Periodic Reporting for period 4 - ArtifiCell (Synthetic Cell Biology: Designing organelle transport mechanisms)
Periodo di rendicontazione: 2020-03-01 al 2021-08-31
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 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 exocytosis
Synucleinopathies 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 pore
Using 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.
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 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 (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 (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).