Post-docs and students Details: Kim Doré, Post-doc
Website: www.greenspine.ca
Research : Fluorescence lifetime imaging (FLIM) of protein interactions in the synapses of hippocampal neurons and development of a high-resolution microscope based on Stimulated emission depletion (STED) in order to do ultrastructural localization of these interactions.
Each neuron in the brain forms thousands of excitatory synapses onto tiny, micron-sized spines that contain an extremely high density of signaling proteins, just apposed to the pre-synaptic neurotransmitter release site. CaMKII, a calcium/calmodulin-dependent kinase, is one of the most abundant proteins of the post-synaptic density. We are studying the dynamic interactions between CaMKII and glutamate receptors, such as the NMDA receptor, as well as other proteins present in spines, as mounting evidence suggests that those interactions play an essential role in forms of synaptic plasticity involved in learning and memory. By tagging proteins of interest with fluorescent proteins and expressing them in neurons by gene transfer, we can use Förster Resonance Energy Transfer (FRET) to reveal interacting partners. To quantify FRET in neurons, we are imaging the fluorescence lifetime (FLIM) of the donor fluorophore. For example, by using neurons co-transfected with a mCherry-tagged CaMKII along with a GFP-tagged NMDA receptor (subunit NR1), we find that the lifetime of GFP is reduced, following glutamate stimulation, revealing an interaction between the NMDA receptor and CaMKII. However, the spatial resolution of optical microscopy is limited to approximately 200 nm, which is clearly insufficient for studying nanometer range interactions within the micron-sized spines. By incorporating into our custom-built FLIM microscope a new technique, Stimulated Emission Depletion (STED), which can improve the optical resolution of fluorescence microscopy to the 20-70 nm domain, we will be able to perform ultrastructural localization of the studied protein-protein interactions in living cells.
FLIM of hippocampal neurons expressing GFP-NR1 (NMDAR sub-unit) and mCherry-CaMKII. Color coding indicates GFP-NR1 lifetime, red color regions are where the CaMKII-NMDAR interaction is maximal A) Unstimulated neurons B) Neurons after 1 min stimulation with glutamate and glycine (NMDAR agonists). |
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