REGULATION OF AXON GUIDANCE BY ENA/VASP PROTEINS

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