Final Report Summary - OPTICALBULLET (Studies of neurosecretion by remote control of exocytosis and endocytosis with ligt)
In this project we used light to selectively control the fundamental processes of secretion in living cells: exocytosis and endocytosis. Light is a convenient means of manipulating biological processes because it can be patterned in space and time, it is non-invasive and can be used in addition to pharmacological and electrophysiological techniques.
We achieved the control exocytosis with light in chromaffin cells, by exploiting the calcium permeability of the photoswitchable glutamate receptor LiGluR. We applied this method to control neurotransmission with light in hippocampal neurons, and found that expression of LiGluR enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the colour of illumination. However, we noticed that the efficacy of light-mediated exocytosis is not as high as that of the physiological process, and could be improved by enhancing the molecular targeting of LiGluR (specific promoters and/or protein interaction motifs).
In order to control endocytosis with light, we developed photoswitchable peptide inhibitors of clathrin-mediated endocytosis. We call them TrafficLights peptides because they can send ‘stop’ and ‘go’ signals to vesicle traffic through cellular membranes. We have identified several peptides with light-regulated affinity to the target protein (the adaptor protein AP-2 involved in clathrin-mediated endocytosis). TrafficLights peptides can also penetrate the cell membrane, which makes them directly usable in living cells. In this context, we have been able to reversibly manipulate clathrin-mediated endocytosis with light, both in large cell ensembles and at the molecular level. We have achieved a very reliable design methodology that can be generalized to other protein-protein interactions of wide biochemical interest or therapeutic relevance. The selective, spatiotemporally-patterned inhibition of such protein-protein interactions with light can be used to study the interaction dynamics in living cells, and to remotely regulate therapeutic action sites and dosages.
We achieved the control exocytosis with light in chromaffin cells, by exploiting the calcium permeability of the photoswitchable glutamate receptor LiGluR. We applied this method to control neurotransmission with light in hippocampal neurons, and found that expression of LiGluR enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the colour of illumination. However, we noticed that the efficacy of light-mediated exocytosis is not as high as that of the physiological process, and could be improved by enhancing the molecular targeting of LiGluR (specific promoters and/or protein interaction motifs).
In order to control endocytosis with light, we developed photoswitchable peptide inhibitors of clathrin-mediated endocytosis. We call them TrafficLights peptides because they can send ‘stop’ and ‘go’ signals to vesicle traffic through cellular membranes. We have identified several peptides with light-regulated affinity to the target protein (the adaptor protein AP-2 involved in clathrin-mediated endocytosis). TrafficLights peptides can also penetrate the cell membrane, which makes them directly usable in living cells. In this context, we have been able to reversibly manipulate clathrin-mediated endocytosis with light, both in large cell ensembles and at the molecular level. We have achieved a very reliable design methodology that can be generalized to other protein-protein interactions of wide biochemical interest or therapeutic relevance. The selective, spatiotemporally-patterned inhibition of such protein-protein interactions with light can be used to study the interaction dynamics in living cells, and to remotely regulate therapeutic action sites and dosages.