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.

Public Health Relevance

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 during cell division lead to cells acquiring an abnormal number of chromosomes ? a pathological cellular state called aneuploidy. By characterizing fundamental cell division processes, the proposed research will provide fertile ground for improving disease treatment and prevention.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Gindhart, Joseph G
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University of Massachusetts Amherst
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Ye, Anna A; Verma, Vikash; Maresca, Thomas J (2018) NOD is a plus end-directed motor that binds EB1 via a new microtubule tip localization sequence. J Cell Biol 217:3007-3017
Ye, Anna A; Maresca, Thomas J (2018) Measuring mitotic forces. Methods Cell Biol 144:165-184
Ye, Anna A; Cane, Stuart; Maresca, Thomas J (2016) Chromosome biorientation produces hundreds of piconewtons at a metazoan kinetochore. Nat Commun 7:13221
Ye, Anna A; Torabi, Julia; Maresca, Thomas J (2016) Aurora A Kinase Amplifies a Midzone Phosphorylation Gradient to Promote High-Fidelity Cytokinesis. Biol Bull 231:61-72
Ye, Anna A; Maresca, Thomas J (2016) Generating a ""Humanized"" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5. J Vis Exp :e53594
Ye, Anna A; Maresca, Thomas J (2015) It's all relative: Centromere- versus pole-based error correction. Cell Cycle 14:3777-8
Drpic, Danica; Pereira, António J; Barisic, Marin et al. (2015) Polar Ejection Forces Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes. Cell Rep 13:460-468
Ye, Anna A; Deretic, Jovana; Hoel, Christopher M et al. (2015) Aurora A Kinase Contributes to a Pole-Based Error Correction Pathway. Curr Biol 25:1842-51
Cane, Stuart; Maresca, Thomas J (2014) Cell division: the prehistorichore? Curr Biol 24:R529-32
Cane, Stuart; McGilvray, Philip T; Maresca, Thomas J (2013) Insights from an erroneous kinetochore-microtubule attachment state. Bioarchitecture 3:69-76

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