The long-term goal of this project is to understand how dynein and microtubules control the position of the mitotic spindle in budding yeast cells. We discovered that microtubules bind to the cortex and then slide along it, pulling the mitotic spindle into the mother / bud neck. We discovered that sliding depends on dynein and dynactin, and that dynein and dynactin are targeted to the distal plus ends of cytoplasmic microtubules. Based on this localization, we formulated a model for dynein function in yeast, termed the """"""""offloading"""""""" model. In this model, dynein at the plus end of a cytoplasmic microtubule is offloaded and anchored to the cortex when the end of a dynamic microtubule contacts a cortical attachment site. Active dynein then moves toward the minus end of the microtubule, causing microtubule sliding. We discovered that LIS1/Pac1 is necessary for targeting dynein to plus ends and for dynein function. Dynactin complex is necessary for dynein function but not targeting. Dynein is found in stationary complexes at the cell cortex, but dynactin is not, suggesting that dynactin mediates offloading. The protein Num1 appears to be an essential component of the cortical attachment site to which dynein is proposed to offload. We propose to further our understanding of how dynein functions by continuing our studies of the molecular basis of the processes that compose the model, including plus-end targeting, offloading, and cortical attachment. We will identify and characterize new components of the dynein pathway, study the interactions among dynein pathway components, and elucidate their functional roles in the various processes. We will focus on defining molecular mechanisms for these processes and understanding how they are regulated. ? ? ?
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