The cell nucleus of higher eukaryotic cells undergoes a dynamic process of disassembly and reassembly during the open mitosis. Failing to reform a single nucleus encasing the entire set of chromosomes often results in small extranuclear bodies, referred to as micronuclei. Micronuclei are prone to irreversible rupture and catastrophic DNA damages, which has been largely implicated in cancer. Recent works have suggested that barrier-to-autointegration factor (BAF/BANF1) crosslinks DNA to a single mass, thereby guiding nuclear membranes to bridge across chromosomes. However, further investigation is necessary to understand detailed biophysical mechanism of action by which BAF mediates nuclear membranes. Previous studies and preliminary data led to the hypothesis of this study that BAF may form fiber-like structures, beyond simple dimers, that connect and congress distant chromosomes. The goal of the proposed study is to investigate this possibility and its implications in nuclear envelope formation. To this end, the first aim is to develop a biophysical assay using Xenopus egg extract and micro-patterned DNA to scrutinize the bridging of nuclear envelope across gaps between chromosomes, and quantitatively dissect the role of BAF complex during this process. In the next aim, 3D dynamics of the fiber-like structures of BAF during nuclear formation will be analyzed in living cells, using lattice light-sheet microscopy and molecular perturbation techniques. Finally, DNA binding and polymerization dynamics of BAF revealed in the extract system and living cells will be reconstituted in a simplest system of purified components to further investigate the biophysical basis. Consequently, the proposed study will offer the mechanistic insight into nuclear formation and micronucleation and establish the foundation for mathematical modeling of these processes. Furthermore, the results have the potential to expand our understanding of cancer micronuclei, thereby aiding in the development of novel cancer therapy and diagnostics. Conducting the proposed study, the applicant aims to deepen his expertise in quantitative biology and microscopy, while at the same time, expanding the breadth of his skills and intellectual horizon to reconstituted cell-free systems and translational research. This project will benefit from the highly interdisciplinary research environment of the Systems Biology department at Harvard Medical School along with the sponsor?s strong support.
The proposed study will perform biophysical analysis and state-of-the-art live cell microscopy imaging to elucidate the mechanism of cell nucleus formation occurring at the end of cell division. Specifically, this study will investigate how nuclear envelope bridges across chromosomes to enclose a single nucleus, preventing the emergence of micronuclei, an aberrant nuclear morphology implicated in the genomic instability of cancer cells. The proposed study will contribute to fundamental knowledge of nuclear reassembly and micronucleation with potential impact on understanding of cancer and development of new therapeutics.
