This research aims to figure out mechanistic causes of the 3-D reorganizations of filamentous actin and myosin II that occur in phase with mitotic cycles during pseudo cleavage furrow formation in the syncytial Drosophila blastoderm. Preliminary observations and drug experiments in Drosophila implicate rapidly changing arrays of microtubules as causing periodic reorganization of F-actin and cytoplasmic myosin II (among other biologically important cytoskeletal constituents). Interactions between these key cytoskeletal filaments in cells, if verified and understood, have broad applications in cell biology and in particular in explaining how the contractile ring is positioned to cause cell division. A new combination of embryo fixation techniques and computer-implemented analysis of laser scanning confocal microscope data yields 3-D imaging of the complex cytoskeletal geometry inside large deep cells. These new techniques facilitate high resolution 3-D assessment of perturbations of normal cytoskeletal dynamics caused in Drosophila by maternal effect mutations gnu (giant nuclei), mav (mavericks), and sponge as well as perturbations caused by drug microinjections. These will be analyzed to help verify or reject competing hypotheses about what mechanistic interactions between cytoskeletal elements cause wildtype cytoskeletal kinematics, of which we will generate the definitive 3-D description at closely spaced time points through the mitotic cycle. These new techniques will be extended to make a similar description of wildtype F-actin/myosin II/microtubule dynamics in early blastomeres of C. elegans embryos, discovering to what extent cytoskeletal dynamics in these two study organisms match, and setting the stage for future analysis of mutationally caused perturbations in C. Elegans. A mathematical/computer simulation model will be developed, using a large system of differential equations enforcing the laws of Newtonian mechanics, to compute what cell-level cytoskeletal reorganizations are caused by competing hypotheses about molecular-level interactions between key cytoskeletal constituents. This mathematical model will be used to ascertain whether actin/microtubule and myosin/microtubule molecular-level interactions our experimental analyses imply can actually bring about the cell-level cytoskeletal reorganizations required for early morphogenesis of syncytial Drosophila embryos and cytokinesis in C.elegans blastomeres.
