Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties

Abstract

SummaryThe fusion pore is the first crucial intermediate formed during exocytosis, yet little is known regarding the mechanisms that determine the size and kinetic properties of these transient structures1. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pores. To address this, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores, and enabled the escape of larger cargos. To determine whether this was due to changes in nascent pore size versus stability, we developed a novel approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords μsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, trans-SNARE pairing was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable, SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, that mimics the action of botulinum neurotoxin A, dramatically reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determine fundamental properties of individual pores.

Toward a unified picture of the exocytotic fusion pore

“…Surprisingly most pores had stable, ion channel‐like conductances , in contrast to earlier work by us using 16–17 nm MSP NDs fusing with engineered cells (Fig. ).…”

SNARE ‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle

“…This movement should be hindered by the stiffness and the length of the SNARE complex, as well as by its attachment to HOPS, which is anchored in both membranes (Ho & Stroupe, ; Lürick et al , ). It is therefore plausible that a pore lined by multiple SNARE complexes displays a larger stability against hemifission, in line with experimental observations (Shi et al , ; Bao et al , ). Furthermore, the large volume of the HOPS complex deforms the fusion site.…”

“…This suggests that expansion of fusion pores might be rate‐limiting, reversible, and give rise to a potentially long‐lived intermediate. In line with this, in vitro data show that the number of trans‐SNARE complexes around a fusion pore and SNARE‐associated proteins influences the diameter and dynamics of that pore (Shi et al , ; Lai et al , ; Bao et al , ). Also in vivo , exocytic fusion pores are highly dynamic and their properties depend on the expression level of exocytic SNAREs and membrane tension in vivo (Bao et al , ; Shin et al , ).…”

“…In line with this, in vitro data show that the number of trans‐SNARE complexes around a fusion pore and SNARE‐associated proteins influences the diameter and dynamics of that pore (Shi et al , ; Lai et al , ; Bao et al , ). Also in vivo , exocytic fusion pores are highly dynamic and their properties depend on the expression level of exocytic SNAREs and membrane tension in vivo (Bao et al , ; Shin et al , ). Despite this dynamics, exocytic fusion pores usually remain relatively short‐lived (sub‐second range) (Bao et al , ; Shin et al , ).…”

The fusion pore, 60 years after the first cartoon

“…The increased pore dilation with increasing v‐SNARE copies was not saturated at 15 v‐SNARE copies per face, which was the maximum capacity of the 23 nm NDs used in these experiments . Consistent with these results, fusion pore size increased from ~ 1 to ~ 3 nm with increasing SNARE copy numbers from 3 to 7, based on electrophysiological ND‐planar membrane fusion pore conductance measurements .…”