Download SynCAM2a ΔPDZ Δ4.1B ΔPDZ - University of Oregon (SPUR)

The role of of SynCAM2a in synapse
formation
 Sofia Nakhnikian-Weintraub, Smith College 2012
 Mentor: Courtney Easley-Neal, PI: Phil Washbourne
Synapse Formation
Axonal growth
cone filopodia
SynCAMs
dendrite
Adapted from Washbourne et al 2004
SynCAM1 can induce synapse formation
in vitro
axon
non neuronal
cell
non neuronal cell
transfected with
syncam1
non neuronal cell
transfected with syncam1
labeled with presynaptic
markers
•Hippocampal neurons form synaptic terminals onto
SynCAM- expressing non-neuronal cells
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
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
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.
Zebrafish development
16hpf
19hpf
17hpf
Spontaneous
contractions
begin
22hpf
25hpf
21hpf
Touch
response
begins
Adapted from Kimmel et al 1995
SynCAM2a expression in RB cells
involved in the touch response
Touch response
circuitry
In situ 24 hpf
SynCAM2a
expression
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.
Syncam2a deletion constructs are missing one or
both protein interaction domains
Transmembrane
domain
4.1B domain
PDZ domain
SynCAM2a ΔPDZ
Δ4.1B
ΔPDZ/
Δ 4.1B
Steps for making S2a fl and deletion
constructs
 Insert: PCR, TOPO cloning,
miniprepping, restriction
digest
 Vector: Digest,
dephosphoryolation
Insert
Vector
backbone
 Ligation, transformation
 Miniprepping – isolating the
DNA from bacteria.
Complete
plasmid
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.
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 UASsignal sequence-mkate-S2a (coding sequence)-full
length
 This adds several steps to the cloning process
Antibody staining (IHC) of pre and post
synaptic proteins
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