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Synthetic Cell Biology: Designing organelle transport mechanisms

Periodic Reporting for period 3 - ArtifiCell (Synthetic Cell Biology: Designing organelle transport mechanisms)

Reporting period: 2018-09-01 to 2020-02-29

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.
During these first 18 months, we have focused on 3 aspects: 1. Exocytosis in PC12 cells; 2. DNA-induced fusion; 3. Quantitative characterization of the effect of a fusion regulator on the intermembrane interactions.
1. Exocytosis
Wotking with PC12 cells, we have characterized the rate of vesicle docking and exocytosis under various conditions.
2. DNA-induced fusion
We have forced vesicles in close contact and sometimes fusion using DNA origami and DNA tethers.
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, using different membrane composition, buffers and mutant.
4. Theoretical predisction of the kinetics of fusion under the action of SNARE proteins.
5. Development of a microfluidic setup allowing the formation of a membrane perfectly mimicking the lipid composition of the plasma membrane.
The following achievementst resulted from the first 54 months of the project:
1. Placing and shaping vesicles, and forcing them to fuse with DNA origami (related to publication 1 in Nature Chemistry): we have controlled the assembly, arrangement and remodelling 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 predictable
outcomes, 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 (related to 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 efficiencies
corresponding 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 (related to publication 3 in Angewandte Chemie): we designed DNA-origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA-coate 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. The DNA-origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle-deforming DNA structures.
4. We predicted through a mechanical model that SNARE proteins need to cooperate for sub-millisecond fusion. Fusion speed is maximum for 3-6 SNARE compelxes.
5. We measured the energies invvolved in conformation changes of the calcium sensor in neurotransmission, Synapotagmin.This shed light on the organization of the prefusion protein complex.
6. We developped a new setup which allowes to mimic exocytosis in vitro with high fidelity. In the coming months, we will use it to characterize the kinetics of the fusion pore.

We have also written, published and shared a novel software to analyze single vesicle fusion on model membranes (see publication 4 in Langmuir).