The Function of Dynein in Budding Yeast

描述(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发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，然后沿着它滑动，将有丝分裂纺锤体拉入母/芽颈部。我们发现滑动依赖于动力蛋白和动力蛋白，并且动力蛋白和动力蛋白定位于细胞质微管的远端加末端。基于这一定位，我们建立了一个酵母动力蛋白功能的模型，称为“卸载”模型。在这个模型中，当动态微管的末端接触皮质附着部位时，细胞质微管正端的动力蛋白被卸载并锚定到皮质。然后，活性动力蛋白向微管的负端移动，导致微管滑动。我们发现，Lis1/Pac1对于将dynein靶向正端和dynein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。在细胞皮质的固定复合体中发现了动力蛋白，但动态肌动蛋白没有，这表明动态肌动蛋白介导了卸载。Num1蛋白似乎是Dynein要卸载到的皮质附着部位的重要组成部分。我们建议通过继续我们对组成模型的过程的分子基础的研究来加深我们对动力蛋白功能的理解，包括加端靶向、卸载和皮质附着。我们将识别和表征动力蛋白途径的新成分，研究动力蛋白途径成分之间的相互作用，并阐明它们在不同过程中的功能作用。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控的。

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.