Regulation of dynein activity during spindle positioning in budding yeast

2020 Spring.Includes bibliographical references.Cytoplasmic dynein is a minus-end directed, microtubule motor that is highly regulated to ensure it is targeted to the correct location at a specific time for its function in cells. This is particularly important for the process of spindle positioning during mitosis. Dynein is targeted to the cell cortex and activated to pull on astral microtubules attached to spindle poles to move the spindle into position at the site of cytokinesis. The position of the spindle dictates the plane of division and influences whether a cell divides asymmetrically or symmetrically- an important distinction during embryonic development and homeostasis. Using the model organism budding yeast, we confirmed that dynein is held in an inactive state before reaching its destination at the cell cortex by identifying a key factor in dynein activation- the cortical receptor Num1. We determined that the mechanism of activation involves enhancing dynein-dynactin interaction and releasing the recruitment factor, Pac1/Lis1. Additionally, I determined the role of another regulator in the dynein pathway, Ndl1/NudE. Ndl1/NudE aids the recruitment factor, Pac1/Lis1 in targeting dynein to astral microtubule plus ends that then deliver the motor to the cortex. Interestingly, it appears Ndl1/NudE may have another function that competes Pac1/Lis1 off dynein in a specific context. Next, I explored the two possible mechanisms of Num1-mediated dynein activation. First, I established an in vitro motility assay to observe how the regulators dynactin, Num1 and Pac1/Lis1 may coordinate to affect dynein activity. I determined the purification conditions for complete dynactin complexes as well as Num1 constructs to test whether Num1 acts as an adapter to activate the dynein-dynactin complex. Finally, I examined the second mechanism of Num1-mediated activation by initiating the release of Pac1/Lis1 from dynein complexes. I predicted that Num1 may influence the conformational changes of dynein during its mechanochemical cycle in conjunction with dynein engaging the microtubule that could induce Pac1/Lis1 release. To test this, I mutated dynein in a way that restricted conformation changes and observed how this affected Pac1/Lis1 interaction. The results show that Pac1/Lis1 binding is profoundly affected by dynein structure. Further, Num1 can still initiate Pac1/Lis1 release despite restriction in conformational changes, which suggests Num1 may initiate Pac1/Lis1 disassociation in another way. Together these data reveal important details of how regulatory proteins coordinate to spatially and temporally regulate dynein during spindle positioning