To investigate how cytoplasmic dynein orients the mitotic spindle, we are using the budding yeast S. cerevisiae as a model cell system. This system offers facile genetics, ease of protein manipulation, and a simple astral microtubule organization enabling the dissection of dynein regulation during asymmetric cell divisions, which provides physiological relevance to human stem cell biology, development, and cancer. In previous studies, we found that dynein is targeted to the dynamic astral microtubule plus ends and is delivered via tip-tracking to its cortical anchoring protein, Num1. At the cortex, dynein uses its minus end-directed motor activity to pull an astral microtubule and the connected spindle toward the site of dynein anchorage. We discovered the critical domain in Num1 responsible for dynein interaction and function, and found She1 as a potent inhibitor of dynein motility along the astral microtubules. We will investigate how these cortical and microtubule-associated components mediate spatial and temporal regulation of dynein activity, thereby ensuring directional spindle movement into the yeast bud cell. Aim 1. Identify the mechanism responsible for dynein inhibition along astral microtubules. To dissect the molecular basis of She1-mediated dynein regulation, we will determine how She1 interacts with dynein and analyze the subcellular localization of She1, testing the hypothesis that it is asymmetrically targeted to different areas of the cell to restrict dynein activity. We will search for specific Sh1 binding partners, investigate the functional roles of these proteins, and test the significance of their interactions with She1. Aim 2. Determine the effect of dynein-cortex interaction on dynein motility. We hypothesize that cortical Num1 enhances dynein motility enabling dynein-dynactin to pull the mitotic spindle into the narrow bud neck. Using multiple approaches encompassing biochemistry, genetics, and in vitro single-molecule motility assays, we will dissect the molecular basis underlying the interaction between dynein, dynactin, and Num1. We will investigate the potential roles of Num1 in triggering dynein offloading or stimulating dynein minus end-directed motility.