Nascent fusion pore opening monitored at single-SNAREpin resolution. Heo P, Coleman J, Fleury JB, Rothman JE, Pincet F. Proc Natl Acad Sci U S A. 2021 Feb 2;118(5). doi: 10.1073/pnas.2024922118. PubMed PMID: 33495324. Download from HAL-INSERM
Using our recently designed microfluidic setup, we investigated the early stage of SNAREpin-induced fusion. We discovered
the existence of subsecond transient fusion pores with a well-defined subnanometer size that occur when one or two SNAREpins are mediating vesicle fusion. In contrast, when vesicle fusion is mediated by three SNAREpins, the fusion pore
reaches a diameter larger than 1.5 nm and expands spontaneously and indefinitely. These results quantitatively explain the need for a complex machinery to ensure a submillisecond neurotransmitter release after the arrival of the action potential during synaptic transmission.
Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.
Keywords: SNAREs; membrane fusion; metastable states; suspended bilayer.
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