Neuron- & Tissue-Specific Clathrin Light Chain Function

描述（由申请人提供）：网格蛋白包被囊泡（CCV）调节受体介导的内吞作用（RME）和神经传递，这是调节神经元生长和神经系统功能的两个关键事件。然而，CCV周期如何适应神经元功能仍然不清楚。拟议的研究将探索和破坏网格蛋白轻链（LC）的功能，网格蛋白动力学的关键调节因子，以了解CCV贩运如何介导神经元分化和功能。有趣的是，两种不同的LC（LCa和LCb）在神经元中的表达与其他细胞类型不成比例，这可能是为了促进RME以外的神经传递。此外，神经元主要由选择性剪接的LC组成。为了区分LCa，LCb及其神经元对应物的功能，酵母双杂交系统将筛选特异性结合这些物种的蛋白质。候选蛋白的分析将阐明它们各自在调节神经元和非神经元细胞中的网格蛋白动力学中的作用。此外，将在斑马鱼和小鼠中破坏LCa和LCb表达，以区分各自LC在神经元发育和神经系统功能中的体内功能。将分析无效基因型的膜运输、突触和认知缺陷。最终，这些互补的方法将揭示LC调控的CCV运输如何指定组织和神经元功能。 候选人Newmyer博士在研究培养的非神经元细胞中组成型CCV介导的运输方面具有丰富的经验。拟议的研究将扩大她的膜运输专业知识，研究神经元内和斑马鱼和小鼠内的CCV运输。Newmyer博士与UCSF建立了合作关系，以分析小鼠和斑马鱼中LC调节的网格蛋白功能。拟议的研究将确定蛋白质和产生细胞系，以建立一个强大的研究计划，确定调节组织特异性CCV运输的分子和细胞机制。最终，这个项目将使候选人发展成为一名神经科学家，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.