REGULATION OF AXON GUIDANCE BY ENA/VASP PROTEINS

18. 奖学金培训目标及职业名称(最后，第一，机构高尔盖特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SUNY hc - syracuse神经生物学威斯康星大学麦迪逊分校神经生物学威斯康星大学麦迪逊分校导师吉野君Katherine Kalil导师/雇主Karina Meiri博士Katherine Kalil博士我的职业目标是在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，我将研究哺乳动物中枢和外周神经系统轴突引导和可塑性的细胞骨架动力学和信号级联。为了帮助我实现这个目标，这个奖学金将支持以下活动。我将获得与转基因小鼠一起工作的专业知识，并利用表征良好的分子技术操纵神经元。我还将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。再加上我在博士预科和博士培训期间获得的尖端荧光和明场成像技术的专业知识，这些新的技术和智力方法将极大地增强我开发和追求独立研究项目的能力。赞助商19。姓名与学位Frank Gertler博士职位/职级副教授研究方向：细胞生物学，神经生物学I(Nk),"_！我:1 (ol我_1l (O)”dO_ ' _ ' L。22. 本应用程序的总体目标是阐明通过蛋白激酶A （PKA）磷酸化Ena/VASP在轴突生长和中枢神经系统发育中的指导作用。首先，生成Ena/VASP-null小鼠。如果由于早期产前致死而无法创建三重敲除小鼠，则将采用Cre/lox重组系统的条件敲除策略。其次，将Ena/VASP蛋白磷酸化突变体引入Ena/VASP缺失的神经元，并评估其对生长锥运动和轴突生长的影响。为了测试Ena/VASP磷酸化对轴突引导皮质神经元的功能，将磷酸化突变体暴露于netrin和BDNF梯度下；两种已知通过PKA发出信号的分子。Ena/VASP蛋白也与A激酶锚定蛋白（AKAPs）的结合有关。为了确定皮质神经元中哪些akap与Ena/VASP蛋白相关，将与PKA的II型调节亚基进行共免疫沉淀和凝胶覆盖。发现结合Ena/VASP蛋白的akap将被克隆并用YFP标记。荧光共振能量转移（FRET）将与CFP-Ena/VASP蛋白进行，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蛋白在中枢神经系统发育中的功能对于理解定向神经元迁移和生长受损的人类中枢神经系统疾病非常重要。PHR 4 tr-1 (RAy 1;) / f:三Fnrm P_nA _ RB CC个人NRSA应用程序目录 ======================================== 节结束 ===========================================

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===========================================