dc.description.abstracteng | 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. | de |