OPTICALBULLET · Studies of neurosecretion by remote control of exocytosis and endocytosis with ligt

Optical switches are photoisomerizable compounds that allow to remotely controlling the activity of proteins, cells and entire organisms with light. These tools are revolutionizing research in biology with their high selectivity and spatiotemporal resolution. Here we propose to develop and apply optical switches to investigate the fundamental processes of secretion, exocytosis and endocytosis, in a way that is non-invasive, acute, and orthogonal to pharmacological and electrophysiological techniques. The optical control of exocytosis will be carried out by means of photoswitchable, Ca2+-permeable channels (LiGluR and Channelrhodopsin-2) which allow triggering vesicle fusion at single synaptic terminals. This procedure will allow studying vesicle release kinetics and the differences between synapses of the same neuron. The photocontrol of endocytosis will be carried out with: (1) inhibitory peptides of the clathrin pathway modified with an azobenzene crosslinker in order to photomodulate their structure and affinity, and (2) photoswitchable synthetic compounds based on chemical inhibitors of dynamin. Photomodulation of endocytosis in chromaffin cells and neurons will allow interfering with the internalisation of membrane receptors with an unprecedented spatial and temporal control. The use of photoswitchable inhibitors of endocytosis would allow for the first time to manipulate reversibly and with subcellular resolution, the vesicular trafficking of the endocytic pathway. In addition, these photoswitches could reveal how endocytosis regulates spatially receptor activation, controlling cell patterning and cell fate.