REGULATION OF AXON GUIDANCE BY ENA/VASP PROTEINS

18.奖学金培训的目标和职业名称(最后，第一个，中间首字母)说明_登特_埃里克W机构高露洁大学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-锡拉丘兹神经生物学大学威斯康星-麦迪逊神经生物学大学导师Jun Yoshino Katherine Kalil导师/雇主Karina Meiri博士Katherine Kalil博士Katherine 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。具体地说，我将研究哺乳动物中枢和外周神经系统的轴突引导和可塑性所涉及的细胞骨架动力学和信号级联。为了帮助我实现这一目标，该奖学金将支持以下活动。我将在转基因小鼠的工作中获得专业知识，并利用具有良好特征的分子技术来操纵神经元。我还将加强我在生物化学方面的培训。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，将使我接触到许多研究发育神经生物学的互补的智力方法。再加上我在博士前和博士后培训期间获得的尖端荧光和亮场成像技术方面的专业知识，这些新的技术和智力方法将极大地增强我开发和从事独立研究计划的能力。19.赞助商姓名及学位(S)葛德乐博士20.职位/职级副教授研究方向/领域细胞生物学、神经生物学I(NK，“_！：1I(ol I_1l(O]”do_‘_’L.22.说明(不超过所提供的篇幅)本申请的总体目标是阐明蛋白激酶A(PKA)的Ena/Vasp磷酸化在轴突生长和在发育中的中枢神经系统中的指导作用。首先，将产生Ena/Vasp缺失的小鼠。如果由于早期的产前死亡而不可能产生三重基因敲除小鼠，则将通过重组的Cre/lox系统来采用有条件的基因敲除策略。其次，将Ena/Vasp蛋白的磷酸化突变体引入Ena/Vasp缺失神经元，并评估它们对生长锥运动和轴突生长的影响。为了测试Ena/Vasp在轴突导向上的磷酸化功能，含有磷酸化突变体的皮质神经元将暴露于Netrin和BDNF的梯度中，这两个分子通过PKA发出信号。Ena/Vasp蛋白也与锚定L蛋白(AKAP)的A_激酶结合有关。为了确定哪些AKAP与皮层神经元中的ena/Vasp蛋白相关联，将与PKA的II型调节亚基进行免疫共沉淀和凝胶覆盖。已发现与Ena/Vasp蛋白结合的AKAP将被克隆并用YFP标记。荧光共振能量转移(FRET)将与CFP-EnA/Vasp蛋白一起进行，以确定EnA/Vasp蛋白和AKAP在生长锥体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了解人类中枢神经系统疾病的定向迁移和突起生长受损具有重要意义。Phr 4tr-1(射线1；)/f：TRI Fnrm P_NA_RB CC个人非RSA应用目录========================================Section End===========================================

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