Neuron- & Tissue-Specific Clathrin Light Chain Function

描述(申请人提供)：笼状蛋白包裹的囊泡(CCV)调节受体介导的内吞作用(RME)和神经传递，这两个关键事件调节神经元生长和神经系统功能。然而，CCV周期如何适应神经元功能仍不清楚。这项拟议的研究将探索和破坏笼状蛋白动力学的关键调节因子--笼状蛋白轻链(LCS)的功能，以了解CCV转运如何介导神经元分化和功能。有趣的是，两个不同的LC(LCA和LCB)相对于其他类型的细胞在神经元中不成比例地表达，这可能是为了除了RME之外促进神经传递。此外，神经元主要由交替拼接的LCs组成。为了区分LCA、LCB及其神经元的功能，酵母双杂交系统将筛选特定结合这些物种的蛋白质。对候选蛋白质的分析将阐明它们在调节神经细胞和非神经细胞中的网状蛋白动力学中各自的作用。此外，LCA和LCB在斑马鱼和小鼠中的表达将被干扰，以区分各自LCS在神经发育和神经系统功能中的体内功能。零基因将被分析为膜运输、突触和认知缺陷。最终，这些互补的方法将揭示LC监管的CCV运输如何指定组织和神经功能。 候选人Newmyer博士在研究CCV在培养的非神经元细胞中的结构性转运方面拥有丰富的经验。这项拟议的研究将把她的膜转运专业知识扩展到研究神经元内以及斑马鱼和小鼠体内受调控的CCV转运。纽迈尔博士已经在加州大学旧金山分校建立了合作关系，分析小鼠和斑马鱼体内LC调控的网状蛋白功能。拟议的研究将识别蛋白质并产生细胞系，以建立一个强大的研究计划，识别调节组织特异性CCV运输的分子和细胞机制。最终，这个项目将使候选人能够发展成为一名神经学家，并追求学术终身教职。

DESCRIPTION (provided by applicant): Clathrin-coated vesicles (CCVs) modulate receptor-mediated endocytosis (RME) and neurotransmission, two critical events that regulate neuronal growth and nervous system function. However, how the CCV cycle is adapted to neuronal function remains obscure. The proposed studies will explore and disrupt the functions of clathrin light chains (LCs), key regulators of clathrin dynamics, to learn how CCV trafficking mediates neuronal differentiation and function. Interestingly, two divergent LCs (LCa and LCb) are disproportionately expressed in neurons relative to other cell types, possibly to facilitate neurotransmission in addition to RME. Additionally, neurons are largely composed of alternatively spliced LCs. To distinguish the functions of LCa, LCb and their neuronal counterparts, a yeast two-hybrid system will screen proteins that specifically bind these species. Analysis of candidate proteins will elucidate their respective roles in modulating clathrin dynamics in the neuronal and non-neuronal cell. Additionally, LCa and LCb expression will be disrupted in zebrafish and mouse to distinguish the in vivo functions of the respective LCs in neuronal development and nervous system function. The null genotypes will be analyzed for membrane trafficking, synaptic and cognitive deficiencies. Ultimately these complementary approaches will reveal how LC-regulated CCV trafficking specifies tissue and neuronal function. The candidate Dr. Newmyer has extensive experience in studying constitutive CCV-mediated trafficking in cultured non-neuronal cells. The proposed research will expand her membrane trafficking expertise to studying regulated CCV trafficking within the neuron and within zebrafish and mouse. Dr. Newmyer has established collaborations within UCSF to analyze LC-regulated clathrin function within mice and zebrafish. The proposed studies will identify proteins and generate cell lines to establish a strong research program that identifies the molecular and cellular mechanisms that modulate tissue-specific CCV trafficking. Ultimately, this project will enable the candidate to develop into a neuroscientist and pursue an academic tenure track faculty position.