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.
The original goal of this project was to determine whether the kinetic properties of microtubule-based motor proteins are sufficient to achieve meaningful spatial non-uniformities within the cell during mitosis. The project design was to 1) prepare purified, fluorescently-labeled motor proteins and measure motor behavior in vitro at the single-molecule level; 2) introduce those same motor proteins into living mitotic cells in the easily-imaged early embryos of sea urchins and other invertebrates; and 3) use computer simulations to test whether the measured properties of individual motor molecules are sufficient to explain the observed behavior in living cells. The basic question is this: are motors fast enough, and do they travel coherently enough, to defeat diffusion in a large cell? Due to a combination of several unforeseen technical limitations, the original goals of the project could not be achieved with the time and support level originally planned. However, this project partially supported parallel studies which address fundamentally equivalent questions using different methods. The original motivation for this project was an interest in animal cell cytokinesis, in which genetic studies have implicated a particular microtubule-based motor in the delivery of key regulatory signals to the cell equator. The idea is that this motor "interprets" the geometry of the cell’s microtubule array to create a spatial pattern of cell surface contractility. Yet, in large cells that divide rapidly (as do the cells in early embryos), it is not clear that known motors have the capacity to achieve the required distribution in the time allowed. Although our original plans were frustrated, we did succeed to directly visualize apparent transport of the relevant motor and its principle cargo in starfish oocytes and embryos. We were able to document the timely accumulation of motor and cargo in direct relation to the "output" of the system, namely the patterned activity of the conserved contractility regulator Rho. This strongly supports the prevailing hypothesis which this project was designed to test. That is, in order to accomplish the observed distribution in the cell, it must be that the combination of motor activity and microtubule dynamics is sufficient to defeat diffusion. What remains so far unfulfilled, importantly, is to test to what extent it is the individual behaviors of motors that account for this, and furthermore how specific motors are adapted or regulated to accomplish distinct roles within the cell. Although the funding for this project is exhausted, we continue our efforts to fluorescently tag this and other motors with photoconvertible labels to enable quantitative tracking of defined cohorts within living cells. In addition to the direct results of the project’s research activities, this grant provided partial support for a cell and molecular biology laboratory and imaging instruments at a marine field station. This support, in turn, facilitates use of the marine station by other scientists for unrelated research, both by maintaining infrastructure and by keeping research expertise locally available. This grant also partially supported research training opportunities for students. Finally, this project, despite the failure to achieve the original goals, contributed to development of techniques for investigating molecular dynamics in live cells.
