Expansion of the fusion stalk and its implication for biological membrane fusion

Over the past 20 years, it has been widely accepted that membrane fusion proceeds via a hemifusion step before opening of the productive fusion pore. An initial hourglass-shaped lipid structure, the fusion stalk, is formed between the adjacent membrane leaflets (cis leaflets). It remains controversial if and how fusion proteins drive the subsequent transition (expansion) of the stalk into a fusion pore. Here, we propose a comprehensive and consistent thermodynamic understanding in terms of the underlying free-energy landscape of stalk expansion. We illustrate how the underlying free energy landscape of stalk expansion and the concomitant pathway is altered by subtle differences in membrane environment, such as leaflet composition, asymmetry, and flexibility. Nonleaky stalk expansion (stalk widening) requires the formation of a critical trans-leaflet contact. The fusion machinery can mechanically enforce trans-leaflet contact formation either by directly enforcing the trans-leaflets in close proximity, or by (electrostatically) condensing the area of the cis leaflets. The rate of these fast fusion reactions may not be primarily limited by the energetics but by the forces that the fusion proteins are able to exert.Membrane fusion is a fundamental process in cell biophysics, being involved in viral infection, endo- and exocytosis, and fertilization. The textbook example of membrane fusion comprises three experimentally observed metastable lipidic structures—namely, the rhombohedral stalk (1, 2), the hemifusion diaphragm (HD) (3–5), and the toroidal fusion pore (6). These structures represent (local) free energy minima that are connected via transient states (free energy barriers) within the fusion pathway. How the stalk transitions (expands) into the fusion pore remains controversial (7–9). Different pathways have been proposed based on experimental observations (3, 5, 10–13), molecular simulations (8, 9, 14–20), continuum elastic models (15, 21, 22), and self-consistent field theory (23, 24).Arguably, the best-studied fusion reactions are the ones mediated by influenza hemagglutinin and soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) molecules. Hemagglutinin-mediated fusion displays an unusual sensitivity toward point mutations in its amphiphilic fusion peptide. Here, even single point mutations can selectively trap the fusion reaction in a hemifused state (25). Hemagglutinin therefore very likely plays an active, essential role in the subsequent evolution of hemifusion intermediates (20). In contrast, it remains unclear if SNARE molecules play a role therein. SNARE molecules subject force on the membrane via the ends of the transmembrane domains (TMDs) (26). The X-ray–resolved structure of the postfusion neuronal SNARE complex suggests that TMDs come together during the fusion reaction, and may actively drive fusion up to the expansion of the fusion pore (27). However, in vitro and in vivo experiments, where the TMD was either replaced by a lipid anchor or partly truncated, provided mixed results concerning the essence of subsequent driving forces after initial membrane merger (28, 29).To discern whether the fusion machinery plays an active role after stalk formation, we estimated the lower bound of the free energy barrier against expansion of the stalk. If stalk expansion faces a substantial free-energy barrier even under extremely fusogenic conditions (i.e., the lower bound of the free energy barrier), then fast, in vivo fusion reactions, such as synaptic fusion, likely involve an active mechanism.We performed coarse grained molecular dynamics simulations, where computational efficiency is enhanced by representing several atoms by a single interaction site, to study the progression of a stalk formed between two highly curved “dimples” with a curvature of –1/10 nm⁻¹. We modeled such a scenario by studying the fusion process of a 20-nm–sized vesicle with its own periodic image (Fig. 1). Such an extreme curvature approaches the upper bound of membrane curvature that fusion proteins, such as synaptotagmin, can generate when being overexpressed on the membrane (30). The exaggerated curvature stress of the here-modeled fusion site should approach the lower bound of the in vivo expansion barrier (31, 32). In addition, we explored the effect of phosphatidylethanolamine (PE) lipids and cholesterol, the two abundant “fusogens” in the plasma membrane, on the expansion of the metastable stalk.Open in a separate windowFig. 1.(A) A 20-nm–sized vesicle, mimicking the protein-free cap of a (protein) induced dimple or nipple, fuses with its own periodic image. To ensure free flow of lipid material (tensionless conditions), two artificial pores with a radius 1.6 nm are present at the vesicle’s foot. The simulation box can freely adjust its length in the Z dimension, as well as in the XY dimension. The green circles depict the hydrophilic probes which mimic the squeezing action of one or multiple SNARE complexes. (B) Example of a scenario where widening of the stalk is driven by the presence of multiple neuronal SNARE complexes (26, 51). The C termini of the transmembrane domains (green circles) exert a squeezing force on both the trans-leaflets and thereby drive thinning and widening of the stalk. Lipids: tails are shown in gray, head groups (spheres) in tan, cholesterol in brown. SNAREs: syntaxin-1A in red, synaptobrevin-2 in green, and SNAP-25 in green. Transmembrane domains are shown in yellow.In total we performed more than 500 simulations of 1.6 μs each to study four relevant scenarios: (i) The leaflets of the surrounding bulk membrane are in equilibrium and material freely flows between the bulk membrane and the dimple; (ii) the flow of material between the dimple’s cap and the surrounding bulk membrane is restricted/inhibited due to the crowding of nearby fusion proteins; (iii) a tension difference between the leaflets is induced by, e.g., an asymmetric ion concentration; and (iv) the presence of shape-stabilizing matrix proteins on the trans-leaflets of viral envelopes (11) or a membrane adhered to a solid support (supported bilayers).To estimate the free energy required for (protein-mediated) expansion of the stalk, we placed a hydrophilic probe consisting of eight bundled solvent beads in each vesicle. We alter the equilibrium distance between the probes within the simulations (Fig. 2 and SI Appendix, Methods). Such a scenario mimics, e.g., the hydration shell(s) of the charged TMD ends (C termini) of SNARE molecules (Fig. 1B) that come together during the fusion reaction (27), and thereby exert a squeezing force on the stalk (26).Open in a separate windowFig. 2.The expansion barrier of the stalk—standard hemifusion mechanism. The figures show the fusion reaction between the membranes (at 310 K) in response to pulling two hydrophilic probes (green) toward each other through the center of the stalk. This process mimics the action of SNARE zipping where the hydrophilic transmembrane domain C termini are pulled together. (Upper) The required free energy for facilitating such a process. (Lower) Corresponding formation of the hemifusion diaphragm. The initial stalk (I) expands linearly (II) before expanding radially (III and IV). Once the barrier (III) of 17–24 k_BT is overcome, the subsequent formation/expansion of the hemifusion diaphragm (IV) becomes spontaneous (plateau region). The addition of 40% POPE does not significantly affect the barrier of stalk widening. The addition of 30% cholesterol marginally increases the barrier against stalk widening (from 17 to 24 k_BT). Trans-leaflets are shown in yellow, cis leaflets in gray. The example shows the situation for the pure POPC membrane (PC head groups are shown in tan).First, we considered a scenario where the leaflets of the dimple are in equilibrium and the chemical potential of the cis and trans-leaflets remains constant during the fusion process. Free lipid exchange is facilitated between all four leaflets by placing two artificial 1.6-nm–sized pores at the foot of the dimple, which facilitate a lipid flip-flop rate of 0.4 ns⁻¹ (33, 34) (Fig. 1A and SI Appendix, Fig. S1). Because the pores allow a free exchange of lipids and solvent, and because the x-, y-, and z-dimensions of the simulation box are semi-isotropically coupled to the same external pressure bath (1 bar), we can assume no lateral tension is present in the leaflets.     

Chemical and Swelling Evaluations of Amino Group Crosslinking in Gelatin and Modified Gelatin Matrices

Purpose. To determine the extent of amino group crosslinking in gelatin matrices by chemical assay, and to compare these results to crosslinking evaluations from swelling measurements. Methods. Matrices crosslinked with a water soluble carbodiimide (EDC/G), glutaraldehyde (GTA/G), as well as a GTA crosslinked matrix prepared from gelatin modified to contain 230% greater crosslinking sites (GTA/Mod) were evaluated. Crosslinking extent, X _c, was determined by a UV assay of uncrosslinked amino groups before and after crosslinking, and was used to obtain crosslinking densities. Equilibrium swelling ratios, Q _m at 37°C in isotonic pH 7.4 were used to calculate crosslinking degree from the Flory equation for swelling of ionic polymers for comparison to the chemically determined crosslinking densities. Results. Of the original 33 × 10^–5 moles -amino groups/g gelatin, 91 to 95% were crosslinked in EDC/G and GTA/G. GTA/Mod lost 95% of the original 108 × 10^–5 moles amino groups/g gelatin. Crosslinking densities were 4.1 × 10^–4 and 4.2 × 10^–4 moles/mL for EDC/G and GTA/G, respectively. The value for GTA/Mod increased to 14.2 × 10^–4 moles/mL. Values of Q _m followed the same trend. The Flory crosslinking degrees for both gelatin matrices were 12 × 10^–4 and 13 × 10^–4 moles/mL, respectively. The value for the more extensively crosslinked GTA/Mod was 280 × 10^–4moles/mL. Conclusions. The swelling and chemical evaluations of crosslinking are in general agreement for matrices with the lower of two crosslinking levels. The chemical determination appears suitable for evaluating amino group crosslinking in gelatin and it may be suitable for other proteinaceous materials.