SNARE machinery is optimized for ultrafast fusion

Abstract

SNARE proteins zipper to form SNAREpins that power vesicle fusion with target membranes in a variety of biological processes. A single SNAREpin takes about 1 second to fuse two bilayers, yet a handful can ensure release of neurotransmitters from synaptic vesicles much faster, in a 10th of a millisecond. We propose that, similar to the case of muscle myosins, the ultrafast fusion results from cooperative action of many SNAREpins. The coupling originates from mechanical interactions induced by confining scaffolds. Each SNAREpin is known to have enough energy to overcome the fusion barrier of 25-35 k B T, however, the fusion barrier only becomes relevant when the SNAREpins are nearly completely zippered and from this state each SNAREpin can deliver only a small fraction of this energy as mechanical work. Therefore they have to act cooperatively and we show that at least 3 of them are needed to ensure fusion in less than a millisecond. However, to reach the pre-fusion state collectively, starting from the experimentally observed half-zippered metastable state, the SNAREpins have to mechanically synchronize which takes exponentially longer time as the number of SNAREpins increases. Incorporating this somewhat counter-intuitive idea in a simple coarse grained model results in the novel prediction that there should be an optimum number of SNAREpins for sub-ms fusion: 3-6 over a wide range of parameters. Interestingly, in situ cryo-electron microscope tomography has very recently shown that exactly six SNAREpins participate in the fusion of each synaptic vesicle. This number is in the range predicted by our theory.

Symmetrical organization of proteins under docked synaptic vesicles

“…It may seem surprising that a handful of SNAREpins – if they are synchronously released – could achieve sub‐millisecond fusion pore opening. However, recent modeling that includes the concept of mechanical coupling predicts exactly this . Membranes are rigid on the length scale of a fusion pore, and as a result, SNAREpins will necessarily be mechanically coupled.…”

“…At this stage, each SNAREpin still has ~ 5 k B T of mechanical work to deliver toward overcoming the bilayer fusion barrier (~ 25 k B T for physiologically relevant lipid compositions ). For a single SNAREpin, this barrier is overcome in about 1 s Therefore, each added SNAREpin reduces this waiting time for fusion by a factor of about 10 2 (calculated as e (−5 kBT) ), so as the extraordinary consequence of mechanical coupling just three SNAREpins will reduce the time for vesicle release to ~ 0.1 ms .…”

“…Another feature of mechanical coupling is that, together with the measured energy landscape of the SNAREpin zippering , it also predicts that there is an optimum number of SNAREpins that maximizes fusion rate . The reason is that fusion cannot occur until the last SNAREpin fully zippers, and when there are a large number of them this becomes rate limiting.…”

“…Though small for any one of the SNAREpin, the probability that one or more SNAREpins are 'left behind' increases exponentially with the number of SNAREpins [40]. The tradeoff between these opposing processes predicts an optimum of 4-6 SNAREpins is required to achieve submillisecond release as needed for synaptic transmission, depending on the exact choice of various parameters [40]. It is remarkable that this optimum, predicted from first principles, should align so closely with what molecular imaging of synaptic-like vesicles in situ suggests.…”

Independent Yet Synergistic Roles of Synaptotagmin-1 and Complexin in Calcium Regulated Neuronal Exocytosis

“…Indeed, recent modeling studies considering the concept of mechanical coupling have predicted that an optimum of 4-6 SNAREpins is required to achieve sub-millisecond vesicular release (Manca et al 2019). Our data suggests that Cpx irreversibly arrests SNAREpin assembly (Figure 4 Supplement 1) and raises the intriguing possibility that the Cpx clamp is not necessarily reversed during the Ca 2+ -activation process.…”