Errors in chromosome segregation result in a pathological cellular condition called aneuploidy. Aneuploidy causes a majority of miscarriages in the first trimester, birth defects and has been linked to tumorigenesis and metastasis. It has long been appreciated that the accuracy of cell division depends on chromosomes becoming bioriented, a configuration where each sister chromatid is attached to microtubules from opposing spindle poles. Force and the tension that it produces are integral inputs to the regulation of chromosome biorientation. In fact, properly bioriented attachments are stabilized by tension generated across the kinetochore - the protein complex that assembles during cell division on the centromeres of each sister chromatid and links chromosomes to microtubules. Despite its central importance to genomic integrity, chromosome biorientation is not an assured outcome. In fact, erroneous attachments are common during cell division and they must be corrected to avoid aneuploidy. Error correction requires the selective destabilization of kinetochore-microtubule (kt-MT) interactions on improperly attached chromosomes. Current knowledge of the mechanisms responsible for de- stabilizing incorrect kt-MT attachments is far from complete. The long-term goal is to describe the fundamental molecular properties of cell division and, in doing so, to identify cellular processes that can be targeted by therapies to control aneuploidy. The objective of this proposal is to characterize novel aspects of error correction by combining in vitro biochemical techniques with live-cell assays in D. melanogaster tissue culture cells. The central hypothesis is that error correction occurs via two pathways: a centromere-based system and a spindle pole-based mechanism, each of which is impacted by forces that produce tension at kinetochores. The rationale underpinning the research is that determining the mechano-molecular basis of error correction will in- form the development of novel therapies that modulate error correction pathways to regulate aneuploidy. The central hypothesis will be tested with three specific aims.
Aim 1 will focus on the functional contribution of a tension-dependent structural change, called intrakinetochore stretch, to kt-MT attachment stability. The goal of aim 2 is to describe a novel error correction pathway that is hypothesized to be mediated by pole-based kinase gradients.
Aim 3 will address the mechanical basis of polar ejection force generation by kinesin-10. A battery of stable cell lines and imaging techniques have been developed and implemented to an extent that completion of the work is both feasible and expected to significantly advance the understanding of the essential process of error correction and the contribution of force to its regulation. The approach is innovative because it unites molecular engineering with high- and super-resolution microscopy techniques both in vitro and in living cells to define the molecular foundations of a critical cellular proces. The research is significant because it is expected to identify exploitable access points to the correction machinery that could be therapeutically targeted to treat and prevent a range of human diseases.
Cellular mechanisms that ensure faithful transmission of the genome during cell division are vital to normal embryonic development and the maintenance of healthy tissues through adulthood. Mistakes in cell division lead to cells acquiring an incorrect set of chromosomes, which causes birth defects, miscarriages and is implicated in tumorigenesis and cancer metastasis. Thus, by characterizing fundamental cell division processes, the proposed research will provide a foundation for understanding, treating and preventing an array of human diseases.
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