The Function of Dynein in Budding Yeast

描述（由申请人提供）：该项目的长期目标是了解动力蛋白和微管如何控制出芽酵母细胞中有丝分裂纺锤体的位置。我们发现微管与皮质结合，然后沿着皮质滑动，将有丝分裂纺锤体拉入母芽颈。我们发现滑动取决于动力蛋白和动力蛋白，而动力蛋白和动力蛋白的目标是细胞质微管的远端+端。基于这种定位，我们制定了酵母动力蛋白功能的模型，称为“卸载”模型。在这个模型中，当一个动态微管的末端接触皮层附着位点时，细胞质微管正端的动力蛋白被卸载并锚定在皮层上。然后主动动力蛋白向微管的负端移动，引起微管滑动。我们发现LIS1/Pac1对于将动力蛋白靶向到正端和动力蛋白功能是必需的。动力蛋白复合体是动力蛋白功能所必需的，但不是靶向性的。动力蛋白存在于细胞皮层的固定复合体中，但动力蛋白不存在，这表明动力蛋白介导了卸载。蛋白Num1似乎是皮质附着位点的重要组成部分，动力蛋白被提议卸载到皮质附着位点。我们建议通过继续研究构成该模型的过程的分子基础，包括+端靶向、卸载和皮质附着，来进一步了解动力蛋白的功能。我们将识别和表征动力蛋白通路的新组分，研究动力蛋白通路组分之间的相互作用，并阐明它们在各种过程中的功能作用。我们将专注于定义这些过程的分子机制，并了解它们是如何被调节的。

DESCRIPTION (provided by applicant): 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.