1. The role of of SynCAM2a in synapse formation Sofia Nakhnikian-Weintraub, Smith College 2012 Mentor: Courtney Easley-Neal, PI: Phil Washbourne

2. Synapse Formation Axonal growth cone filopodia dendrite Adapted from Washbourne et al 2004  SynCAMs

3. SynCAM1 can induce synapse formation in vitro non neuronal cell axon non neuronal cell transfected with syncam1 labeled with presynaptic markers Hippocampal neurons form synaptic terminals onto SynCAM- expressing non-neuronal cells

4. SynCAM structure SynCAMs are present in mice, zebrafish and humans All contain 3 immunoglobulin (IG) domains, a transmembrane domain, and a 4.1 binding and PDZ binding domain

5. Experimental Plan Subclone 4 SynCAM2a constructs (full-length, and 3 deletions: 4.1B, PDZ, and 2X [both protein binding domains] deletions into a vector with a fluorescent tag. Microinject zebrafish embryos with the 4 constructs. Stain known pre and postsynaptic proteins through immunohistochemistry (IHC) to determine whether SynCAM is present at synapses in the zf spinal cord, and to assess whether colocalization occurs between synaptic proteins and SynCAM

6. Why use Zebrafish? Zebrafish, like humans, are vertebrates. Zebrafish develop rapidly, and have functional nervous systems within 1 day. The embryos are fertilized externally and there is no parental care, allowing for collection and manipulation of eggs. Zebrafish are transparent for the first few days of life, allowing in vivo experimentation, and live imaging.

7. Zebrafish development Adapted from Kimmel et al 1995 Spontaneous contractions begin 17hpf Touch response begins 21hpf 16hpf19hpf22hpf25hpf

8. SynCAM2a expression in RB cells involved in the touch response In situ 24 hpf SynCAM2a expression Touch response circuitry

9. Restricting expression of SynCAM2a with transgenic lines We have a line that expresses gal4 in the RBs, I will inject with a UAS SynCAM2a full length and dominant negative construct.

10. Syncam2a deletion constructs are missing one or both protein interaction domains SynCAM2a ΔPDZ Δ4.1B ΔPDZ/ Δ 4.1B Transmembrane domain 4.1B domain PDZ domain

11. Steps for making S2a fl and deletion constructs Insert: PCR, TOPO cloning, miniprepping, restriction digest Vector: Digest, dephosphoryolation Ligation, transformation Miniprepping – isolating the DNA from bacteria. Insert Vector backbone Complete plasmid

12. Microinjection is a method used to introduce DNA constructs into embryos A capillary needle is used to inject 3-4 nL of the DNA constructs into one cell embryos.

13. New cloning strategy The fluorescent tag was inhibiting protein function, so we will attach the tag to the other end of the protein. Subclone in pieces: signal sequence, coding sequence and fluorescent tag Instead of UAS S2a full length-make we will make UAS- signal sequence-mkate-S2a (coding sequence)-full length This adds several steps to the cloning process

14. Antibody staining (IHC) of pre and post synaptic proteins

16. Future Directions Finish cloning the new constructs Inject embryos with the constructs Confirm presence of SynCAM2a at synapses with full length construct Assess affect of the dominant negative constructs on protein recruitment and synaptic function Behavioral analysis of fish expressing full-length and dominant negative constructs