Membrane fusion and synaptic transmission

Frédéric Pincet – Team leader

Membrane fusion and synaptic transmission

At the synapse, the signal coming from a neuron must be transmitted to the next cell, which can be another neuron or a muscle. This signal is conveyed by synaptic vesicle containing molecules called neurotransmitters. These vesicles dock and fuse with the presynaptic plasma membrane to release the neurotransmitters in less than 1 msec. The fusion of the synaptic vesicle is the most critical event of the whole neurotransmission process. The engine for fusion is the assembly of cognate proteins called SNAREs, v-SNARE in the synaptic vesicle and t-SNARE in the target pre-synaptic plasma membrane. The formation of the SNARE complex, the SNAREpin, forces the two membranes together, disrupts their cohesion to overcome the activation energy for fusion. This picture does not explain how neurotransmission can be so fast and so finely tuned in space and time. To address this, we have recently focused on five specific questions: (i) what is the number of SNAREpins in the fusion process and are there cooperative effects? (ii) What are the various intermediate states, the associated energies and molecular arrangements, as well as their functional role? (iii) How is the fusion process regulated: what are the actors and how do they affect the intermediate states? (iv) What is the energy to overcome to induce fusion? (v) How can we reproduce of SNAREs and other regulators by alternate molecules?

We have addressed these questions by developing original in vitro approaches. For instance, we inserted a controlled number of SNAREs into nanodiscs (~10nm suspended membranes, templated by proteins) and found that one SNAREpin is sufficient to start fusion but three are required to expand the nascent fusion pore. We have also developed a new sensitive technique that freezes the intermembrane distance thereby blocking the SNAREpins in intermediate states. With highly sensitive optical imaging, we have characterized the molecular conformation of the SNAREs in these intermediate states and measured the involved energies. We discovered that the initial assembly of the SNAREs is actually extremely slow because of a high activation energy, and found several ways to bypass this slow process, e.g. by pre-structuring the t-SNARE. In vivo, this is potentially achieved by a chaperon protein, probably Munc18, which prebinds both v- and t-SNAREs. We have also performed the first measurement of the energy that has to be provided to fully fuse two lipid bilayers. We found that it is in the lower range of the predicted values (30-35 kBT). Physiologically this value makes perfect sense because it is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion on demand.

Five recent results

  1. Intermediate states during SNAREpin formation prior to membrane fusion and neurotransmission
  2. Comparison between nanodisc and liposome fusion
  3. Energy for membrane fusion
  4. DNA Origami to control single fusion events
  5. Accelerating SNARE-mediated Membrane Fusion by DNA-lipid Tethers

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