Cells live in noisy environments and must integrate multiple biochemical, mechan- ical and geometric signals during development. Cells use these signals to make critical decisions such as de- termining the orientation of the division plane. Orientation of the division plane is dictated by the position of centrosomes, small nucleus-associated organelles that nucleate microtubule (MT) arrays. MTs interact with the motor protein dynein, and the forces generated by these interactions position the centrosomes. In polarized cells, where speci?c factors are segregated to distinct areas of the cell, centrosome positioning is especially important: centrosomes aligned along the polarity axis of the cell will produce unequal daughter cells (asymmetric division, involved in cell fate speci?cation), while centrosomes oriented orthogonal to the polarity axis will produce identical daughter cells (symmetric division, associated with proliferation). Impaired centrosome positioning can also be a hallmark of disease states: cancer cells often exhibit abnormal centrosome positioning. In this project, we will demonstrate that biochemical, mechanical, and geometric signals act interdependently to position centrosomes in polarized cells prior to division. We will investigate the role of these features using mathematical modeling, which can deal with complex multiscale interactions. To ensure biological realism, we will experimentally test our models using early embryos of the nematode worm Caenorhabditis elegans, which polarize and orient their centrosomes along the long axis of the cell resulting in an asymmetric ?rst division. The proteins involved in polarization and centrosome positioning are highly conserved across organisms, making insights applicable to other biological contexts. To determine the role of biochemical signaling, we will develop a model that tracks spatial and temporal protein dynamics, producing patterns over long space scales as a result of fast, local interactions. We will test the model by perturbing protein localization and quantifying changes in dynein localization or centrosome movement. We have observed mechanical asymmetries in the centrosome-associated MT arrays. We will determine the origin and consequence of this mechanical asymmetry by developing a model of centrosome maturation, which takes place over short time and space scales. We will compare model results to ?uorescent protein dynamics during centrosome maturation, and use a novel ?uorescent protein timer to determine if this asymmetry is related to centrosome age. To investigate the role of cell geometry, which operates over large time and space scales, we will use a biologically based whole cell model along with simpli?ed energy models to determine the most favorable centrosome position over a range of volume and aspect ratio values. We will experimentally perturb the size and shape of the embryos to demonstrate whether geometry alone can induce or compensate for defects in force generation during centrosome positioning. The powerful combination of theoretical and experimental approaches will yield a fundamental understanding of dynein-mediated centrosome movement in response to biochemical, mechanical and geometric features.
Proper centrosome positioning is critical during development, and is regulated by highly conserved biochemical, mechanical and geometric cues. This project utilizes theoretical and experimental ap- proaches spanning small scale molecular interactions to cellular scale organization to elucidate the mechanisms regulating centrosome positioning prior to mitosis. Since proper centrosome positioning is critical for a variety of developmental processes, this understanding could lead in the long term to comprehensive identi?cation of defects due to impaired centrosome positioning and development of targeted therapies.
