Seeing is believing: the stalk intermediate.

Membrane biophysicists have sought the molecular nature of biomembrane fusion for more than 30 years. In this time, two conflicting hypotheses have evolved to explain very different observations. One, based on electrophysiological measurements on patch-clamped cells (1xStructure and function of fusion pores in exocytosis and ectoplasmic membrane fusion. Lindau, M. and Almers, W. Curr. Opin. Cell Biol. 1995; 7: 509–517Crossref | PubMed | Scopus (186)See all References)(1), is the proteinacious pore hypothesis. This model, popular among neurobiologists, contends that the initial fusion pore consists of a single proteinacious channel that later opens into a full fusion pore due to dissipation of the initial protein pore components into lipid bilayer. Recent mutational studies of the trans-membrane domain of the synaptic fusion protein syntaxin claim to support this hypothesis and propose that several syntaxin trans-membrane domain’s may form the initial pore (2xTransmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis. Han, X., Wang, C.T., Bai, J., Chapman, E.R., and Jackson, M.B. Science. 2004; 304: 289–292Crossref | PubMed | Scopus (203)See all References)(2). The alternative hypothesis is the lipidic pore hypothesis. It contends that the fusion pore derives from nonlamellar lipid structures and proceeds through an initial partially fused structure in which lipids mix although aqueous compartments do not. The partially fused state is termed hemifusion. A large number of observations offer support for this view for viral, exocytotic, and model systems (3.xLysolipids reversibly inhibit Ca(2+)-, GTP- and pH-dependent fusion of biological membranes. Chernomordik, L.V., Vogel, S.S., Sokoloff, A., Onaran, H.O., Leikina, E.A., and Zimmerberg, J. FEBS Lett. 1993; 318: 71–76Abstract | Full Text PDF | PubMed | Scopus (140)See all References, 4.xSecretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers. Lee, J. and Lentz, B.R. Proc. Natl. Acad. Sci. USA. 1998; 95: 9274–9279Crossref | PubMed | Scopus (68)See all References, 5.xHemifusion in SNARE-mediated membrane fusion. Xu, Y., Zhang, F., Su, Z., McNew, J.A., and Shin, Y.K. Nat. Struct. Mol. Biol. 2005; 12: 417–422Crossref | PubMed | Scopus (151)See all References, 6.xTrans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion. Reese, C., Heise, F., and Mayer, A. Nature. 2005; 436: 410–414PubMedSee all References, 7.xSNAREs can promote complete fusion and hemifusion as alternative outcomes. Giraudo, C.G., Hu, C., You, D., Slovic, A.M., Mosharov, E.V., Sulzer, D., Melia, T.J., and Rothman, J.E. J. Cell Biol. 2005; 170: 249–260Crossref | PubMed | Scopus (92)See all References, 8.xNeuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion. Dennison, S.M., Bowen, M.E., Brunger, A.T., and Lentz, B.R. Biophys. J. 2006; 90: 1661–1675Abstract | Full Text | Full Text PDF | PubMed | Scopus (103)See all References). Despite the preponderance of evidence for the lipidic pore hypothesis, no one has until now actually observed in a biological system a lipidic pore or one of the presumed lipidic intermediates thought to precede the lipidic pore. An article in this issue of the Biophysical Journal(9xConical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused. Zampighi, G.A., Zampighi, L.M., Fain, N., Lanzavecchia, S., Simon, S.A., and Wright, E.M. Biophys. J. 2006; : 2910–2918Abstract | Full Text | Full Text PDF | PubMed | Scopus (52)See all References)(9) dramatically alters this situation.The lipidic pore hypothesis is most often expressed in terms of the “stalk model”. This proposes specific structures for the intermediates that lead to a fusion pore. After examining bilayers supported on mica sheets (10xBiomembrane fusion: a new concept derived from model studies using two interacting planar lipid bilayers. Chernomordik, L.V., Melikyan, G.B., and Chizmadzhev, Y.A. Biochim. Biophys. Acta. 1987; 906: 309–352Crossref | PubMed | Scopus (194)See all References)(10), the stalk structure was proposed to consist of merged contacting or trans monolayers and unfused cis monolayers (11xMembrane fusion: overcoming of the hydration barrier and local restructuring. Leikin, S.L., Kozlov, M.M., Chernomordik, L.V., Markin, V.S., and Chizmadzhev, Y.A. J. Theor. Biol. 1987; 129: 411–425Crossref | PubMed | Scopus (81)See all References)(11), as illustrated in Fig. 1 of Zampighi et al. (9xConical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused. Zampighi, G.A., Zampighi, L.M., Fain, N., Lanzavecchia, S., Simon, S.A., and Wright, E.M. Biophys. J. 2006; : 2910–2918Abstract | Full Text | Full Text PDF | PubMed | Scopus (52)See all References)(9). Siegel proposed a “modified stalk model” in which another type of intermediate structure, the trans-membrane contact (TMC), might also be energetically possible (12xThe modified stalk mechanism of lamellar/inverted phase transitions and its implications for membrane fusion. Siegel, D.P. Biophys. J. 1999; 76: 291–313Abstract | Full Text | Full Text PDF | PubMedSee all References)(12). Kinetic studies of PEG-mediated vesicle fusion established two intermediate states (13xEvolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Lee, J. and Lentz, B.R. Biochemistry. 1997; 36: 6251–6259Crossref | PubMed | Scopus (152)See all References)(13). Simple calculations based either on a macroscopic materials model (14xEnergetics of vesicle fusion intermediates: comparison of calculations with observed effects of osmotic and curvature stresses. Malinin, V.S. and Lentz, B.R. Biophys. J. 2004; 86: 2951–2964Abstract | Full Text | Full Text PDF | PubMedSee all References)(14) or on a field theoretical treatment with course-grained models of lipid and water molecules (15xField theoretic study of bilayer membrane fusion. I. Hemifusion mechanism. Katsov, K., Muller, M., and Schick, M. Biophys. J. 2004; 87: 3277–3290Abstract | Full Text | Full Text PDF | PubMed | Scopus (105)See all References)(15) showed that both the stalk and TMC were candidate structures for these intermediates.The first structural evidence for the lipidic pore model came from x-ray scattering studies by Yang and Huang of pure lipid systems in which under extreme dehydration and elevated temperature, a stable rhombohedral lipid phase consisting of hexagonally packed stalk structures was documented (16xA rhombohedral phase of lipid containing a membrane fusion intermediate structure. Yang, L. and Huang, H.W. Biophys. J. 2003; 84: 1808–1817Abstract | Full Text | Full Text PDF | PubMedSee all References)(16). Although this demonstrates that stalk-like structures are stable under certain conditions, it does not demonstrate these structures in fusing biomembranes. Zampighi et al. (9xConical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused. Zampighi, G.A., Zampighi, L.M., Fain, N., Lanzavecchia, S., Simon, S.A., and Wright, E.M. Biophys. J. 2006; : 2910–2918Abstract | Full Text | Full Text PDF | PubMed | Scopus (52)See all References)(9) report in this issue of the Biophysical Journal conical thin sectioning and electron tomography analysis of samples of rat cortical synapse “active zones” that provides the first evidence for a stalk or TMC structure at the point of contact of fusing membranes. They observe both “docked” (within 15 nm of the presynaptic membrane active zone) and undocked synaptic vesicles, with docked vesicles existing in three possible states: 1), distinct (i.e., with a membrane clearly distinct from that of the presynaptic membrane, ∼15%), 2), hemifused (having a region where contacting membrane leaflets could not be detected, ∼75%), and 3), fused (having a pore evident in single membrane present in the hemifused region, ∼10%). This is consistent with the observation of different functional pools of synaptic vesicles (17xInhibition of SNARE complex assembly differentially affects kinetic components of exocytosis. Xu, T., Rammner, B., Margittai, M., Artalejo, A.R., Neher, E., and Jahn, R. Cell. 1999; 99: 713–722Abstract | Full Text | Full Text PDF | PubMedSee all References)(17) and with the suggestion that the fast-release pool could consist of hemifused vesicles (18xProtein machines and lipid assemblies: current views of cell membrane fusion. Lentz, B.R., Malinin, V., Haque, M.E., and Evans, K. Curr. Opin. Struct. Biol. 2000; 10: 607–615Crossref | PubMed | Scopus (106)See all References)(18). The resolution of these micrographs (∼4 nm) and the diameter of the hemifused structures (∼8 nm; see Fig. 1 in Zampighi et al. (9xConical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused. Zampighi, G.A., Zampighi, L.M., Fain, N., Lanzavecchia, S., Simon, S.A., and Wright, E.M. Biophys. J. 2006; : 2910–2918Abstract | Full Text | Full Text PDF | PubMed | Scopus (52)See all References)(9)) made it difficult to determine whether stalks or TMC were present in hemifused regions, although the conclusion of a hemifused structure seems irrefutable. Similarly, it is difficult, if not impossible, to discern the location of pores (edge or center of hemifused structures) or to be certain about the location of proteins associated with these structures. Zampighi et al. point out filamentous structures near to but not in the hemifusion structures. Nonetheless, the very small size of the hemifused structures makes it unlikely that there is significant protein material intimately associated with them. Although this uncertainty means that the elegant reconstructed images of Zampighi et al. will not end the controversy between the lipidic- and proteinacious-pore camps, they do provide a quantum jump in structural support for the lipidic pore model.