Investigation of Neuronal Protein Trafficking at the Molecular Scale
Time: Fri 2023-12-15 09.00
Location: Air&Fire, Tomtebodavägen 23, 171 65, Solna
Subject area: Physics, Biological and Biomedical Physics
Doctoral student: Martina Damenti , Biofysik, Ilaria Testa
Opponent: Professor Clive Bramham, University of Bergen
Supervisor: Associate Professor Ilaria Testa, Skolan för teknikvetenskap (SCI)
Neurons are polarized cells that encode information in the nervous system viaelectrochemical connections named synapses. The tuning of synapticconnections is enabled by a plastic protein trafficking system which operates atthe nanoscale to finely tweak the neuronal ultrastructure. Our understanding ofthe neuronal biology has certainly benefited from the advent of live-cellcompatible fluorescence techniques able to reach the molecular level. However,the neuronal trafficking system involves molecular complexes, from organelles tosynaptic modulators, which act with varying dynamics at differentspatiotemporal scales. A single technique struggles to portrait these complexphenomena since it is hard to combine molecular resolution, speed, and lowphototoxicity. Hence, their investigation often demands, together with technicaladvancements, the combination of advanced fluorescence methods withcomplementary features. In this thesis, I explore the neuronal protein traffickingsystem at the molecular scale applying cutting-edge fluorescence microscopy andspectroscopy techniques.The relationship between the geometry and dynamics of the tubular endoplasmicnetwork and the sub-compartment size of neurons is investigated using acombination of STED and parallelized RESOLFT microscopy. In addition, thethree-dimensional dynamic interaction between tubular endoplasmic reticulumand mitochondria is described.The basal activity-driven recycling of synaptic vesicles is, for the first time,monitored via event-triggered STED, an automated method able to initiate STEDimaging upon detection of events such as calcium spikes.Insights into the post-synaptic reorganization of scaffolding and skeletal proteinsupon stimulation is gained by extending the live-cell super-resolution throughputto all the dimensions with multi-foci and 3D parallelized RESOLFT.Lastly, the molecular states of Activity-Regulated Cytoskeleton-Associatedprotein (Arc) involved in distinct aspects of neuronal protein trafficking arestudied. Our observations, obtained combining distinct advanced methods asDNA-PAINT and STARSS, support a previously unexplored Arc mechanism ofaction.