The Dynamic Organization of Proteins in the Synaptic Bouton
by Sofiia Reshetniak
Date of Examination:2021-10-13
Date of issue:2022-09-12
Advisor:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Andreas Janshoff
Persistent Address:
http://dx.doi.org/10.53846/goediss-9435
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Abstract
English
Protein availability is crucial for maintaining cellular processes. In synapses, the identities and functions of proteins involved in neurotransmission are well known. Their spatial distribution, copy numbers, and lifetimes have also been thoroughly investigated. However, little information is available on their mobility and its regulation. In this thesis, I have addressed this issue by analyzing the mobility of 45 diverse proteins in the synapses of primary hippocampal neurons. Relying on the fluorescence recovery after photobleaching (FRAP) technique, I have measured the mobility of proteins in the axons and synapses of living neurons. The FRAP results were then combined with electron microscopy data and in silico simulations to extract detailed information on protein mobility. The results provided diffusion coefficients for the analyzed proteins in various locations of the synapto-axonal compartment, demonstrating generally lower mobility in the synapses compared to the axons, and, as expected, lower mobility for membrane proteins as compared to soluble proteins. The main parameters affecting protein mobility were determined to be synaptic geometry and binding to the synaptic vesicles. I then aimed to further investigate the role of the synaptic vesicles in the regulation of synaptic protein mobility. An in vitro approach was employed to measure the diffusion of purified synaptic proteins in the presence or absence of synaptic vesicles in a cell-free system, using fluorescence correlation spectroscopy (FCS). The results demonstrated a major impact of the synaptic vesicles on protein mobility, but implied that other parameters, such as the spatial organization of the synaptic vesicle cluster, also play a role in determining protein mobility in the synapse. Combined with previous observations, these results suggest a fundamental role of the synaptic vesicle cluster in the organization and maintenance of synaptic morphology and physiology. The generated data provide quantitative information on the mobility (expressed as a fraction of mobile molecules and their diffusion coefficients) and distribution of major synaptic proteins (including key SNAREs, endo- and exocytosis co-factors, and cytoskeletal components) in synapses, the synaptic vesicle cluster, and axons. This represents the most detailed view of the dynamic organization of proteins in the synaptic bouton presently available and was used to generate a representational visual rendering of protein mobility in the synaptic bouton. Additionally, the quantitative data provided here can be used while generating computational models of synaptic physiology, allowing for models of higher precision than were previously possible. For example, these data have been used to computationally analyze the involvement of dynamin and clathrin in the endocytosis of synaptic vesicles. By considering the mobility rates and copy numbers of the corresponding proteins, such analysis explained why clathrin-independent pathways of vesicle retrieval must be employed to maintain the spontaneous network activity. This example demonstrates how the mobility data can be used to obtain additional information on synaptic processes that were difficult to address in the past. The generated database of synaptic protein mobility data will be useful not only for laboratories specializing in the modeling and system biology of synapses, but also for investigations of individual proteins, and the generated visualizations can also be used for educational purposes. As such, the results presented here have a broad spectrum of potential applications.
Keywords: diffusion; protein mobility; synapse; synaptic vesicle
