GENETIC AND IMMUNOLOGIC ANALYSIS OF MICROTUBULE PROTEINS

描述(由申请人提供)：分子马达蛋白在细胞内的运输对许多神经元和其他细胞过程是必不可少的。这些过程似乎包括定位细胞器和组织分泌，以及神经元信号、损伤反应和在神经退行性疾病和其他疾病中失败的过程。我们的重点是动蛋白，它在许多细胞环境中产生沿微管定向运动，包括有丝分裂、囊泡运输和轴突运输。我们实验室和其他实验室的大量工作已经提供了许多关于这些系统的基本性质的信息，但仍然没有回答几个重要的问题，其中两个问题将在即将到来的资金阶段解决：1)运动蛋白利用的逻辑是什么？2)运动蛋白马达是如何调节的，并附着在细胞内的货物上？为了回答这些问题，我们建议主要关注传统的动蛋白，即动蛋白-I。在战术上，我们同时使用老鼠和果蝇，因为它们的独特和互补的特征。为了实现我们的目标，我们将在下一个项目期实现三个具体目标。具体地说，我们建议：1)检验这一假设，即神经元特有形式的kinesin-I执行神经元特有的功能，如神经元特有货物的慢速和快速轴突运输，而无处不在的形式在神经细胞和非神经细胞中执行相同的一般功能。利用Iox-cre系统分析小鼠体内三个运动蛋白重链(KIF5A、KIF5B和KIF5C)以及两个主要运动蛋白轻链基因(KLC1和KLC2)的无效和条件性基因敲除突变。我们将集中于几种具有代表性的细胞类型，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。此外，我们还将检验相关假设，即Kinesin-I在神经细丝的缓慢轴突运输中发挥主要作用。2)验证关于Kinesin轻链(KLC)对货物附着和对Kinesin-I的调节都是必需的假设。这一假设将通过分析缺乏明确KLC亚基的小鼠突变的生化和细胞表型来检验。3)阐明两种不同的Kinesin-I囊泡附着复合体的结构和功能。我们将确定Syd/JIP-3和淀粉样前体蛋白复合体的组成和在Kinesin-I运输中的作用。

DESCRIPTION (provided by applicant): Intracellular transport by molecular motor proteins is essential for many neuronal and other cellular processes. These processes appear to include positioning organelles and organizing secretion, as well as neuronal signaling, damage responses, and processes that fail in neurodegenerative and other diseases. Our focus is on kinesins, which generate directed movements along microtubules in many cellular contexts including mitosis, vesicle traffic, and axonal transport. Considerable work from our laboratory and others has provided much information about the basic properties of these systems, but has left unanswered several important questions, two of which we will attack in the coming funding period: 1) What is the logic of kinesin motor utilization? 2) How are kinesin motors regulated and attached to intracellular cargoes? To answer these questions, we propose to focus primarily on conventional kinesin, kinesin-I. Tactically, we use both mice and Drosophila because of their unique and complementary features. To achieve our goals, we will attack three specific aims in the next project period. Specifically, we propose: 1) To test the hypothesis that neuron specific forms of kinesin-I carry out neuron-specific functions such as slow and fast axonal transport of neuron-specific cargoes, while ubiquitous forms carry out the same general functions in both neural and non-neural cells. Null and conditional knockout mutants in the three kinesin heavy chain (KIF5A, KIF5B, and KIF5C), and the two major kinesin light chain (KLC1 and KLC2) genes in the mouse will be generated and analyzed using the Iox-cre system. We will focus on a few representative cell types including cultured embryonic fibroblasts, hepatocytes, photoreceptors, motor neurons, sensory neurons, and cultured hippocampal neurons. In addition, we will test the related hypothesis that kinesin-I plays a major role in the slow axonal transport of neurofilaments. 2) To test the hypothesis that kinesin light chain (KLC) is required for both cargo-attachment and regulation of kinesin-I. This hypothesis will be tested by analyzing the biochemical and cellular phenotype of mouse mutants lacking defined KLC subunits. 3) To elucidate the structure and function of two different proposed kinesin-I vesicular attachment complexes. We will determine the composition and role in kinesin-I transport of components of the syd/JIP-3 and the amyloid precursor protein complexes.