For those interested in neuromodulators:
Treatment of striatal neurons with a D1 receptor agonist led to an increase in the dendritic staining intensity of NMDA receptor NR2B subunits. There was also an increase in the association of NR2B subunits with PSD-95 — a scaffold protein required for the assembly of NMDA receptors — and in the surface localization of NR2B-containing receptors.
Original article in J. Neurosci. from Dunah and colleagues. An excerpt from the original aricle of a neat application of FRET continues after the jump.
FRET was used to detect where the phosphorylated NR2B was in pervanadate-treated (pervanadate is a non-specific tyrosine phophatase inhibitor… ie. it prevents dephosphorylation) and normal striatal culture:
To confirm the localization of tyrosine-phosphorylated NR2B subunit in intact cells, we applied FLIM, a fluorescence resonance energy transfer (FRET)-based approach to detect protein–protein interactions (Fig. 8A, quantified in B). Untreated and pervanadate-treated striatal neurons at DIV21 were double immunostained under permeabilizing conditions for the NR2B subunit using an antibody to the C terminus (Wang et al., 1995Go) and for phosphotyrosine proteins with the anti-phosphotyrosine antibody; the donor fluorophore (NR2B) was labeled with Alexa Fluor 488 and the acceptor fluorophore (phosphotyrosine proteins) with Cy3. Proximity of the two fluorophores results in resonance energy transfer and shortens the fluorescence lifetime of the donor. We measured the donor lifetime in each image pixel using a pulsed laser and dual-photon confocal system. For negative controls, cells stained with only the donor fluorophore (Alexa Fluor 488) were used.
Using this approach, we detected robust staining for NR2B in both the soma and dendrites of neurons. In the untreated cells, only a small proportion of pixels exhibited a shortened donor lifetime, and these were concentrated along the dendritic regions (Fig. 8Ad,B), indicating the presence of tyrosine-phosphorylated NR2B. Treatment of striatal neurons with the phosphatase inhibitor pervanadate markedly enhanced the number of pixels with a short fluorescent lifetime, indicating a large increase in the extent of tyrosine phosphorylation of the carboxy tail of NR2B. Quantitative analysis of the signals in both the soma and the dendrites revealed that tyrosine-phosphorylated NR2B was increased in both compartments, but the effect was more than six times greater in the dendrites than in the soma (Fig. 8Af,B). These data demonstrate a strong association between tyrosine phosphorylation of NR2B and dendritic localization of the receptor protein.