Final Report Summary - NANOMAP (The Synapse Nanomap)
The organization of the cell determines its biological function. Despite its importance, this aspect has been difficult to investigate, since conventional imaging techniques cannot determine the position of the cellular elements with sufficient precision. Light imaging is normally limited by diffraction to spots of ~200-300 nm in size, while electron microscopy provides unsurpassed resolution, but limited labelling efficiency. We used a diffraction-unlimited technique, stimulated emission depletion (STED) microscopy, which brings the imaging resolution to ~30-60 nm, thus allowing us to pinpoint the position of various cellular elements with nanometer accuracy. Using this technique, we focused on providing a functional map of cellular elements within the synapse – a nanomap of the synapse. We determined the positions proteins summing to almost 50% of the synapse weight, in three different systems: isolated cortical synapses, cultured hippocampal synapses and the mouse nerve-muscle synapse. We used a combination of STED imaging, electron microscopy positioning and in silico modelling to obtain structural and functional information. Moreover, we went beyond imaging technology by employing quantitative biochemistry to investigate the copy numbers for each of these proteins. By a combination of quantitative Western Blotting and modern mass spectrometry approaches we obtained the copy numbers for ~1200 proteins in the synapses, making over 90% of the synapse.
The data was all assembled into the form of a 3D nanomap of the synapse, as proposed in the original application. This map is currently finished and will be soon ready for on-line viewing.
The data was all assembled into the form of a 3D nanomap of the synapse, as proposed in the original application. This map is currently finished and will be soon ready for on-line viewing.