Mechano-molecular regulation of kinetochore function

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PROJECT SUMMARY & ABSTRACT Chromosome mis-segregation results 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 metas- tasis. The accuracy of cell division depends on chromosomes becoming bioriented, a configuration where each sister chromatid is attached to microtubules (MTs) from opposing spindle poles. Force and the tension that it produces are integral to high fidelity transmission of the genome. Bioriented attachments become stabilized by tension generated across the kinetochore (KT) – a large protein complex that fulfills two essential functions as (1) the link between chromosomes and spindle MTs and (2) the regulatory hub for a spindle assembly check- point (SAC) that delays anaphase onset until chromosomes are attached to spindle MTs and bioriented. Intrin- sically disordered proteins (IDPs), which are proteins that do not have reproducible folds or tertiary structures, are abundant at the KT and on the surface of chromosomes. In fact, ~50% of the molecular mass of the Dro- sophila KT is predicted to be intrinsically disordered while an IDP enriched compartment called the perichro- mosomal layer accounts for >30% of the mitotic chromosome mass. This proposal studies the function of “un- structure” – specifically the role of intrinsically disordered proteins (IDPs) in cell division. 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 combine in vitro bi- ochemical and biophysical assays with live-cell experimentation in D. melanogaster and human tissue culture cells to study conserved IDPs involved in cell division. The central hypothesis is that mechano-sensing and force-transducing IDPs, which localize to KTs, centromeres and chromatin, harness force-generation by dy- namic spindle MTs to regulate spindle assembly checkpoint (SAC) signaling and chromosome movement. The rationale underpinning the research is based on the fact that the IDPs of interest are uniquely positioned to ex- perience MT-dependent forces. The central hypothesis will be tested with three specific aims. Aim 1 will focus on regulation of a checkpoint protein-KT interaction that we hypothesize is mechanical in nature. The goal of aim 2 is to characterize a novel cup structure assembled around KTs that is coated with a SAC protein and that we hypothesize is enriched for IDPs. Aim 3 will study the contribution of a very large protein, which is 97% dis- ordered, called Ki-67 to cell division. Completion of these aims is expected to significantly impact basic knowledge of force-transducing IDPs to the fidelity of cell division. The approach is innovative because it pairs cell-based experiments including the use of live-cell force sensors with single molecule biophysical assays on IDPs. The research is significant because it opens new avenues of research into conserved IDPs that could be exploited therapeutically to modulate SAC activity and to target Ki-67 – a protein long-used as a proliferation marker in cancer diagnosis, but whose precise function in cell division remains unclear.