Synthetic Biology: Exocytosis – SynBioExo

Develop methods to deliver PNA to membranes representing the vesicle and plasma membranes of a cell in a reproducible manner. This would enable us to mimic and reproduce in vitro, the main steps of exocytosis, namely tethering/docking and fusion. We will first independently optimize each of these steps and then combine them to engineer the complete exocytosis process with PNA. Once this is achieved, we will try to provide an artificial signal (also based on PNA technology) to switch on exocytosis at desired place and time.
At the end of this proposal, we will be able to fully replace or enhance the tethering/docking step and/or the fusion step using PNA. Exocytosis can then completely be performed, controlled and triggered artificially by synthetic molecules.

We reproduced the tethering/docking stage with DNA tethers. Using DNA origami, we have also produced vesicles of monodisperse size that resemble synaptic vesicles.

After this project is finished, we plan to transfer the technology to in vivo systems and completely reprogram and reengineer exocytos in cells with synthetic molecules.

1. W. Xu, J. Wang, J.E. Rothman, F. Pincet, “Accelerating SNARE-mediated Membrane Fusion by DNA-lipid Tethers”, Angewandte Chemie, 54,14388-14392 (2015).
2. W. Xu, B. Nathwani, C. Lin, J. Wang, E. Karatekin, F. Pincet, W. Shih, J.E. Rothman, «A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion«, J. Am. Chem. Soc., 138: 4439?4447 (2016).
3. F. Pincet, V. Adrien, R. Yang, J. Delacotte, J.E. Rothman, W. Urbach, D. Tareste, “FRAP to Characterize Molecular Diffusion

Exocytosis is one of the fundamental physiological processes in the cell. Diverse and important functions like secretion of hormones or enzymes and release of neurotransmitters at the synapse require exocytosis. So, malfunction of this process can lead to many diseases such as diabetes or neurodegenerative diseases (Alzheimer, etc…). Exocytosis occurs when a vesicle initially in the cytosol and containing cargos (e.g. enzymes, hormones or neurotransmitters) fuses with the plasma membrane, releasing the cargo into the extracellular space. This involves several sequential steps: loose tethering, followed by a tighter docking, which precedes the final step involving the assembly of the SNARE complex which drive membrane fusion. Hence, the basic steps are tethering, docking and fusion. In some specific cases, an extra intermediate is added, vesicle priming, in which the assembly of SNARE complexes is initiated but arrested mid-way. This accounts for a rapid and synchronous release upon a triggering signal (e.g. calcium intake in the case of neurotransmission). Tethering and docking are achieved by proteins that are able to bind when the vesicle is still far from the plasma membrane (tens of nm). Fusion is carried out by cognate SNARE proteins that are present in both the vesicles (v-SNAREs) and target plasma membrane (t-SNAREs). Upon assembly of the SNAREs, the vesicle and plasma membranes are forced in close proximity which induces fusion.
We and others have already shown that the tethering/docking step and fusion process can be artificially reproduced in vitro using synthetic molecules, DNA or peptide nucleic acids (PNA). In this proposal, we plan to utilize these tools to re-engineer cells and create an artificial programmable cellular pathway for controlled release of stored substances i.e. synthetic exocytosis. We will do this in two separate but interdependent phases that will proceed in parallel: (i) in vitro development of a prototype of programmable, switchable PNA based exocytosis and (ii). Develop methods to deliver PNA to the vesicle and plasma membranes of a cell in a reproducible manner. This would enable us to extend the of the prototype developed in vitro phase to in vivo conditions. We want to mimic and reproduce, the main steps of exocytosis, namely tethering/docking and fusion, both in vitro and in vivo. We will first independently optimize each of these steps and then combine them to engineer the complete exocytosis process with PNA. Once this is achieved, we will try to provide an artificial signal (also based on PNA technology) to switch on exocytosis at desired place and time.
At the end of this proposal, we will be able to fully replace or enhance the tethering/docking step and/or the fusion step using PNA. Exocytosis can then completely be performed, controlled and triggered artificially by synthetic molecules. Re-engineering the cell with synthetic molecules would open new perspectives in cell science and medicine. At the fundamental level, it will allow us to better control and understand how each step of exocytosis works. On a more applied touch, it may provide a new way to repair or control hormone secretion or neurotransmitter release in various diseases with a new approach never envisioned before.