness the forces generated by spindle microtubules to drive chromosome segregation is yet to be obtained. To address these important questions requires an approach that integrates structural and functional methods capable of dissecting and probing individual roles of protein interactions mediated at varying timescale. We use molecular biology and biochemical approaches to characterize protein interactions in vitro, X-ray crystallography, Cross-linking/Mass spectrometry, Small Angle X-ray Scattering (SAXS) and Electron Microscopy for structural analysis and a combination of in vitro and cell-based in vivo functional assays using structure-guided mutations for functional characterization.
The specific questions that we aim to address currently are i) What is the molecular basis for the establishment and maintenance of CENP-A nucleosomes at centromeres? ii) How do the outer kinetochore microtubule binding components cooperate to facilitate spindle driven chromosome segregation? and iii) How CPC, a key player required for eliminating incorrect kinetochore-microtubule attachment is targeted to the kinetochore? We address these question by characterizing protein complexes involved in centromere maintenance (Mis18 and Mis18-associated), physically coupling chromosomes to kinetochores (the Ska complexes and other outer kinetochore microtubule binding factors) and error-correction (CPC and its centromere/kinetochore receptors). The structural and functional insights from these studies will also provide new avenues for targeting specific protein-interactions to fight mitosis related human health disorders.
Spiller, F*., Medina-Pritchard, M*., Abad, M. A*., Wear, M. A., Molina, O., Earnshaw, W. C and Jeyaprakash, A. A. (2017). Molecular Basis for Cdk1 Regulated Timing of Mis18 Complex Assembly and CENP-A Deposition. EMBO Rep 18, 894-905. Doi:10.15252/embr.201643564
Subramanian, L*., Medina-Pritchard, B*., Barton, R., Spiller, F., Kulasegaran-Shylini, R., Radaviciute, G, Allshire, R. C and Jeyaprakash A. A. (2016). Centromere Localization and Function of Mis18 Requires ‘Yippee-Like’ Domain-Mediated Oligomerization. EMBO Rep 17, 496-507. Doi:10.15252/embr.201541520. (* equal contribution)
Abad, M. A*., Medina, B*., Santamaria, A*., Zou, J., Plasberg-Hill, C., Madhumalar, A., Jayachandran, U., Redli, P. M., Rappsilber, J., Nigg,
E. A. and Jeyaprakash. A. A. (2014). Structural Basis for Microtubule Recognition by the Human Kinetochore Ska Complex. Nat Commun 5, 2964. doi:10.1038/ncomms3964. (* equal contribution)