Final Report Summary - GLUTRAF (Ultra-high resolution imaging of GluR1 trafficking in neuronal spines)
Aim 1: Molecular mechanisms of AMPA receptor stabilisation
Hippocampal neurons were transfected with different AMPA receptor subunits fused to EOS fluorescent protein. These were either HA-mEos2-GluA1, EOS-TEV-HA-GluA1, myc-GluA2-tdEOS alone or in combination with Homer1c Cerulean, HA-GluA1 or Homer1c GFP. The SPT-PALM results indicated that the localisation and mobility of the subunits in each cases were markedly different. GluA1 subunit of the AMPA receptors showed very low mobility and was immobilised in several 50 - 80 nm clusters which were named as nanodomains. The number of these clusters varied from spine to spine. However, almost 80 % of the spines had at least one nanodomain. Interestingly the overexpressed of GluA2 subunit showed very high mobility and no or very little confinement at the synapses. On co-expressing untagged GluA1 along with GluA2 we were able to restore the clustering, indicating that the stochiometry between various subunits was indeed important for the receptor clustering at the synapse. Furthermore, we showed with complementary high density single molecule technique named universal point acquisition of nanoscale topography (uPAINT) that the endogenous AMPA receptors are also organised to nanodomains. We also used stimulated emission depletion microscopy (STED) to show that surface AMPA receptors show similar distribution indicating a functional organisation on the postsynaptic membrane. As a result of careful analysis of diffusional behaviour of several single molecules in and out of nanodomains, we were able to conclude that these domains are the result of immobilisation dynamics of several receptor molecules. We also found that several of these domains are stable for minutes but there are also the formation and deletion of domains along time. It was also observed that there are three possible mechanistic modes of how the receptors are trafficked along the spatial environment of the receptor. We showed the individual receptor molecules can show strong, weak or no confinement before getting immobilised strongly indicating a very short organisation controlled by local environment at the posysynaptic density.