Chromosomes are the repositories of our genetic material. We consider them to be living, breathing objects whose fluctuations in time and space underlie their most basic functions. By comparing meiotic and mammalian mitotic chromosomes and E.coli nucleoids we seek to identify fundamental commonalities. Meiosis underlies sexual reproduction. Its unique hallmarks are pairing and recombination of maternal and paternal homologs, including the phenomenon of crossover interference in which crossover sites occur with even spacing along the chromosomes. We analyze this patterning process by 4D long timescale visualization in our new C.elegans platform and by cytogenetic studies in the fungus Sordaria. We are probing our new mechanical model and our discovered inter-homolog structure/DNA bridges, concomitantly analyzing new-found players and identifying more. For pairing, with our new low SNR spot detection algorithm and FROS tags in budding yeast, we probe partner searching and homology identification. In Sordaria, our first-ever comprehensive screen of meiotic long noncoding RNAs will identify species involved in patterning and/or pairing. Other studies investigate the evolution of meiosis from mitosis and evolution of stable autopolyploidy. Mitotic chromosomes start in a diffuse but spatially ordered state (G1), but ultimately evolve into compact, side-by-side sister chromatids ready for segregation. We are pursuing our discovery of inter-sister structure/DNA bridges and their emergence via axial torsional stress by quantitative modeling. Using live cell imaging of mammalian chromosomes, including our new 4D long timescale platform for fluorescent speckle microscopy, we are exploring our finding that metaphase chromosomes are folded, not coiled, and will ask when/how G1 chromosomes acquire their disposition, with/without our proposed compaction/expansion cycles. E.coli chromosomes also undergo global compaction/expansion cycles, as we discovered. Now, by high throughput 4D imaging of cells growing in agarose grooves, and of membrane-enclosed L-forms, we are investigating the (supercoiling-dependent) mechanism of these cycles; their roles for sister segregation and cell division; and the roles of nucleoid/membrane interactions in both aspects. We are also working to reconstitute nucleoid cycles in vitro, and are asking if cycles also occur in other bacteria. For many of the above studies, chromosomes can be viewed as mechanical objects, subject to deforming forces (stresses) that drive local and global movement, abrupt changes or, via stress redistribution, spatial patterning. To directly detect and analyze such effects, we are developing ZnS-Mn mechano- luminescent nanocrystals as a non-invasive in vivo stress sensor. Once developed, this tool will be applied to detection of waves and/or other, yet-to-be imagined, stress patterns in mammalian chromosomes. Our unique studies will provide novel entry points into problems of infertility and birth defects (meiosis), genetic instability and cancer (mitotic cells) and antibiotic resistance (E.coli L-forms).
The proposed research takes novel approaches to elucidating the fundamental behaviors and roles of chromosomes, which are the repositories of our genetic material. We study meiosis, aberrancies of which confer infertility and birth defects; we study mammalian chromosome organization and segregation, defects in which underlie cancer; and we study E.coli, including L-forms, a lifestyle that bacteria use to evade antibiotics. Our research will therefore have a broad impact on many fundamental issues that directly impact human health.
