Molecular biology of calcium trigger vesicle fusion in situ.

Calcium ions in the human body serve as second messengers and are responsible for cell homeostasis. Muscle contractions and neuronal communication are supported by electrical activity. Voltage-activated calcium channels are crucial for many cellular events such as secretion of hormones or neuropeptides. In last few years the amount of research on Ca2+ channels has markedly increased, but there are limitations related to physical limitations in the spatial resolution of fluorescence microscopy. This phenomenon results in a loss of information with regard to the true location of a point source that is emitting light. There are a newly developed methods such as Stimulated Emission Depletion Microscopy (STED) or Photoactivated localization microscopy (PALM) that allows to imaging closer to the molecular scale.
The general aims of my project so far were focused on understanding the mechanism of action and distribution of N-type calcium channels by using novel tools I have helped to develop for calcium channel imaging.
To find out if Cav2.2 Ca2+ (Neuronal type) channels are in a clustered conformation or randomly distributed in the cellular plasma membrane, STED was used. After measuring the size of the signals in the STED image data we can conclude that Cav2.2 are patterned in clusters, with regard to the diffraction limit of STED microscope. To support that conclusion and characterize our imaging capability, more experiments using known size, sub-diffraction fluorescent beads and viruses capsids filled with GFP will be conducted.
To quantify the mobility and location of N-type calcium channels PALM was used. Using a photo-uncageable fluorescent dye mono-conjugated with a peptide toxin allows for imaging single channel nano-scale locations. This experiment being optimised to overcome non-specific staining but already is significantly better than the current state-of-the-art.