Super-resolution single molecule imaging techniques have provided tremendous help to decipher molecular and cellular mechanisms. Fluorescent probes tagged to proteins of interest have allowed tracking of single proteins in various membranous compartments such as the plasma membrane. Single molecule imaging has been instrumental in our current understanding of the nanoscale organisation of the plasma membrane which contain small nanoscale domains enriched with various proteins and lipids. Nanodomains cause reduced diffusion of certain molecules via protein-protein, protein-lipid interactions, and cytoskeletal structures. These nanodomains facilitate biological functions occurring on the plasma membrane such as exocytosis, endocytosis and signalling. In this seminar, I will introduce single molecule imaging techniques and the challenges facing the field when dealing with terabytes of data to analyse on a daily basis. I will also present some recent results suggesting that mobility states and nanocluster organisation are dynamically regulated and involved in protein functions [1, 2]. Finally, I will conclude on the development of new technologies allowing single molecule imaging of subdiffractional endocytic structures [3, 4] as well as GFP-tagged and endogenous proteins for the first time.
1. Bademosi, A.T. et al. (2017) In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters. Nat Commun 8, 13660.
2. Kasula, R. et al. (2016) The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming. J Cell Biol 214 (7), 847-58.
3. Joensuu, M. et al. (2017) Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules. Nat Protoc 12 (12), 2590-2622.
4. Joensuu, M. et al. (2016) Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles. J Cell Biol 215 (2), 277-292.