The Use of Super-Resolution Microscopy Techniques to Study the Function of Neuronal Proteins
January 15th, 2018 RAMÓN MARTÍNEZ-MÁRMOL The University of Queensland

For decades, the small size (1 m in diameter) and the crowded environment of synaptic terminals and dendritic spines, together with the organization of many neuronal proteins in clusters that are well below the resolution limit of traditional confocal microscopy, constituted the main limitations to deciphering the molecular mechanisms that govern synaptic physiology. Traditionally, only the use of electrophysiology, amperometry and electron microscopy have been able to overcome these limitations and bring some light to the aforementioned question. However, these powerful techniques were greatly limited by the lack of their ability to provide dynamic information on protein mobility and nanoscale organization. To study the mobility of neuronal proteins under physiological and pathological conditions, with resolutions below the diffraction limits of light, our lab uses a combination of different super-resolution microscopy techniques. In my presentation, I will present our last results using single particle tracking PALM (sptPALM), on the mobility and nanoscale organization of crucial proteins for the progression of Alzheimer’s disease. I will also present new techniques and analysis being developed in the lab. Subdiffractional tracking of internalized molecules (sdTIM), consists on imaging internalized molecules tagged following a pulse-chase using bright fluorescent organic dyes such as Atto. To image synaptic vesicles in hippocampal nerve terminals, we used anti-green fluorescent protein Atto647N-tagged nanobodies trapped in SVs from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)–pHluorin. We could follow multiple SVs simultaneously with high spatiotemporal resolution (30 to 50 nm and 20 msec) and we have characterized both the number of active transport and diffusive states and transitions between these states.

Seminar, January 15, 2018, 16:00. ICFO’s Seminar Room

Hosted by Prof. María García-Parajo