Imaging Molecules at Synapses with Mini-SOG Constructs

Wednesday, November 06, 2013 — Poster Session I
4:00 p.m. – 6:00 p.m.	FAES Academic Center (Upper-Level Terrace)	NINDS	NEURO-3

Authors

X Chen
P Gallant
C Winters
M Lazarou
X Li
R Youle
A Sousa
R Leapman
T Reese

Abstract

Photoconversion of miniSOG is evident at spines in rat hippocampal cultures transfected with a C-terminal fusion PSD-95–miniSOG construct (Chen et al., 2012). Mini-SOG generates singlet oxygen to locally precipitate diaminobenzidine (DAB), which reacts with OsO4 to become visible evident by electron microscopy. The propensity of some DAB photo-reaction products to diffuse (Courtnoy et al., 1983) was manifest in our initial experiments. We have now developed a method to minimize diffusion to more definitively localize expressed PSD-95 at the postsynaptic density (PSD). High concentrations of sucrose were added to the photoconversion medium to slow diffusion. Adding sucrose at 40-60% (w/w) to the DAB incubation medium effectively restricted reaction products to within 30 nm of the PSD. EM tomography of photo-reaction positive synapses resolved the individual vertical filaments thought to contain PSD-95 at the PSD. Moreover, some of the vertical filaments terminated in electron dense structures up to 10 nm in diameter at the distal ends of the vertical filaments, where we expected to find the mini-SOG fusion sites for PSD-95 (Chen et al, 2011). Other mini-SOG constructs expressing synaptic proteins are being developed to compare their localizations to that of PSD-95 at synapses.