Analysis of mitotic protein dynamics and function in Drosophila embryos by live cell imaging and quantitative
Ingrid Brust-Mascher1, Gul Civelekoglu-Scholey, Jonathan M Scholey
1Department of Molecular and Cellular Biology, University of California, Davis, One Shields Avenue, Davis, CA, 95616, USA.
Abstract:
Mitosis depends upon the mitotic spindle, a dynamic protein machine that uses ensembles of dynamic microtubules (MTs) and MT-based motor proteins to assemble itself, control its own length (pole-pole spacing), and segregate chromosomes during anaphase A (chromosome-to-pole motility) and anaphase B (spindle elongation). In this review, we describe how the molecular and biophysical mechanisms of these processes can be analyzed in the syncytial Drosophila embryo by combining (1) time-lapse imaging and other fluorescence light microscopy techniques to study the dynamics of mitotic proteins such as tubulins, mitotic motors, and chromosome or centrosome proteins; (2) the perturbation of specific mitotic protein function using microinjected inhibitors (e.g., antibodies) or mutants to infer protein function; and (3) mathematical modeling of the qualitative models derived from these experiments, which can then be used to make predictions which are in turn tested experimentally. We provide details of the methods we use for embryo preparation, fluorescence imaging, and mathematical modeling.
