REGULATION OF AXON GUIDANCE BY ENA/VASP PROTEINS

18.奖学金培训目标和职业名称（最后，第一，中首字母）说明_ DENT_ ERIK W机构高露洁大学博士威斯康星大学麦迪逊分校博士机构/公司领域神经生物学纽约州立大学HSC-Syracuse神经生物学威斯康星大学麦迪逊分校神经生物学导师Jun Yoshino凯瑟琳Kalil导师/雇主Karina Meiri博士凯瑟琳Kalil博士凯瑟琳Kalil博士我的职业目标是在一所研究型大学获得神经生物学/细胞生物学领域的教职。具体来说，我将研究细胞骨架动力学和信号级联参与轴突的指导和可塑性的中枢和外周神经系统的哺乳动物。为了帮助我实现这一目标，该奖学金将支持以下活动。我将获得专业知识，在与转基因小鼠和操纵神经元与良好表征的分子技术。我也会加强我在生物化学方面的训练。此外，参与麻省理工学院生物系充满活力和多样化的研究社区将使我接触到许多互补的智力方法来研究发育神经生物学。再加上我在博士前和博士培训期间获得的尖端荧光和明场成像技术方面的专业知识，这些新的技术和知识方法将大大提高我开发和追求独立研究计划的能力。19. honeymoon姓名和学位Frank Gertler r Ph.D. 20. 21.第21章.研究兴趣/领域细胞生物学，神经生物学I（Nk，"_！1 I（ol I I（O）“dO "L. 22.本申请的总体目标是阐明蛋白激酶A（PKA）引起的Ena/VASP磷酸化在发育中的中枢神经系统中轴突生长和引导中的功能。首先，将产生Ena/VASP缺失小鼠。如果由于早期产前致死性而不可能产生三重敲除小鼠，则将通过Cre/lox重组系统采用条件性敲除策略。第二，将Ena/VASP蛋白的磷酸化突变体引入Ena/VASP缺失的神经元中，并评估它们对生长锥运动性和轴突生长的影响。为了测试Ena/VASP磷酸化对轴突引导的功能，将含有磷酸化突变体的皮质神经元暴露于netrin和BDNF的梯度;已知通过PKA发出信号的两种分子。Ena/VASP蛋白还涉及结合A _激酶锚定L_鱼藤蛋白（AKAP）。为了确定哪些AKAP与皮质神经元中的Ena/VASP蛋白相关，将用PKA的II型调节亚基进行免疫共沉淀和凝胶覆盖。将克隆发现结合Ena/VASP蛋白的AKAP并用YFP标记。将用CFP-Ena/VASP蛋白进行荧光共振能量转移（FRET），以确定Ena/VASP蛋白和AKAP在生长锥中何时何地相互作用。阐明Ena/VASP蛋白在CNS发育中的功能对于理解其中定向神经元迁移和生长受损的人类CNS疾病将是重要的。PHR 4 tR-1（RAy 1;）/f：tRI Fnrm P_nA _ RB CC单个NRSA应用程序目录=

18. GOALS FOR FELLOWSHIP TRAINING AND CAREER NAME (Last, first, middle initial) instructions_ DENT_ ERIK W INSTITUTION Colgate University Dr. University of Wisconsin-Madison Dr. INSTITUTION/COMPANY FIELD Neurobiology SUNY HSC-Syracuse Neurobiology Univ Wisconsin-Madison Neurobiology Univ Wisconsin-Madison MENTOR Jun Yoshino Katherine Kalil SUPERVISOR/EMPLOYER Dr. Karina Meiri Dr. Katherine Kalil Dr. Katherine Kalil My career goals are to obtain a faculty position in the field of neurobiology/cell biology at a research university. Specifically, I will study the cytoskeletal dynamics and signaling cascades involved in axon guidance and plasticity of the central and peripheral nervous systems of mammals. To help me achieve this goal this fellowship will support the following activities. I will gain expertise in working with transgenic mice and manipulating neurons with well characterized molecular techniques. I will also enhance my training in biochemistry. Furthermore, participation in the dynamic and diverse research community in the biology department at MIT will expose me to many complementary intellectual approaches to studying developmental neurobiology. Coupled with my expertise in cutting edge fluorescent and brightfield imaging techniques acquired during my predoctoral and doctoral training, these new technical and intellectual approaches will greatly enhance my ability to develop and pursue an independent research program. SPONSOR 19. NAME AND DEGREE(S) Frank Gertler r Ph.D. 20. POSITION/RANK Associate Professor 21. RESEARCH INTERESTS/AREAS Cell Biology, Neurobiology I(Nk,"_!:1 I(ol I I _1l(O] "dO_'_'L. 22. DESCRIPTION (Do not exceed space provided) The overall goal of this application is to elucidate the function of Ena/VASP phosphorylation by protein kinase A (PKA) in axon outgrowth and guidance in the developing central nervous system. First, Ena/VASP-null mice will be generated. If it is not possible to create triple knockout mice due to early prenatal lethality, a conditional knockout strategy will be employed by means of the Cre/lox system of recombination. Second, phosphorylation mutants of Ena/VASP proteins will be introduced into Ena/VASP-null neurons and their effects on growth cone motility and axon outgrowth will be assessed. To test the function of Ena/VASP phosphorylation on axon guidance cortical neurons containing phosphorylation mutants will be exposed to gradients of netrin and BDNF; two molecules known to signal through PKA. Ena/VASP proteins have also been implicated in binding A _kinase anchoring l_roteins (AKAPs). In order to determine which AKAPs associate with Ena/VASP proteins in cortical neurons co-immunoprecipitations and gel overlays will be performed with the type II regulatory subunit of PKA. AKAPs discovered to bind Ena/VASP proteins will be cloned and labeled with YFP. Fluorescent resonance energy transfer (FRET) will be performed with CFP-Ena/VASP proteins to determine where and when Ena/VASP proteins and AKAPs interact in growth cones. An elucidation of the function of Ena/VASP proteins in CNS development will be important for understanding human CNS diseases where directed neuronal migration and outgrowth are impaired. PHR 4tR-1 (RAy 1;)/f:tRI Fnrm P_nA _ RB CC Individual NRSA Application Table of Contents ========================================Section End===========================================