An imaging toolbox for understanding the molecular biology of calcium triggered vesicle fusion in situ
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Calcium ions in the human body are responsible for cell homeostasis. In addition, many cell functions, such as synaptic transmission, hormone and neuropeptide release are triggered by calcium. Ca2+ concentration must be therefore carefully regulated by voltagegated calcium channels. Exocytosis is the process of the fusion of the secretory vesicle with the plasma membrane. For effective and fast exocytosis, vesicles must be docked near calcium channels. The probability of the release of synaptic vesicles is hypothesized to increase with the number of proximal calcium channels. N-type (Cav2.2) calcium channels cooperate directly with SNARE proteins and synaptotagmin through the specific synprint site, which is located on the main pore forming subunit. In recent years the amount of research on calcium channels has markedly increased, but there are still limitations in the methods used to study Ca2+. This results in a loss of information with regard to the true location of a point source that is emitting light and therefore the proteins we want to localize. The development of super-resolution techniques allows imaging of these proteins closer to the molecular scale, enabling us to better understand these cellular processes. The results presented in this thesis aim to understand the distribution and behaviour of Ntype calcium channels across the cell membrane, using advanced microscopy techniques to image below the diffraction limit. Our findings revealed that the synprint site has an influence on Cav2.2 calcium channel cluster patterning and behaviour. The results demonstrate no direct interaction between Cav2.2 calcium channels and syntaxin-1A. The experiments with a genetically encoded calcium indicator fused to a SNARE protein together with TIRF microscopy present a promising method to examine the calcium “activity” across the plasma membrane. Findings presented in this thesis introduce a new angle of looking at the interaction of syntaxin-1A with the synprint motif of Cav2.2 calcium channels. Taken together the results can create a novel model of the distribution and behaviour of Cav2.2 calcium channels in the secretory cells and it is recommended that these methods are employed more widely in the future to investigate ion channel distribution and function in cells.