Defining the Mechanistic Impact of PRC1 Overexpression on Mammalian Spindle Mechanics

Project Summary/Abstract Errors in chromosome segregation lead to aneuploidy, a hallmark of cancer. While we know nearly all molecules involved in mammalian cell division, we do not understand how they work together to give rise to the anaphase spindle’s mechanical function, or how molecular changes in disease impact spindle mechanics. This is the big picture question that drives my work. This proposal aims to define the impact of protein regulator of cytokinesis 1 (PRC1) overexpression on mammalian spindle mechanics. PRC1 crosslinks microtubules to form antiparallel bundles (bridging fibers) which are involved in spindle formation, chromosome segregation, and cytokinesis. Recent work revealed that PRC1 overexpression in cancer correlates with increased proliferation, chromosomal instability, and drug resistance. Yet, a mechanistic understanding of how overexpression impacts proper PRC1 and spindle function is missing. While in vivo studies have begun to uncover PRC1’s and bridging fibers’ functions, their underlying mechanics remain poorly understood. Indeed, mechanical perturbations are challenging in vivo and mammalian spindles cannot yet be reconstituted in vitro. To bridge this gap, our lab has recently adapted biophysical tools to probe mammalian metaphase spindle mechanics in vivo, which I have tuned to probe anaphase mechanics. The anaphase spindle’s ability to effectively generate force while also deforming over minutes stems from its tight control over the dynamics and mechanics of its bridging fibers, and of the overall spindle. Here, I propose to determine how PRC1 overexpression disrupts these dynamics and mechanics and impacts the structural integrity of spindles in cancer. I hypothesize that PRC1 overexpression will produce an overall less dynamic and deformable spindle structure. Such behavior would provide insight into why PRC1 overexpression is correlated with increased chromosomal instability in cancer. In Aim 1, I will test how bridging fiber microtubule lifetime and protein kinetics change in response to PRC1 overexpression. I will assess dynamics using live imaging of fluorescently tagged proteins. In Aim 2, I will test how anaphase bridging fibers alter their expansionary sliding behavior in response to applied forces of differing velocities, and how this response changes with PRC1 overexpression. I will apply forces via microneedle manipulation and measure sliding using photoactivatable tubulin marks. In Aim 3, I will determine how PRC1 overexpression impacts overall spindle integrity in healthy cells and cancer cells with natural PRC1 overexpression. I will use PDMS-based cell confinement to apply extracellular force and measure the rate of spindle fracturing events. I will overexpress PRC1 in a non-cancer line to test sufficiency and use siRNA to deplete it in a cancer cell line to test necessity. Together, this work will reveal basic physical principles driving robust anaphase spindle mechanics and may help uncover how accurate chromosome segregation breaks down in cancer cells overexpressing PRC1.