Periodic Reporting for period 4 - ArtifiCell (Synthetic Cell Biology: Designing organelle transport mechanisms) Reporting period: 2020-03-01 to 2021-08-31 Summary of the context and overall objectives of the project Imagine being able to design into living cells and organisms de novo vesicle transport mechanisms that do not naturally exist? At one level this is a wild-eyed notion of synthetic biology. But we contend that this vision can be approached even today, focusing first on the process of exocytosis, a fundamental process that impacts almost every area of physiology. Enough has now been learned about the natural core machinery (as recognized by the award of the 2013 Nobel Prize in Physiology or Medicine to the PI and others) to take highly innovative physics/engineering- and DNA-based approaches to design synthetic versions of the secretory apparatus that could someday open new avenues in genetic medicine. The central idea is to introduce DNA-based functional equivalents of the core protein machinery that naturally form (coats), target (tethers), and fuse (SNAREs) vesicles. We have already taken first steps by using DNA origami-based templates to produce synthetic phospholipid vesicles and complementary DNAbased tethers to specifically capture these DNA-templated vesicles on targeted bilayers. Others have linked DNA oligonucleotides to trigger vesicle fusion. The next and much more challenging step is to introduce such processes into living cells. We hope to break this barrier, and in the process start a new field of research into “synthetic exocytosis”, by introducing Peptide-Nucleic Acids (PNAs) of tethers and SNAREs to re-direct naturally-produced secretory vesicles to artificially-programmed targets and provide artificially-programmed regulation. PNAs are chosen mainly because they lack the negatively charged phosphate backbones of DNA, and therefore are more readily delivered into the cell across the plasma membrane. Future steps, would include producing the transport vesicles synthetically within the cell by externally supplied origami-based PNA or similar cages, and - much more speculatively - ultimately using encoded DNA and RNAs to provide these functions. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have focused on several scientific aspects: 1. Exocytosis in PC12 cells; 2. DNA-induced membrane remodelling, fusion and tubulation; 3. Quantitative characterization of the effect of a fusion regulator on the intermembrane interactions; 4. Theoretical prediction of the kinetics of regulated-exocytosis. 5. Alteration of exocytosis by an external factor, α-Synuclein; 6. Study of the nascent fusion pore: when is it sufficiently large for exocytosis?1. ExocytosisWotking with PC12 cells, we have characterized the rate of vesicle docking and exocytosis under various conditions. 2. DNA-induced fusionWe have forced vesicles in close contact and fusion using DNA origami.3. Quantitative characterization of the effect of a fusion regulator on the intermembrane interactions.We have directly measured interactions of membranes decorated with a fusion regulator, synaptotagmin.4. Theoretical prediction of the kinetics of fusion under the action of SNARE proteins. We have designed a simple mechanical model that predicts the variation of the fusion time with the number of SNAREpins.5. Alteration of exocytosisSynucleinopathies are neurological diseases that are characterized by the accumulation of aggregates of a cytosolic protein, α-synuclein, at the plasma membrane. Even though the pathological role of the protein is established, the mechanism by which it damages neurons remains unclear due to the difficulty to correctly mimic the plasma membrane in vitro. Using a microfluidic setup we developed during this project, we studied the action of α-Synuclein on the plasma membrane.6. Kinetics and dimensions of the nascent fusion poreUsing the same microfluidic setup, we established the kinetics and dimensions of transient states of the nascent fusion pore and determined the conditions when the pore is large enough to ensure cargo release during exocytosis.7. Ultra-fast imaging of exocytosis in nerve terminals Using the iGluSnFR probe, we developped a protocol to image single vesicular release events in single boutons with 4 ms temporal and 75 nm spatial resolution. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The following achievements resulted from the project:1. Placing and shaping vesicles, and forcing them to fuse with DNA origami (publication 1 in Nature Chemistry): we have controlled the assembly, arrangement and remodeling of liposomes by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictableoutcomes, opening up opportunities for the systematic study of membrane mechanics.2. Regulation of synaptic vesicle priming by proline-rich transmembrane protein 2 (PRRT2) in PC12 cells (publication 2 in Cell Reports): We have shown that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrated that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficienciescorresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders.3. Forcing vesicles to tubulate with DNA-nanosprings (publication 3 in Angewandte Chemie): we designed DNA-origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA-coated membrane tubules emerge from spherical vesicles when DNA-origami polymerization or high membrane-surface coverage occurs. Unlike many previous methods, the DNA self-assembly-mediated membrane tubulation eliminates the need for detergents or top-down manipulation.4. We measured the energies involved in conformation changes of the calcium sensor in neurotransmission, Synapotagmin (publication 5 in PNAS).5. We showed through a mechanical model that SNARE proteins need to cooperate for sub-millisecond fusion. Fusion speed is maximum for 3-6 SNARE complexes (publication 6 in PNAS).6. We developed two new setup which allows to mimic exocytosis in vitro with high fidelity. One is based on a pore spanning membrane (publication 4) and the other relies on a home-made microfluidic chip (related to publication 7).7. We have shown that -Synuclein can freeze and perforate the membrane it is acting on. This action can significantly impair exocytosis (publication 8).8. We observed that the nascent fusion pore induced by SNAREs can have various discrete sizes that are directly linked to the number of SNAREpins pulling at the edge of the pore (publication 9). This is a fundamental issue during exocytosis as it implies that there is a minimum number of simultaneously acting SNAREpins to allow exocytosis. This minimum depends on the hydrodynamic radius of the cargo.9. We measured a state-of-the-art imaging tools which allow us to directly investigative the regulation of vesicular release at single presynaptic boutons across synaptic populations with unprecedented resolution. We have also written, published and shared a novel software to analyse single vesicle fusion on model membranes (publication 4 in Langmuir). Imaging of different modes of vesicular release in a single bouton using iGluSnFR imaging
