Collaborative Research: Motor-driven Pattern Formation during Cell Division

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

Abstract Intellectual Merit: During cell division, the cytoskeleton reorganizes itself rapidly to align and separate chromosomes, and then cleave the cell in two. Cell division itself is mediated by contractile proteins, whose assembly is controlled by the signaling protein Rho. The mitotic apparatus, which sorts the chromosomes, is based on microtubules, and somehow provides the spatial information to pattern Rho activity, and hence contractile protein assembly, in space and time. A long-standing hypothesis proposes that molecular motors carry signals along microtubules of the mitotic apparatus to tell the cell surface where to contract. This project will directly test this hypothesis by investigating the quantitative conditions under which motors of the kinesin family can translate the information immanent within the rapidly-changing microtubule array into a pattern of Rho signaling at the cell surface. To do this, the investigators will create hybrids between motor proteins and photoactivatable fluorescent proteins and measure their biochemical properties. They will measure motor motility parameters by observing single motors attaching to and moving along single microtubules in vitro. These same hybrid motor probes will be introduced into cells, and live imaging will be used to measure their dynamic behavior during cell division at high resolution. Meanwhile, advanced 3D computer simulations will be developed to predict how these same quantified agents should behave in the context of the whole cell, either normally or when the geometry of the microtubule array is experimentally altered; in turn, assays in live cells will test these predictions. By combining live-cell imaging, single-molecule measurement, and computer simulation of hypothetical outcomes, the project will produce an account of the physically-plausible conditions under which the cell's toolkit of molecular motors and cytoskeletal assembly regulators could add up to a mechanism for robust spatial pattern formation during cell division. Broader impacts: This project will train undergraduate and graduate students in high-resolution live-cell microscopy. The research also involves development of a sophisticated agent-based computer simulation program that is expected to have broad application to many other fundamental problems in cell biology, beyond the specific research goals of this project. This software and computer code will be made freely available to other researchers. Likewise, the research is expected to produce several useful molecular probes that will facilitate broader studies of the behavior of the cytoskeleton by other researchers. These probes will be distributed freely to researchers using Addgene.org, a non-profit plasmid repository which makes constructs available for the cost of shipping. Finally, this research will generate, as by-products of the specific experiments conducted, numerous microscope images and videos which the investigators will make freely available via both lab websites and public collections for educational and other research use.