GENETIC AND IMMUNOLOGIC ANALYSIS OF MICROTUBULE PROTEINS

我们的长期目标是了解细胞内转运的分子基础和逻辑，这对正常的神经元和其他细胞功能至关重要，并可能在神经退行性变和其他疾病过程中发挥重要作用。我们的重点是动蛋白，它在包括有丝分裂、囊泡运输和轴突运输在内的各种细胞环境中产生沿微管的定向运动。具体地说，我们想回答两个一般性的问题：运动蛋白利用的逻辑是什么？动蛋白马达是如何调节和附着到细胞内的货物上的？为了回答这些问题，我们建议主要关注传统的Kinesin(Kinesin-I)的功能，因为在运动功能领域正在进行的许多感兴趣的问题可以在对这种经过充分研究的单个马达及其组件的研究中得到解决。在战术上，我们将在我们的实验中同时使用果蝇和老鼠，因为它们具有独特和互补的优势。因此，果蝇将被用来识别编码潜在调控蛋白或附着蛋白的新基因，并提供有关附属组件功能的基本信息。小鼠将被用来对首次在果蝇中识别或分析的基因进行详细的生理、细胞生物学和生化分析。为了实现这些总体目标，我们将在下一个项目期间达到三个具体目标：1)通过分析小鼠三个不同的Kinesin重链亚基(KIF5A、KIF5B和KIF5C)中的突变，了解传统Kinesin(Kinesin-I)的功能范围。这项工作将通过使用lox-cre系统产生系统性和条件性基因敲除突变体来进行。这些突变将主要在六种不同的细胞类型中进行分析，包括培养的胚胎成纤维细胞、肝细胞、光感受器、运动神经元、感觉神经元和培养的海马神经元。2)验证运动蛋白轻链(KLC)是货物附着或调节运动蛋白-I所必需的假说。这一目标将通过分析缺乏每个KLC亚基的系统性和条件性突变小鼠的生化和细胞表型来实现。3)识别和分析调节组件和货物附件组件中的新运动。这些新的成分将在果蝇中被鉴定和克隆，然后使用遗传学、细胞学和生化方法进行深入的表征。

Our long-term goal is to understand the molecular basis and logic of intracellular transport, which is crucial for normal neuronal and other cellular functions, and may play important roles in neurodegenerative and other disease processes. Our major emphasis is on kinesins, which generate directed movements along microtubules in a variety of cellular contexts including mitosis, vesicle traffic, and axonal transport. Specifically, we want to answer two general questions: What is the logic of kinesin motor utilization? How are kinesin motors regulated and attached to intracellular cargoes? To answer these questions, we propose to focus primarily on the functions of conventional kinesin (kinesin-I), because many of the issues of interest that are playing out in the motor function field can be attacked in studies of this well-studied single motor and its components. Tactically, we will use both Drosophila and mice in our experiments because of their unique and complementary advantages. Thus, Drosophila will be used to identify new genes encoding potential regulatory or attachment proteins and to provide basic information about accessory component functions. Mice will be used for detailed physiological, cell biological, and biochemical analyses of genes first identified or analyzed in Drosophila. To achieve these general goals, we will attack three specific aims in the next project period: 1) To understand the range of functions of conventional kinesin (kinesin-I) by analyzing mutants in the three different kinesin heavy chain subunits in mice (KIF5A, KIF5B, and KIF5C). This work will be carried out by generating systemic and conditional knockout mutants using the lox-cre system. These mutants will be analyzed primarily in six different cell types including cultured embryonic fibroblasts, hepatocytes, photoreceptors, motor neurons, sensory neurons, and cultured hippocampal neurons. 2) To test the hypothesis that kinesin light chain (KLC) is required for the cargo-attachment or regulation of kinesin-I. This aim will be achieved by analyzing the biochemical and cellular phenotype of systemic and conditional mouse mutants lacking each KLC subunit. 3) To identify and analyze new kinesin regulatory and cargo-attachment components. These new components will be identified and cloned in Drosophila and then characterized in depth using genetic, cytological, and biochemical methods.