GLUTRAF · Ultra-high resolution imaging of GluR1 trafficking in neuronal spines

Ultra-high resolution imaging of GluR1 trafficking in neuronal spines

Molecular organisation of the postsynaptic membrane is the core machinery involved in the functioning of an excitatory chemical synapse. Of the multiple components which orchestrate the reception of neurotransmitter release, AMPA receptors are the key molecules involved in the fast synaptic transmission. Over the years efforts have been made to understand the localisation and correlate subsequent function of these receptors along with molecules associated with them in neuronal synapses. It has been shown that AMPA receptors are recruited, incorporated and recycled in a transient manner and this mobility has been identified as a decisive step in controlling the synaptic plasticity. Thus the equilibrium between the synaptic and extra synaptic AMPA receptor number is crucial in controlling basal transmission and synaptic response. This balance is known to be regulated by the subunit composition of these receptors and by interacting intracellular scaffold proteins. However, how this machinery is organised in postsynaptic density or in response to the reception of chemical signal is still unclear. The major goal of our study during the funding period was be to analyse with the highest possible spatio-temporal resolution the molecular mechanisms involved in the recruitment and redistribution of AMPARs in the synapses. The major objectives for the funding period were devoted to understand molecular mechanisms of AMPAR stabilisation and their mobility and also AMPAR trafficking between different subcellular regions. The technological challenge was to try to acquire this information at the highest possible subcellular resolution from living cells. The focus of the first half of the funding period was the instrumentation and calibration of a new high density single molecule detection system based on photoactivation localisation microscopy in combination with single particle tracking. After these the newly developed single particle tracking photoactivated localisation microscopy (SPT-PALM) microscope was used for routine observation of physiological questions addressed in the proposed project, which will be summarised below. 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.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз