The goal of the proposed research is to understand how cleavage planes are specified in differential cell divisions of animal cells; divisions that produce daughters of differing size and developmental potential. When an animal cell divides, the advancing cleavage furrow bisects the mitotic apparatus. The orientation of the furrow is determined by the orientation of the mitotic apparatus. In all cases that have been studied, cells become polarized prior to a differentiative division and cytoplasmic components become segregated along the axis of polarity. In many organisms it has been shown that a microtubule-based motor system aligns the mitotic spindle along the axis of polarity by localized capture and shortening of astral microtubules, thereby ensuring the segregation of the portioned components into individual daughter cells. We propose to continue our studies of the mechanisms that specify the orientation of the mitotic apparatus (and hence the cleavage plane) in developing embryos of the nematode Caenorhabditis elegans. In the previous grant periods, we discovered that dynactin is required for rotational alignment of the spindle and that it is localized to a discrete focus on the cell cortex where it acts to capture astral microtubules during rotational alignment of the mitotic apparatus. In the next grant period we are proposing to explore two complementary research avenues. Firstly, using a combination of reverse genetics, drug inhibition studies and live imaging, we propose to identify the mechanisms that bring about the accumulation of the dynactin focus at an appropriate position in the cell cortex. Secondly, we propose to explore the mechanism by which the sperm defines the axis of polarity (and hence the cleavage plane) of the fertilized zygote and brings about the partitioning of the PAR-3 complex and PAR-2 (the primary determinants of embryonic polarity) into two non-overlapping domains. Our strategy is to attempt to repolarize the apolar mutant, spd-2 using experimentally induced localized concentrations of microtubules, and to use live imaging and drug inhibition strategies to study the partitioning of the PAR-3 complex from PAR-2. [unreadable] [unreadable]