Function suggests nano-structure: electrophysiology supports that granule membranes play dice

Abstract

Cellular communication depends on membrane fusion mechanisms. SNARE proteins play a fundamental role in all intracellular fusion reactions associated with the life cycle of secretory vesicles, such as vesicle-vesicle and vesicle plasma membrane fusion at the porosome base in the cell plasma membrane. We present growth and elimination (G&E), a birth and death model for the investigation of granule growth, its evoked and spontaneous secretion and their information content. Using a statistical mechanics approach in which SNARE components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation behind granule growth and secretion. Results from experimental work, mathematical calculations and statistical modelling suggest that for vesicle growth a minimal aggregation of three SNAREs is required, while for the evoked secretion one SNARE is enough. Furthermore, the required number of SNARE aggregates (which varies between cell types and is nearly proportional to the square root of the mean granule diameter) affects and is statistically identifiable from the size distributions of spontaneous and evoked secreted granules. The new statistical mechanics approach to granule fusion is bound to have a significant changing effect on the investigation of the pathophysiology of secretory mechanisms and methodologies for the investigation of secretion.

Figure 1.

A model of the vesicle-associated multimeric SNARE–ring interactions, where each rosette ring is a K-SNARE complex. Membrane fusion requires transient structural reorganization of at least some lipids with two optional pathways (I and II) [37,38]. During membrane fusion, the outer membrane leaflets are loosely connected by SNAREs (step A) and brought into close proximity (step B), whereas the distal membrane leaflets remain separate until the opening of a fusion pore (step C). The current concept correlates the initiation of hemifusion ((e), step D) with the formation of a contact stalk-like zone between the membranes in which the two proximal monolayers are connected by a SNARE aggregate. The stalk forms a hydrophobic ‘narrow bridge’ between contacting vesicle outer leaflets [–39]. The initial local stalk may evolve to a fusion pore (pathway I), or expand to hemifusion (pathway II). This transitional hemifusion stage is a critical metastable intermediate for membrane fusion. Interactions (figure 1b) can be homotypic (granule–granule fusion) or heterotypic (granule–plasma membrane fusion). The curvature of secretory vesicles dictates the potency and efficacy of the granule fusion capacity [28]. Stationarity requires K_γ ≤ K_β + 1. K_γ ≈ K_β = K is proportional to the inner SNARE rosette perimeter 2πa (radius, a = [(h(D–h)]^0.5). The SNARE interaction patch diameter is estimated to be 2.4 nm ([40], (c)) and membrane width (approximately 2h, (d)) is within 3.4–4.5 nm, depending on the lipid composition [–43]. (e) (i) Shows the linear correlation between the maximal rosette size and the granule size (square root of diameter) for various h values. Inset, a 12-example (see the electronic supplementary material) scattergram of K_γ and K_β (the broken line indicates diagonal K_γ = K_β). (e) (ii) Shows inset data correlated with theoretical granule size bounds (open symbols) and experimental data (solid symbols). (Online version in colour.)

Figure 2.

Morphological insight from the model. The effective area of the porosome estimated at 3.52% of the granule surface area [26]. The plot of the vesicle diameter versus the patch size (figure 1e) suggests the relation that enables the estimation of the distance between neighbouring SNAREs at about L ≈ 1.46 K_β nm.

Figure 3.

The number of granules needed for the detection of the evoked mode, in terms of the burst rate. Each vertical bar represents all examples in the benchmark with the given MGS. Secretion magnification rates from 8 to 32 yield similar results.