In situ cryo-electron tomography of actively firing neuronal synapses
Final Report Abstract
The fusion of synaptic vesicles with the presynaptic active zone membrane displays the fundamental principle of neurotransmitter release – and thus of neuronal communication. Although first electron micrographs of vesicle fusion were captured more than 50 years ago, we are still far from understanding all molecular, biophysical, and kinetic aspects of the fusion process. In the here presented project, I have developed a workflow to characterize the fusion of vesicles with unprecedented structural and temporal resolution. In detail, I have combined optogenetic stimulation of neurons with plunge freezing for cryofixation, which allowed me to immobilize cellular processes only 2-5 ms after an action potential. To assess whether the stimulation was successful and to identify synapses that released neurotransmitters, I utilized the glutamate sensor iGluSnFR for cryo-confocal microscopy. Glutamate that is released during vesicle fusion can bind to iGluSnFR, which induces a conformational change and an increase in fluorescence intensity of the biosensor. During the combined optogenetic stimulation and cryofixation, iGluSnFR was arrested in its glutamate-bound state, which means that the high fluorescence intensity could be preserved and used as a marker for synaptic activity. On these active neurons, I performed cryo-electron tomography. With this 3D microscopy method, artefacts from fixation or staining can be avoided, resulting in best-possible sample preservation and structural resolution. In my optogenetically stimulated neuronal samples, I was able to identify different stages of synaptic vesicle fusion including tethering, the formation of a fusion pore, a dilated fusion pore, and the integration of vesicle membrane into the membrane of the presynaptic active zone. With these findings, I could confirm that my workflow has successfully been established. In the future, the combination of optogenetics and cryo-electron tomography may be key to approaching open questions in the field of synaptic transmission. Beyond that, also other research fields using photosensitive proteins will benefit from the here developed optogenetic plunge freezer.
