In Vivo Analysis of Membrane Fusion

Abstract

Membranes provide a barrier that allows chemical reactions to be isolated from the environment. The plasma membrane, for example, delineates self from nonself, and thus must have played an essential role in the evolution of life. Yet under numerous circumstances it is equally important that membranes be breached. Numerous forces oppose the spontaneous fusion of membranes; thus, specialised proteins have evolved to fuse membranes. The most well‐understood fusion proteins are the viral fusion proteins and the SNARE proteins used in the secretory pathway. Recent discoveries have added to the list of fusogens for which necessity and sufficiency have been demonstrated. The list now includes the fusion family proteins, used in cell–cell fusion, and the dynamin‐related proteins, used in organelle fusion. Despite, the diverse structures of fusion proteins, it is likely that they all function to drive membranes through a series of common lipid intermediates. In this article, we review the mechanisms of fusion for biological membranes, and highlight the similarities and differences in these processes.

Key Concepts

  • Fusion proteins can be divided into three major classes: the secretory pathway uses SNARE proteins, cell–cell fusion uses proteins related to viral fusogens and organellar fusion is driven by dynamin‐related GTPases.
  • Fusion proteins are thought to lead membranes through the common set of lipid intermediates: lipid stalk, hemifusion diaphragm and pore formation.
  • Fusion proteins, either SNAREs or viral proteins, play an active role in all steps of the fusion process.
  • Multimerisation of fusogens improves efficiency and in many instances is a requirement for fusion.
  • Diverse proteins act as the fusogens in cell–cell fusion; many remain undiscovered.

Keywords: membrane fusion; virus; SNARE ; lipid; fusogen

Figure 1. Common lipid intermediates during fusion. The proximal leaflets of the plasma bilayers are brought into close proximity forming the hourglass‐like structure known as a lipid stalk. This stalk is then expanded forming a hemifusion diaphragm. In the hemifusion diaphragm the distal leaflets of the bilayer are now in direct contact. A rupture in the hemifusion diaphragm leads to the initial opening of a fusion pore that is then expanded leading to full fusion. The insert shows the shape of lysophosphatidylcholine (LPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). LPC induces positive curvature, whereas PE induces negative curvature. Adding lipids with positive curvature to the proximal leaflets inhibits stalk formation (red lipid – ‘stalk’ and ‘two membranes’ steps). Adding lipids with positive curvature to the distal leaflets promotes fusion (red lipid – ‘pore step’). Arrowheads point to areas where the lipid must adopt a net negative curvature.
Figure 2. Viral fusion using the influenza HA fusion protein and the E fusion protein from flavivirus as the primary examples. Structural rearrangements of the E fusion protein from flavivirus are shown to illustrate commonalities and differences between type I and type II fusion proteins. (a) The influenza virus enters the host by being taken up through the endocytic pathway. On endocytosis, the acidic environment in the endosome activates the HA fusion proteins and the viral capsid is released into the cytoplasm. Structural rearrangements (detailed in c) lead to the fusion of viral and host membranes releasing the contents of the influenza virus into the cytoplasm. (b) While the primary sequence can vary widely, viral fusogens can clearly be classified into three structural classes – for simplicity only the postfusion trimers are shown. The illustrated examples are influenza HA (type I), tick‐borne encephalitis virus TBEV‐E (type II) and vesicular stomatitis virus VSV‐G (type III). Arrowheads indicate the position of the fusion‐peptides/fusion loops in the postfusion structures. Despite the structural differences, viral fusogens undergo many common rearrangements to catalyse fusion (compare c and d). (c and d) The schematics illustrate the rearrangements that HA and TBEV‐E undergo during fusion. Initially, HA and TBEV‐E are present only on the viral membrane (1). On activation, in this case by low pH, the fusion peptide (in red) is exposed and inserted into the host membrane (2). In the case of the type II E fusion protein, E dimer go through a monomeric state to then come together to form a trimer after low pH activation. By contrast, the type I HA fusion proteins exist as trimers before activation. The transition through a monomeric state is still controversial. After activation, the viral and host membranes are then brought into close proximity (3). Zippering of the HA and E proteins (in which the two ends of the fusion protein fold back on each other) induces lipid stalk formation in which the proximal leaflets of the membranes are fused (4). Full zippering induces pore formation, in which the distal leaflets have now become one (5). The postfusion HA and E proteins are left on the membranes of the host having accomplished their function – catalysing the release of the viral genome into the host cell (6). While they are based on experimental evidence, some individual rearrangements are by necessity hypothetical – for instance, the presence of a monomeric transition state in HA‐based fusion. Structures illustrated in (b) were rendered by Thien Vu.
Figure 3. Secretory vesicle fusion using neurotransmission as the example. (a) Synaptic vesicles fuse at synapses and release neurotransmitters into the synaptic cleft. SNARE proteins are present on synaptic vesicles (synaptobrevin – blue) and on the plasma membrane (syntaxin – red and SNAP25 – green). Structural rearrangements in the SNARE proteins are described in detail in (d). (b) The structure of the fully assembled SNARE complex. The example shown is of the SNAREs used in neurotransmission; however, fusion reactions in the secretory pathway all use related SNARE proteins. These proteins form a four‐helix bundle during fusion – the so‐called core complex. Syntaxin contains an additional inhibitory domain known as the Habc domain. (c) The four SNARE family members are characterised by conserved residues that face into the centre of the four‐helix bundle. Illustrated is a crosswise slice through the core complex showing the interactions between arginine on the R SNARE (synaptobrevin) and the three glutamines of the Qa‐SNARE (syntaxin) and the Qbc‐SNARE (SNAP25). In many fusion complexes the Qb and Qc helices are located on separate proteins rather than on a single molecule, as is the case with SNAP25. (d) Unlike viruses, SNAREs are initially located on both membranes destined to fuse (1). Before assembly into the core complex, the SNARE domains are unstructured except for syntaxin which adopts a closed confirmation where the inhibitory Habc domain folds over the SNARE motif. The plasma membrane SNAREs are thought to form an acceptor complex in which syntaxin and SNAP25 partially assemble (2). Synaptobrevin then joins the complex, by making initial contact at the ‐terminal membrane‐distal portion (3). The SNAREs become progressively more structured in an ‐ to ‐terminal direction as they zipper up. The zippering of the SNAREs pulls the proximal leaflets of the membranes together so that they fuse and form a lipid stalk (4). The continued zippering opens the initial fusion pore (5) – the distal leaflets of the membranes have now become one. Post fusion, all of the SNARE proteins are located in the plasma membrane (6). Like the viral fusogens, there is still considerable debate about the exact interaction of the SNAREs and the lipid membranes; thus, while the schematic diagram is based on experimental evidence, numerous individual rearrangements are by necessity hypothetical.
Figure 4. Cell–cell fusion reactions are catalysed by an assortment of unique fusion proteins. The cell–cell fusion reactions are divided into broad categories of those that sculpt organ and cell (left) and those that catalyse gamete fusion (right). The cell–cell fusogens of organ and cell sculpting are better understood – both EFF‐1 and the syncytins have been validated as fusogens. By contrast, the gamete fusogens are less well characterised. Indeed, at present, not a single gamete fusogen has been validated as function in heterologous systems. The gamete fusion molecules represent only the best guesses for the fusogen and in some case have instead been shown to play supporting roles (such as the tetraspanin molecules TET9, 11 and 12 used in ). (a) EFF‐1 is the primary cell–cell fusogen of , it was identified by virtue of its role in the formation of the hypodermis. The schematic represents a embryo at approximately the 1.5‐fold embryonic stage. At this stage the cells destined to become the hypodermis (shaded in purple) remain unfused. EFF‐1 mediates their eventual fusion. EFF‐1 catalyses homotypic fusion – it must be present on both membranes destined to fuse. It is initially upright in the membrane (Figure represents the structural rearrangements it undergoes during fusion). EFF‐1 is necessary and sufficient for fusion. (b) EFF‐1 also catalyses self fusion (that is, fusion of cell to self rather than fusion of cell to neighbour). This is dramatically illustrated in its role in sculpting the PVD neurons. (c) Along with EFF‐1 the other well understood fusion event is the cell–cell fusion underlying placental formation. Between child and mother, the placenta forms a large syncytium known as the syncytiotrophoblast. Mediating this large scale cell–cell fusion event is a class of fusogens, the syncytins, derived from endogenous retroviruses. Syncytins are structurally homologous to the type I viral fusogens. Moving to gamete fusion, (d) the best candidates for sperm–egg fusion in mammals are Izumo, localised on sperm, and its egg‐localised binding partner Juno. Izumo encodes a member of the immunoglobulin superfamily; Juno encodes a member of the folate receptor family. CD9 has also been experimentally linked to the fusion event. CD9 is a member of the tetraspanin superfamily. It is not known how Izumo, Juno and CD9 might catalyse fusion. (e) A more well conserved (and therefore more evolutionarily ancient) gamete fusion family are the asymmetrically localised HAP2/GSC1 proteins first identified in plants. Gamete fusion in involves two sperm being delivered via a pollen tube that enters the embryo sac. The two male gametes (sperm) fuse to the two female cells (egg and central cell). Like mammalian gamete fusion, tetraspanin molecules are also involved (TET9, 11 and 12); they are almost certainly acting to spatially organise a fusogen rather than being the fusogen themselves. (f) Yeast haploid cells fuse during mating. Molecules suggested to mediate yeast mating cell fusion are Prm1p, Fig1p and Fus1p. Prm1p is multipass membrane protein whose expression is induced by the mating pheromone. Fus1p is a single‐pass membrane protein in whose absence the fusion pore opening is delayed and inefficient. Fig1p is a multipass membrane protein. Fig1p fusion defects can be rescued by increasing calcium concentration.
Figure 5. EFF‐1 and syncytin – metazoan fusogens with viral origins. (a) The structure of EFF‐1 is strikingly similar to type II viral fusogens. The postfusion trimers of EFF‐1 and the simian foamy virus E1 fusogen are placed side by side for comparison (also compare to tick‐borne encephalitis virus TBEV‐E from Figure). Interestingly, EFF‐1 lacks the fusion‐loops (open arrowheads) that characterise type II viral fusogens (closed arrowheads). This is consistent with the observation that EFF‐1 is needed in both membranes destined to fuse unlike viral fusogens that begin life in a single membrane and need a mechanism to anchor to the host membrane. (b) The structure of syncytin is strikingly similar to type I viral fusogens. The postfusion trimers of syncytin 2 and the moloney murine leukaemia virus type I fusogen are shown for comparison (also compare to influenza HA from Figure). (c) Schematic of the structural intermediates that EFF‐1 undergoes during fusion. EFF‐1 begins life as an upright monomer present in both membranes destined to fuse (1). These monomers make initial contact across the space between the two cells (2). After initial contact, the monomers start to assemble into higher‐order trimers bringing the two membranes into closer proximity (3). Zippering of EFF‐1 (in which the two ends of the fusion protein fold back on each other) induces lipid stalk formation in which the proximal leaflets of the membranes are fused (4). Full zippering fuses the distal leaflets opening an initial fusion pore (5). The postfusion EFF‐1 trimers are left on the membranes facing out of the cell (6). Like in Figures and the schematic is based on experimental evidence, but also contains a fair degree of speculation partially derived from comparisons to viral fusion (Figure). Structures illustrated in (a and b) were rendered by Thien Vu.
Figure 6. Numerous organelles within cells undergo nonSNARE based fusion. Both mitochondria and endoplasmic reticulum (and potentially other organelles) undergo fusion that is catalysed by a family of membrane anchored GTPases collectively known as dynamin‐related proteins (DRPs) (a) Mitochondria are unique in that they contain an inner and outer membrane whose fusion must be coordinated. Fusion of the outer membrane is catalysed by fuzzy onions (Fzo1p), known as mitofusins in mammals. Fzo1p contains two transmembrane domains anchoring it in the outer membrane. The transmembrane domains are separated by a loop that extends into the space between outer and inner membranes and may play a role in coordinating the fusion of these two membranes. Facing into the cytoplasm, Fzo1p contains a GTPase domain (green oval) and α‐helices (yellow rods) that form coiled‐coils between two mitochondria destined to fuse. Inner membrane fusion is catalysed by Mgm1p, known as Opa1 in mammals. The dynamin‐related Mgm1p contains a GTPase domain (purple oval) and is localised to the inner membrane. In yeast the coordination of outer and inner membrane fusion might be accomplished by Ugo1p, however, Ugo1p is not conserved outside of yeast. (b) Endoplasmic reticulum (ER) membrane fusion is catalysed by DRPs. The ER fusion DRPs include: atlastin (mice and flies), RDH3 (plants) and Sey1p (yeast). They have been shown to be necessary and sufficient for fusion. Much like Fzo1p/mitofusin and Mgm1p/Opa1, they also contain two transmembrane domains (blue) a series of α‐helices (yellow rods), a cytoplasmic facing GTPase domain (purple oval), and a ‐terminal extension that might behave as a lipid disrupting domain (red rods). (c) The crystal structure of the postfusion atlastin dimer. (d) Schematic of the structural intermediates that atlastin undergoes during fusion. Atlastin begins life in both membranes destined to fuse (1). It is initially bound by GTP, and exists in a compact orientation. An unknown trigger leads to opening of atlastin (2). On opening, the GTPase domains form an initial interaction tethering the two membranes destined to fuse (3). This initial interaction between atlastins is converted to a stronger association that involves the α‐helices (yellow rods) (4). The association of the α‐helices trans brings the membranes into close proximity and potentially leads to proximal membrane fusion. The lipid disrupting domains (red rods) are now coordinated to open an initial fusion pore (5). The continued dilation of this initial fusion pore eventually completes the full fusion of the two membranes leaving the postfusion dimers in the fused membrane with the GTPase domains facing into the cytoplasm (6). The exact step at which GTP is hydrolysed to GDP is still a matter of significant debate and therefore, it is not indicated exactly at which step it might occur. The structures of atlastin illustrated in (c) were rendered by Thien Vu.
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Further Reading

Aguilar PS , Baylies MK , Fleissner A , et al. (2013) Genetic basis of cell‐cell fusion mechanisms. Trends in Genetics 29 (7): 427–437.

Baquero E , Albertini AA , Vachette P , et al. (2013) Intermediate conformations during viral fusion glycoprotein structural transition. Current Opinion in Virology 3 (2): 143–150.

Escobar‐Henriques M and Anton F (2013) Mechanistic perspective of mitochondrial fusion: tubulation vs. fragmentation. Biochimica et Biophysica Acta 1833 (1): 162–175.

Harrison SC (2008) Viral membrane fusion. Nature Structural Molecular Biology 15: 690–698.

Hoppins S and Nunnari J (2009) The molecular mechanism of mitochondrial fusion. Biochimica et Biophysica Acta 1793: 20–26.

Jahn R and Fasshauer D (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490 (7419): 201–207.

McNew JA , Sondermann H , Lee T , Stern M and Brandizzi F (2013) GTP‐dependent membrane fusion. Annual Review of Cell and Developmental Biology 29: 529–550.

Podbilewicz B (2014) Virus and cell fusion mechanisms. Annual Review of Cell and Developmental Biology 30: 111–139.

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Palfreyman, Mark T, and Jorgensen, Erik M(Jul 2015) In Vivo Analysis of Membrane Fusion. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0020891.pub2]