Mechanism of Ca 2+ -Regulated Membrane Fusion

Transcription

1 Mechanism of Ca 2+ -Regulated Membrane Fusion Thomas C. Südhof $ Dept. of Molecular and Cellular Physiology, and # Howard Hughes Medical Institute, Lorry Lokey SIM1 Building Room G1021, 265 Campus Drive, Stanford University School of Medicine, CA 94305, USA 1

2 Ever since Bernhard Katz's pioneering work, Ca 2+ is known to trigger neurotransmitter release by stimulating synaptic vesicle exocytosis, thereby initiating synaptic transmission. Katz described the beautiful precision and extraordinary speed of Ca 2+ - triggered synaptic vesicle exocytosis, but how this precision and speed could possibly be achieved by a plausible molecular mechanism remained unknown even the most basic events underlying membrane fusion were uncharacterized. Two decades ago, we set out to study this fundamental problem, which is comprised of two related questions: How do synaptic vesicles and plasma membranes undergo fusion, and how does Ca 2+ control this fusion process? Together with the contributions of others, our work revealed a general mechanism of membrane fusion that is applicable to all intracellular membrane traffic except for mitochondrial fusion, and that operates by the interaction of SNARE and SM proteins. Moreover, our work identified a universal mechanism by which Ca 2+ controls membrane fusion during exocytosis that likely accounts for all Ca 2+ -induced exocytosis reaction, from neurotransmitter release to hormone secretion, fertilization, and mast cell degranulation, and that operates by Ca 2+ -binding to synaptotagmins. Different SNARE, SM and synaptotagmin proteins mediate distinct types of Ca 2+ - triggered exocytosis, with a precise correspondence between the biochemical activities of synaptotagmins and the Ca 2+ -triggering properties of the respective type of exocytosis. Thus, our work delineated a molecular framework that provides insight into the molecular basis of all intracellular membrane fusion, and accounts for the astounding speed and precision of synaptic exocytosis during synaptic transmission. 1. Where we started On a Monday morning in October 1986, I walked for the first time into my newly opened laboratory at UT Southwestern in Dallas, Texas, fresh out of my postdoctoral training with Mike Brown and Joe Goldstein. After surveying the biological landscape, I had just decided to try to study how neurotransmitters are released at synapses. At that time, the work of Bernhard Katz, Victor Whittaker and others had established that neurotransmitter release occurs by Ca 2+ - triggered exocytosis of synaptic vesicles. Moreover, exquisitely detailed electrophysiological studies had revealed that release occurs within a few hundred microseconds after Ca 2+ entered 2

3 a nerve terminal, exhibits amazing plasticity, and depends on Ca 2+ in a non-linear fashion. How neurotransmitters are released by exocytosis, however, was unknown. In 1986, the importance of understanding neurotransmitter release was unquestioned it alone initiates synaptic transmission. Besides Ca 2+, however, not a single molecule important for release had been identified. Screens by Sidney Brenner, Randy Schekman, and their colleagues had isolated gene mutations that disrupt synaptic transmission in C. elegans (Brenner, 1974) or block processing of proteins in the secretary pathway in yeast (Novick et al., 1980), but the nature of the corresponding genes and the function of the encoded proteins were not clear. It was realized that solving neurotransmitter release represented an interdisciplinary problem that intersected the fields of neuroscience, cell biology, and physiology. Neurotransmitter release was known to involve membrane fusion during exocytosis, similar to other membrane trafficking reactions (Whittaker et al., 1972; Heuser and Reese, 1973), and to represent one half of synaptic transmission, thus making it essential for all brain function (Katz, 1969). Moreover, it became increasingly apparent that neurotransmitter release resembled other processes involving Ca 2+ -regulated exocytosis, such as hormone secretion, fertilization, and mast cell degranulation (Nagasawa et al., 1970; Orci et al., 1973; Röhlich et al., 1971; Kanno et al., 1973). How all of these processes are related to each other, however, was unclear, nor were mechanisms envisioned that would confer specific properties onto the various types of exocytosis. Thus, despite the unquestioned importance and fascinating properties of Ca 2+ -regulated exocytosis, little was known about its mechanisms, or those of membrane traffic in general. It was this situation that prompted me to decide, at the very beginning of my career, to search for a molecular handle on how membranes undergo fusion, and how this fusion is controlled by Ca 2+. We chose a simple initial approach: to isolate and clone any possibly important protein in synaptic vesicle exocytosis, solely based on the localization of a protein to presynaptic terminals. At the beginning, we focused on the synaptic vesicles themselves because they could be isolated at high yield and purity, and performed all studies in close collaboration with Reinhard Jahn s laboratory. Later on, we expanded this approach to include the presynaptic active zone and plasma membrane. In the initial part of the project, the goal was simply to achieve a description of the presynaptic terminal, a kind of molecular anatomy, as a starting point for a later functional dissection of the proteins thus identified (Südhof and Jahn, 1991). 3

4 The initial phase of the project dealing with synaptic vesicles lasted for about 5 years, although additional vesicle proteins continued to be discovered afterwards. Together with the work of other laboratories, this phase resulted in the molecular characterization of the most important synaptic vesicle proteins. We purified and cloned the first synaptic vesicle protein, synaptophysin-1, in 1987 (Südhof et al., 1987), followed rapidly by cytochrome-b561 (Perin et al., 1988), synaptobrevin/vamp (Südhof et al., 1989a; note that the Scheller lab independently cloned this protein [Trimble et al., 1988]), synapsins (Südhof et al., 1989b), Rab3A (Fischer v. Mollard et al., 1990), proton pump subunits (Südhof et al., 1989c; Perin et al., 1991), and synaptotagmins (Perin et al., 1990; Geppert et al., 1992). This initial phase produced a fairly comprehensive description of synaptic vesicles, and was followed by a second phase pursuing an analysis of the presynaptic active zone and presynaptic plasma membrane. After the success of the descriptive analysis of synaptic vesicles became apparent, similar approaches were pursued for other neuronal and non-neuronal structures, in particular the postsynaptic density (Cho et al., 1992; Kim et al., 1997). As the molecular shape of synaptic vesicles emerged, the question of how it may interact with the presynaptic plasma membrane became interesting. Early on, Mike Wilson had cloned an abundant presynaptic plasma membrane protein named SNAP-25 that was later identified as a presynaptic plasma membrane SNARE protein (Oyler et al., 1989). Subsequently, syntaxins were cloned under the names of HPC-1 (Inoue et al., 1992) and epimorphin (Hirai et al., 1992), and later named 'syntaxins' by Richard Scheller when he found that syntaxins connect synaptotagmin-1 on synaptic vesicles to Ca 2+ -channels in the presynaptic plasma membrane (Bennett et al., 1992). Quickly afterwards, we identified Munc18-1 by purifying it as a syntaxinassociated protein, resulting in the proposition that Munc18-1 together with the SNARE proteins constitutes the fusion machine of synaptic vesicles (Fig. 1; Hata et al., 1993; see also Garcia et al., 1994; Pevsner et al., 1994). At the same time, we became interested in potential mechanisms linking the presynaptic plasma membrane to postsynaptic specializations. We searched for a receptor of α-latrotoxin, a potent neurotoxin that induces neurotransmitter release, with the notion that this receptor must be a specifically presynaptic cell-surface protein with a function related to synaptic transmission. This search led to the discovery of neurexins as presynaptic cell-adhesion molecules (Ushkaryov et al., 1992), and spawned a series of followup studies which, among others, showed that neurexins form a trans-synaptic junction with postsynaptic cell-adhesion molecules called neuroligins (Ichtchenko et al., 1995) and LRRTMs 4

5 (Ko et al., 2009), and that these molecules are centrally implicated in autism and schizophrenia (Südhof, 2008). Thus, at the end of 1993 most components of the synaptic fusion machinery had been identified, from SNARE and SM proteins to synaptotagmins and synaptic cell-adhesion molecules. As we will describe in the next section, however, it took several years until a plausible model emerged describing how SNARE and SM proteins mediate fusion, even though the collaborative role of these proteins in fusion had been proposed early on (Hata et al., 1993; Fig. 1). 2. Elucidating the mechanism of synaptic membrane fusion Four independent lines of evidence led to our current understanding of how SNARE and SM proteins mediate membrane fusion: a. Genetics in C. elegans and yeast identified genes encoding SNARE and SM proteins as essential for synaptic transmission and membrane traffic, respectively (Brenner, 1974; Novick et al., 1980). b. Studies on how clostridial neurotoxins block synaptic fusion identified SNARE proteins as essential components of the fusion machinery (Link et al., 1992; Schiavo et al., 1992; Blasi et al., 1993a and 1993b; Binz et al., 1994; Fig. 2). c. In vitro reconstitution of fusion led to the identification of a chaperone complex containing NSF (for 'N-maleimide sensitive factor') that is essential for repeated rounds of fusion, resulting in the finding that SNARE proteins form tight complexes that are dissociated by said chaperone complex (Söllner et al., 1993a and 1993b) d. Biochemical studies followed by functional tests identified SM proteins as essential components of the fusion machinery that interact with SNAREs in all fusion reactions (Hata et al., 1993) Each of the four lines of evidence depended on the others to be interpretable, but occurred independently, with the realization of the importance of SM proteins coming last because confusing biochemical evidence obscured their universal function (see below). Together, these studies identified a general mechanism in which a protein complex that is composed of SNARE and SM proteins constitutes the minimal machinery for membrane fusion (Fig. 3), as suggested first in the description of Munc18-1, the first SM protein implicated in fusion (Hata et al., 1993; 5

6 Fig. 1). During fusion, SNARE and SM proteins undergo a cycle of association and dissociation that is maintained by chaperones supporting SNARE-complex assembly (CSPs and synucleins) and disassembly (NSF and SNAPs; Fig. 3). The SNARE/SM protein cycle catalyzes membrane fusion. A model of how the collaboration of SNARE and SM proteins universally mediates eukaryotic membrane fusion is shown in Fig. 3. Although the physical chemistry of fusion is still not completely understood, the underlying principle of SNARE and SM protein function is simple: SNARE proteins are membraneanchored and contain a 60-residue sequence motif (the SNARE motif ). Four SNARE motifs assemble into a parallel four-helical bundle, as first shown by electron microscopy (Hanson et al., 1997). There are four canonical types of SNARE motifs, referred to as Qa, Qb, Qc, and R (Jahn et al., 2003). The four SNARE motifs are usually present in four separate SNARE proteins, except in exocytosis where the plasma membrane SNARE protein SNAP-25 and its homologs contain two SNARE motifs (a Qb and Qc motif) thus only three separate SNARE proteins, syntaxin-1, synaptobrevin/vamp, and SNAP-25, mediate synaptic exocytosis (Fig. 3). SNARE proteins are present on both membranes destined to fuse with each other. When membranes undergo fusion, the SNARE proteins on the two membranes form a trans-complex that involves a progressive zippering of the four-helical SNARE-complex bundle in an N- to C- terminal direction (step 1 in Fig. 3). The progressive zippering of trans-snare complexes forces the two fusing membranes into close proximity. It is thought that the energy released during such trans-snare complex assembly drives fusion, and provides the force that breaks the energy barrier to fusion by the negatively charged phospholipid headgroups (step 2, Fig. 3). Full assembly of the trans-snare complex (together with the action of the SM protein, see below) opens the fusion pore, which then expands (step 3, Fig. 3). Fusion-pore expansion transforms the initial 'trans'-snare complexes into 'cis-snare complexes, as the initially separate membranes now merge completely. cis-snare complexes then become substrates for the ATPase NSF, which binds to SNARE complexes via the α/β/γ-snap adapter proteins (no relation to SNAP-25; McMahon and Südhof, 1995), and dissociates SNARE complexes into monomers (Söllner et al., 1993b), thereby completing the cycle. The essential role of SNARE proteins in fusion was first realized when all three synaptic SNARE proteins were identified as substrates for clostridial neurotoxins that block synaptic fusion (Link et al., 1992; Schiavo et al., 1992; Blasi et al., 1993a and 1993b). Subsequently, a plethora of 6

7 independent evidence, ranging from genetic studies to liposome fusion assays, confirmed the central role of SNARE proteins in fusion (reviewed in Südhof and Rothman, 2009). Moreover, recent findings further embellished the cycle to include a novel set of chaperones that link the synaptic SNARE/SM protein cycle to neurodegenerative diseases. These chaperones are the synaptic vesicle proteins CSPα (for cysteine string protein-α) and synucleins (Chandra et al., 2005; Burre et al., 2010; Sharma et al., 2011). CSPα is a classical co-chaperone that forms a complex with Hsc70 and SGT (Tobaben et al., 2001), and maintains SNAP-25 in a SNAREcomplex assembly competent state under ATP hydrolysis (Sharma et al., 2011). Synucleins, in contrast, are peripheral membrane proteins that bind to nascent SNARE complexes via synaptobrevin, and promote the rate of SNARE-complex assembly by a non-enzymatic mechanism (Burre et al., 2010). Unlike NSF and SNAPs, CSPα and synucleins are not required for regular rounds of SNARE-complex assembly and disassembly, but are essential for longterm maintenance of nerve terminal function. Deletions of these proteins cause severe neurological phenotypes and premature mortality (Fernandez-Chacon et al., 2004; Chandra et al., 2005; Burre et al., 2010). Thus, the SNARE/SM protein cycle exhibits a satisfying symmetry in that both assembly and disassembly of SNARE complexes involve chaperones. SM proteins are essential co-agonists for SNARE proteins in membrane fusion. Although SNARE-complex assembly represents an intuitively persuasive mechanism for driving membrane fusion, innumerable lines of evidence showed that SNARE-complex assembly is not sufficient to mediate fusion (Südhof and Rothman, 2009). Nevertheless, our initial proposal that the SM protein Munc18-1 is an essential co-agonist of SNARE proteins in synaptic fusion (Fig. 1), and that by extension, SM proteins generally act as co-agonists for SNARE proteins in fusion, met with strong skepticism because biochemical data appeared to argue against this hypothesis. Specifically, it was suggested that Munc18-1 inhibits fusion instead of promoting it because excess Munc18-1 appeared to inhibit SNARE-complex assembly (Pevsner et al., 1994; Wu et al., 1998). Indeed, in a rewarding collaboration with Josep Rizo s laboratory we identified two conformational states of syntaxin-1, a closed state in which its N-terminal Habc-domain folds back onto the SNARE motif, rendering it unable to form SNARE complexes, and an open state in which the SNARE motif is free to participate in SNARE complexes (Fig. 4). We showed that Munc18 binds to the closed state of syntaxin-1 that is unable to participate in SNARE complexes (Dulubova et al., 1999). 7

8 This observation offered an explanation for the puzzling inhibition of SNARE-complex assembly by excess Munc18-1, but was difficult to reconcile with an essential role of Munc18-1 in synaptic fusion, a role that among others was documented in knockout mice in which deletion of Munc18-1 produced a much more severe phenotype than deletion of the SNARE protein synaptobrevin-2 (Verhage et al., 2000; Schoch et al., 2001; Fig. 5). Moreover, Peter Novick's found in yeast that the SM protein Sec1p binds to assembled SNARE complexes, not syntaxin (Carr et al., 1999), whereas Richard Scheller's laboratory was again unable, using syntaxin-1 proteins containing N-terminally fused tags, to detect any binding of Munc18 to assembled SNARE complexes, but only confirmed the binding to closed syntaxin (Yang et al., 2000). The resolution to these puzzling and often contradictory observations came from our studies examining the interaction between SM and SNARE proteins that are involved in other intracellular membrane fusion reactions (Yamaguchi et al., 2002; Dulubova et al., 2002 and 2003). In these studies we observed again in collaboration with Josep Rizo s laboratory that a previously ignored, conserved N-terminal sequence of syntaxins mediates attachment of SM proteins to these syntaxins, independent of the conformational state of the syntaxins, but with a high degree of specificity (see example in Fig. 6). These results suggested that in intracellular fusion reactions, SM proteins interact with syntaxins when syntaxins are assembled in SNARE complexes. Strikingly, we observed that synaptic syntaxin-1 contains an N-terminal sequence similar to that of intracellular syntaxins. This prompted the discovery of a second Munc18-binding mode to SNARE proteins, besides the previously established binding of Munc18-1 to syntaxin in the closed conformation (Dulubova et al., 1997). The new second binding mode showed that Munc18-1 is attached to syntaxin-1 in the open conformation assembled in SNARE complexes via the N-terminal syntaxin-1 sequence (Dulubova et al., 2007; independently found by Rothman's laboratory [Shen et al., 2007]). Note that the first binding mode does not require the syntaxin N-terminus, and operates with syntaxin-1 fusion proteins, whereas the second binding mode depends on a free syntaxin N-terminus, accounting for the inability of syntaxins lacking a free N-terminus to support this binding (Yang et al., 2000). Subsequent physiological experiments confirmed that both binding modes are essential for normal synaptic exocytosis (Khvotchev et al., 2007; Gerber et al., 2008; Deak et al., 2009; Rathore et al., 2010). In particular, knockin mice in which syntaxin-1 is rendered constitutively 'open' revealed that Munc18-binding to closed syntaxin is crucial for regulating the initiation of 8

9 fusion (Gerber et al., 2008). In contrast, peptide-injection experiments into calyx synapses, transfection studies on cultured cells, and rescue experiments with Munc18-1 knockout mice and syntaxin-mutant C. elegans demonstrated that the second mode of Munc18-binding to syntaxin is centrally involved in fusion as such, consistent with the fact that this binding mode is shared among all SNARE and SM proteins (Khvotchev et al., 2007; Gerber et al., 2008; Deak et al., 2009; Rathore et al., 2010). The importance of Munc18-1 is further highlighted by the fact that even heterozygous Munc18-1 mutations in humans produce severe impairments (Saitsu et al., 2008). Thus, the overall picture that emerges from these studies is that SM proteins are universally required as collaborators of SNARE proteins in fusion. 3. Understanding how Ca 2+ controls synaptic membrane fusion All intracellular fusion is regulated in space and time, with the spatial and temporal precision of synaptic exocytosis being the most extreme example. When Ca 2+ enters a presynaptic nerve terminal via voltage-gated channels, it triggers synaptic vesicle fusion in less than a millisecond, accelerating the spontaneous rate of synaptic vesicle fusion several thousand fold, with precise targeting of vesicle fusion to the presynaptic active zone (Meinrenken et al., 2003). No other membrane fusion reaction exhibits the same speed or spatial precision. Ca 2+ -triggering of synaptic fusion underlies all synaptic transmission, and enables all brain function it controls neuronal activity from reflex reactions to consciousness and emotions. Moreover, Ca 2+ regulates many other types of exoctyosis, such as hormone secretion, mast cell degranulation, and fertilization. Although these types of exocytosis are less precisely regulated in space and time than synaptic exocytosis, recent work reveals that the mechanisms mediating these types of exocytosis are similar to those governing Ca 2+ -triggering of synaptic vesicle fusion. As we will describe below, all of these events are controlled by a family of Ca 2+ -sensor proteins called synaptotagmins that we identified in 1990 (Perin et al., 1990). Biochemical properties of synaptotagmins. When we purified synaptotagmin-1 (Syt1; Perin et al., 1990), it had been known as a 65 kda immunoreactive band associated with synaptic vesicles (Matthew et al., 1981). Cloning of Syt1 revealed that it is composed of a short N- glycosylated intravesicular sequence, a single transmembrane region, and two cytoplasmic C 2 - domains (referred to as the C 2 A- and C 2 B-domains; Fig. 7A). We subsequently cloned 7 additional synaptotagmin isoforms (Geppert et al., 1992; Li et al., 1995; von Poser et al., 1997; von Poser and Südhof, 2000), and others identified 8 further isoforms (Mizuta et al., 1994; 9

10 Hilbush and Morgan, 1994; Hudsun and Birnbaum, 1995; Craxton and Goedert, 1995; Thompson, 1996; Babity et al., 1997; Fukuda, 2003a and 2003b; Herrero-Turrión et al., 2006), resulting in 16 synaptotagmins in the mammalian genome. Besides these synaptotagmins, defined as proteins containing both a transmembrane region and two C 2 -domains, mammalian genomes encode synaptotagmin-related proteins containing a similar C 2 -domain architecture but lacking a transmembrane region (such as rabphilin), or containing more than two C 2 - domains with a transmembrane region (such as E-Syts and ferlins; reviewed in Pang and Südhof, 2010). Interestingly, synaptotagmins emerged at the very beginning of animal evolution, with cnideria genomes encoding multiple synaptotagmin isoforms, whereas other C 2 -domain proteins, such as E-Syts (Min et al., 2007), are even more ancient evolutionarily. When we cloned Syt1 and found that it contained two C 2 -domains, nothing was known about C 2 -domains except that they represented the "2 nd constant sequence motif" in classical protein kinase C isozymes (Coussens et al., 1986). The structure of Syt1 and the known interaction of protein kinase C with lipids suggested to us that the Syt1 C 2 -domains may bind to lipids, and function in the regulation of fusion (Perin et al., 1990). Indeed, we found that Syt1 binds to phospholipids (Perin et al., 1990) and interacts with phospholipids in a Ca 2+ -dependent manner (Brose et al., 1992). Arguably the most important advance, however, was the demonstration that a single C 2 -domain of Syt1 was capable of Ca 2+ -dependent phospholipid binding (Fig. 7B), which revealed that C 2 -domains are autonomously folded domains that bind Ca 2+ (Davletov and Südhof, 1993). This was the first demonstration that a C 2 -domain binds Ca 2+, and was surprising because C 2 -domains exhibit no sequence homology to EF-hands, which were the standard Ca 2+ -binding motifs at the time (Kretsinger, 1976). The observation that Syt1 C 2 -domains bind Ca 2+ suggested that C 2 -domains may generally function as Ca 2+ -binding modules (Davletov and Südhof, 1993), a hypothesis that has now been widely confirmed. To elucidate how Syt1 C 2 -domains bind Ca 2+, and whether and how other C 2 -domains do so as well, we collaborated first with Steve Sprang and then with again Josep Rizo. The resulting atomic structures of the Syt1 C 2 A-domain revealed a β-sandwich containing flexible top and bottom loops, with Ca 2+ exclusively binding to the top loops (Fig. 7C; Sutton et al., 1995; Ubach et al., 1998). This was the first structure of a C 2 -domain; subsequent work from our laboratory and others revealed that all C 2 -domains exhibit a similar structure, and bind Ca 2+ exclusively via their top loops (to cite papers from our own laboratory, see Shao et al., 1996, 1997, and 1998; Ubach et al., 1998 and 1999; Fernandez et al., 2001; Gerber et al., 2001; Dai et al., 2004 and 2005; Garcia et al., 2004; Lu et al., 2006; Guan et al., 2007; Shin et al., 2010; Xue et al., 2010). 10

11 Systematic mapping of the Ca 2+ -binding site architecture of the Syt1, Munc13, rabphilin, and piccolo C 2 -domains revealed a canonical set of Ca 2+ -binding residues (see references cited above). At present, C 2 -domains are recognized as the second most abundant Ca 2+ -binding motif in eukaryotic cells, with hundreds of C 2 -domain proteins encoded by mammals, preceded in prevalence only by EF-hand Ca 2+ -binding motifs. Moreover, most C 2 -domains bind phospholipids, and phospholipid binding is now thought of as a general function of C 2 -domains in diverse proteins. Just as for EF-hand motifs, however, C 2 -domains do not always bind Ca 2+ and/or phospholipids. The canonical Ca 2+ -binding residues are lacking in many C 2 -domains, including many synaptotagmin isoforms. Moreover, even synaptotagmin C 2 -domains that contain the canonical Ca 2+ -binding residues do not always bind Ca 2+, since small shifts in the relative positions of the β-strands can block Ca 2+ -binding as shown for Syt4 (see Dai et al., 2004). In addition to phospholipid-binding modules, C 2 -domains can also function as protein-interaction modules. This again was first shown for Syt1 C 2 -domains which bind to syntaxin-1 and SNAREcomplexes in Ca 2+ -dependent and Ca 2+ -independent reactions (Li et al., 1995; Pang et al., 2006b). Subsequently, many other C 2 -domains were found to be protein-interaction domains, as most clearly revealed in the structure of the complex of the Munc13 C 2 A-domain (which lacks Ca 2+ -coordination sites) with the RIM zinc-finger domain (Lu et al., 2006). As a result, the biochemical and biophysical studies on Syt1 initiated recognition of C 2 -domains as a widespread protein module that mediates the Ca 2+ -regulation of many membrane traffic and signal transduction events, and additionally functions as a Ca 2+ -independent phospholipid and protein interaction module throughout evolution. Proof that synaptotagmin is the Ca 2+ -sensor for neurotransmitter release. The biochemical properties of Syt1, starting with its phospholipid- and Ca 2+ -binding capacity (Perin et al., 1990; Brose et al., 1992; Davletov and Südhof, 1993), suggested that Syt1 constitutes the long-sought Ca 2+ -sensor for neurotransmitter release (Katz, 1969). Initial experiments in C. elegans and Drosophila, however, disappointingly indicated that Syt1 is not required for neurotransmitter release, although these experiments were consistent with a synaptic function for Syt1 (DiAntonio et al., 1993; Littleton et al., 1993; Nonet et al., 1993). Only subsequent studies exploiting the higher resolution of electrophysiological analyses in mammalian synapses and analyzing Syt1 knockout mice revealed that deletion of Syt1 abolishes all fast synchronous synaptic exocytosis in forebrain neurons, but leaves other types of exocytosis intact (Fig. 8; Geppert et al., 1994). Specifically, although normal action-potential evoked release was largely 11

12 ablated, both sucrose-evoked synaptic exocytosis (which is Ca 2+ -independent) and a secondary form of Ca 2+ -triggered synaptic exocytosis mediating slow asynchronous release were fully maintained in Syt1 knockout neurons, accounting for the earlier results in C. elegans and Drosophila that Syt1 is not required for synaptic exocytosis. These experiments established that Syt1 was essential for fast Ca 2+ -triggered release, but not required for fusion as such. The Syt1 knockout analysis strongly supported the hypothesis that Syt1 is the Ca 2+ -sensor for fast synaptic exocytosis, but did not exclude the possibility that Syt1 positions vesicles next to voltage-gated Ca 2+ -channels, with Ca 2+ -binding to Syt1 performing a function unrelated to triggering exocytosis (Neher and Penner, 1994). To directly test whether Ca 2+ -binding to Syt1 triggers neurotransmitter release, we used homologous recombination in mice to introduce a point mutation into Syt1 that decreases its apparent Ca 2+ -binding affinity approximately 2-fold, and used a similar but innocuous point mutation as a negative control (Fig. 9; Fernandez- Chacon et al., 2001). Electrophysiological recordings of synaptic responses from these mice revealed that the point mutation which decreased the apparent Ca 2+ -affinity of Syt1 approximately 2-fold caused a corresponding decrease in the apparent Ca 2+ -affinity of neurotransmitter release, formally proving that Syt1 is the Ca 2+ -sensor for release (Fernandez- Chacon et al., 2001). Moreover, experiments in chromaffin cells (where Syt1 functions as a Ca 2+ -sensor for chromaffin granule exocytosis) using microfluorometric measurements of Ca 2+ and photolysis of caged Ca 2+ confirmed this result for another system (Sorensen et al., 2003). Finally, measurements of spontaneous vesicle exocytosis showed that it is largely Ca 2+ - dependent, and demonstrated that the knockin mutation which decreases the apparent Ca 2+ - affinity of Syt1 also decreases spontaneous release accordingly, indicating that not only evoked exocytosis, but also spontaneous exocytosis is triggered by Ca 2+ -binding to Syt1 (Xu et al., 2009). Spontaneous exocytosis thus likely is induced by stochastic openings of Ca 2+ -channels and/or Ca 2+ -sparks, and is not truly spontaneous. As described above, biochemically Syt1 binds to both phospholipids and to SNARE proteins as a function of Ca 2+. Which of these two activities mediates Ca 2+ -triggering of release? Moreover, why does Syt1 (and other synaptotagmins) have two Ca 2+ -binding C 2 -domains? Finally, can the apparent Ca 2+ -cooperativity of neurotransmitter release (~5 Ca 2+ -ions; Bollimann et al., 2000; Schneggenburger et al., 2000) be accounted for by the 5 Ca 2+ -binding sites in Syt1 (Fig. 7A)? A series of knockin and rescue experiments provided answers to these additional questions. Knockin mutations that selectively alter either phospholipid- or SNARE-binding revealed that both binding modes contribute to Ca 2+ -triggering of release (Pang et al., 2006), suggesting that 12

13 Syt1 essentially acts as a Ca 2+ -dependent bridge during fusion that activates fusion pore opening by connecting phospholipids and SNARE proteins. Rescue experiments with Syt1 mutants lacking Ca 2+ -binding to either the C 2 A- or the C 2 B-domain originally showed in Drosophila that Ca 2+ -binding to the C 2 B-domain is essential for Syt1 function, whereas Ca 2+ - binding to the C 2 A-domain is not (Mackler and Reist, 2001). Higher resolution electrophysiology, however, demonstrated that block of Ca 2+ -binding to the C 2 A-domain also dramatically impairs Syt1 function, and significantly decreases the apparent Ca 2+ -cooperativity of release (Shin et al., 2009). Viewed together, these results establish that Ca 2+ -binding to both Syt1 C 2 -domains triggers release, accounts for the Ca 2+ -cooperativity of release, and stimulates both phospholipid- and SNARE protein-binding to trigger release. One surprise of the Syt1 knockout experiments was that there was a phenotype at all, because 16 different synaptotagmins are expressed in mouse brain, of which 8 synaptotagmins bind Ca 2+. To address which synaptotagmins are Ca 2+ -sensors for synaptic and/or neuroendocrine exocytosis, we performed further knockout and rescue experiments. Strikingly, systematic rescue experiments of Syt1 knockout neurons from mouse cortex revealed that only two synaptotagmins besides Syt1, Syt2 and Syt9, mediate fast synaptic vesicle exocytosis (Fig. 10A; Xu et al., 2008). The three synaptotagmins that function as Ca 2+ -sensors in synaptic vesicle exocytosis exhibit dramatically different kinetic properties, with Syt2 showing the fasted onset and decline in exocytosis, and Syt9 the slowest (Figs. 10B and 10C). Syt2 and Syt9 are not expressed in most forebrain neurons, explaining why the Syt1 knockout produced such a dramatic phenotype (Pang et al., 2007; Xu et al., 2008). Syt2 is primarily expressed in brain stem and spinal cord synapses that rely on fast synaptic information processing, such as the calyx of Held in the auditory pathway (Pang et al., 2006b), whereas Syt9 is primarily present in neurons of the limbic system involved in emotional and instinctive behaviors (Xu et al., 2008). Thus, the kinetic properties of the three synaptotagmins that function as synaptic Ca 2+ -sensors correspond to the functional needs of the synapses containing them, providing an example for how isoform diversification in evolution creates functional specializations. Parallel experiments in neuroendocrine cells revealed that Syt7 also functions as a Ca 2+ -sensor for exocytosis (see below), whereas the functions of the other 4 Ca 2+ -binding synaptotagmins (Syt3, Syt5, Syt6, and Syt10) remain unknown. Moreover, the significance of the Ca 2+ -independent synaptotagmins is also unclear fertile ground for future studies. The Syt1 knockout analysis revealed that not all Ca 2+ -triggered exocytosis is absent in Syt1- deficient neurons, but an 'asynchronous' component remains (Geppert et al., 1994); similar 13

14 results were obtained for Syt2 and Syt9 (Sun et al., 2007; Xu et al., 2008). Moreover, in Syt1- or Syt2-deficient synapses, spontaneous exocytosis is activated, and asynchronous exocytosis is unclamped (Maximov and Südhof, 2005; Pang et al., 2006b; Sun et al., 2007). The availability of Syt2 knockout mice enabled a biophysical characterization of asynchronous exocytosis in the calyx of Held synapse, which allows precise correlations of Ca 2+ -concentration and exocytosis measurements (Bollmann et al., 2000; Schneggenburger et al., 2000). These experiments demonstrated that asynchronous exocytosis is triggered by Ca 2+ with a much slower kinetics and lower apparent Ca 2+ -cooperativity (~2 Ca 2+ -ions) than synchronous exocytosis (~5 Ca 2+ - ions; Sun et al., 2007). Although it remains unknown which Ca 2+ -sensor activates asynchronous release, its different Ca 2+ -cooperativity indicates that it is likely not a synaptotagmin isoform. Indeed, knockout of all Ca 2+ -dependent synaptotagmins fails to block Ca 2+ -dependent asynchronous release (unpublished observation). How synaptotagmin controls SNARE function: Enter complexin. During our studies on how SNARE proteins mediate membrane fusion, we identified a small protein that tightly bound to SNARE complexes, prompting us to name this protein complexin (McMahon et al., 1995). Initial experiments suggested that complexin regulates SNARE complexes, but only during analysis of complexin-deficient neurons did we realize that it represents an essential mediator for synaptotagmin-triggered fusion (Reim et al., 2001; Maximov et al., 2009). Strikingly, complexindeficient neurons exhibit a milder phenocopy of Syt1 KO mice, with a selective suppression of fast synchronous exocytosis and an increase in spontaneous exocytosis (Reim et al., 2001; Maximov et al., 2009; Yang et al., 2010), suggesting that complexin and synaptotagmins operate in the same pathway and are functionally interdependent. However, complexin is not only a sidekick for synaptotagmins, but acts upstream of synaptotagmins to boost priming of synaptic vesicles, a process we refer to as superpriming. Complexin-deficient neurons exbhibit a decrease in the size of the readily-releasable pool of vesicles, whereas synaptotagmindeficient neurons do not (Yang et al., 2010). Overall, these studies led to a model of complexinand synaptotagmin-function in which complexin both superprimes and clamps SNAREcomplexes that form the subsequent substrate of synaptotagmin action (Fig. 11). How does a small molecule like complexin, composed of only ~130 residues, perform such a feat? Just as for synaptotagmin, atomic structures we obtained in collaboration with Josep Rizo paved the way for an understanding (Chen et al., 2001). The structure revealed that complexin, when bound to assembled SNARE complexes, contains two short α-helices flanked by flexible sequences (Fig. 11). One of the α-helices is bound to the SNARE complex, and is essential for 14

15 all complexin function. The second α-helix, referred to as accessory, is dispensable for the superpriming activity of complexin, but is required for its clamping function (Maximov et al., 2009; Yang et al., 2010). The flexible N-terminal sequence of complexin, conversely, is required for superpriming but is dispensable for clamping (Xue et al., 2007; Maximov et al., 2009). Thus, even a small molecule like complexin has a modular structure. Strikingly, complexin biochemically competes with Syt1 for SNARE-complex binding, at least as far as its SNARE-binding α-helix is concerned (Tang et al., 2007). This observation suggested a facile mechanism by which Syt1 exploits the clamped but superprimed state produced by complexin binding to assemblying trans-snare complexes. This model suggests that complexin clamps exocytosis by preventing assemblying trans-snare complexes from completing full assembly, and simultaneously superprimes the complexes by stabilizing them in an energized state (Tang et al., 2007; Yang et al., 2010). During an action potential, Ca 2+ - binding to Syt1 induces Syt1-binding to the trans-snare complexes that are clamped and energized by complexin, thereby dislodging the complexin clamp, and triggering completion of SNARE-complex assembly and fusion pore opening. From neurotransmitter release to organismal biology synaptotagmins as universal Ca 2+ -sensors for exocytosis. The fact that asynchronous exocytosis persists in synaptotagmin-deficient synapses shows that other Ca 2+ -sensors for exocytosis must exist, raising the question of whether non-synaptic forms of Ca 2+ -stimulated exocytosis, such as hormone secretion or mast cell degranulation, are mediated by a synaptotagmin-dependent mechanism, or by a mechanism corresponding to asynchronous release. Studies of hormonal secretion and mast cell degranulation in synaptotagmin knockout mice addressed this question, revealing unequivocally that Syt1, Syt2, Syt7, and Syt9 mediate most Ca 2+ -dependent secretion (Gustavsson et al., 2008 and 2009; Schonn et al., 2008; Melicoff et al., 2009). Moreover, we recently observed roles for other synaptotagmins in other Ca 2+ -triggered and in Ca 2+ - independent fusion reactions (unpublished). Although many Ca 2+ -dependent types of exocytosis remain to be studied in detail, it thus seems likely that the synaptotagmin-dependent mechanism of controlling Ca 2+ -triggered fusion is paradigmatic for nearly all types of Ca 2+ - stimulated exocytosis. One fascinating question will be whether the generality of the synaptotagmin paradigm extends to the role of complexins (which are also widely expressed in non-neuronal cells; McMahon et al., 1995), further suggesting that all Ca 2+ -induced exocytosis is mechanistically similar except for asynchronous exocytosis. 15

16 4. Outlook where do we go from here, 25 years later? Our work, together with that of others, has uncovered a plausible mechanism explaining how membranes undergo fusion during membrane traffic in eukaryotic cells, and how such fusion is regulated by Ca 2+. Our work started with a specialized, albeit particularly important example of Ca 2+ -controlled fusion, namely synaptic exocytosis, but its results extend beyond this example to provide insight into diverse biological processes. Nevertheless, many new questions emerged that are not just details but of fundamental importance for biology. Such questions range from the need for a precise physicochemical understanding of fusion to that for insight into the role of the fusion mechanism we outlined in diseases such as Parkinson's disease and mental retardation. Most important, however, may be the question of how the fusion and Ca 2+ -triggering machinery that we describe is embedded into the overall cellular organization. For example, synaptic exocytosis is strictly localized to presynaptic active zones, and positioned into close proximity to presynaptic Ca 2+ -channels, allowing fast coupling of Ca 2+ -influx to Ca 2+ -sensing. Studies over recent years have begun to make advances in understanding the supermolecular organization of membrane at the synapse and elsewhere. For example, we discovered that an active zone protein called RIM (Wang et al., 1997) tethers synaptic vesicles close to Ca 2+ - channels, and recruits Ca 2+ -channels to the active zone via direct interactions with Ca 2+ - channels and synaptic vesicles (Kaeser et al., 2011; Han et al., 2011). Although only the beginning, studies like this promise to delineate the overall organization of synapses in space and time in future. ACKNOWLEDGEMENTS I would like to thank my colleagues and collaborators over the years for invaluable advice, guidance, and support, without which my laboratory would not have been able to make any progress. I am particularly indebted to Drs. Reinhard Jahn (Göttingen), Robert E. Hammer (Dallas), and Josep Rizo (Dallas) for many important discussions and innumerable contributions without these scientists, our understanding of synaptic membrane fusion would not be where it is today, and their contributions both to our collaborative studies and to the field in general are invaluable. 16

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26 FIGURES and FIGURE LEGENDS Figure 1 Our 1993 model for the protein complex mediating synaptic membrane fusion (from Hata et al., 1993). The fusion complex was proposed to be composed of the SNARE proteins synaptobrevin/vamp, SNAP-25, and syntaxin, and the SM-protein Munc18. The original caption of the figure is shown below the figure to illustrate the relatively advanced biochemical understanding of the fusion complex in 1993, although the precise nature of the protein interactions only emerged 5 years later. This speculative model was thus proposed in the absence of a true structural understanding, which emerged only later when the biophysical properties of SNARE complexes were elucidated (see text). 26

27 Figure 2 Demonstration that tetanus toxin cleaves synaptobrevin/vamp in synaptosomes to inhibit neurotransmitter release (Link et al., 1992). The image on the left depicts optical measurements of glutamate release from synaptosomes stimulated by high K + (arrow) in the presence of Ca 2+ or EGTA, after preincubation with increasing amounts of tetanus toxin. The image on the right depicts an immunoblot of synaptosomes that were incubated with control buffer (lane A) or tetanus toxin (lane B); proteins were blotted for the indicated proteins to demonstrate the selective loss of synaptobrevin in the synaptosomes. Note that Schiavo et al. (1992) obtained the same result simultaneously. 27

28 Figure 3 Current model of the SNARE/SM protein assembly/disassembly cycle, using synaptic vesicle fusion as an example. Prior to fusion, SNARE proteins are thought to be not bound to each other, although SNAP-25/syntaxin heretodimeric complexes may be present. In the case of exocytotic membrane fusion, the SM protein (a Munc18 isoform) forms a complex with the closed conformation of the respective syntaxin. Step 1 mediates priming of membranes for fusion, involving partial assembly of trans-snare complexes, with opening of the closed conformation of syntaxin and a switch of the Munc18-binding mode of syntaxin from the closed to the open conformation. Step 1 is facilitated by recently discovered chaperones, CSP's and synucleins, that operate by interacting with SNAP-25 and synaptobrevin, respectively, to enhance SNARE complex assembly (Burre et al., 2010; Sharma et al., 2011). Step 2 mediates fusion pore opening, with completion of trans-snare complex assembly. Step 3 produces fusion pore expansion, converting trans-snare complexes into cis-snare complexes, and resulting in the full integration of the two membranes. Step 4, finally, mediates SNARE complex disassembly by the chaperones NSF and SNAPs, and recycling of the vesicles. The cycle shown here for synaptic vesicle fusion is paradigmatic for all fusion reactions in cells except for mitochondrial fusion, although details differ; for example, often four instead of three SNARE proteins are involved, and SM proteins do not always bind to a closed form of a syntaxin. 28

29 Figure 4 Original identification of closed and open states of syntaxin-1 (from Dulubova et al., 1999). The image depicts speculative models of the two syntaxin conformations (numbers refer to residue numbers of rat syntaxin-1a). Note that in the closed conformation, the linker separating the Habc-domain of syntaxin-1 and the N-terminal half of its SNARE motif are proposed to be folded into α-helices (dark shading, left), thereby occluding the ability of the SNARE motif to engage in SNARE complexes (right, refered to as core complex). Initial studies showed that Munc18 binds to the closed conformation of syntaxin in a heterodimeric interaction (Dulubova et al., 1999), whereas later studies revealed that Munc18 and other SM proteins additionally bind to the open conformation of syntaxins via an interaction that requires the N- terminal peptide sequence of syntaxins (see below; Yamaguchi et al., 2002; Dulubova et al., 2002 and 2008). 29

30 A B Figure 5 Demonstration that the Munc18-1 knockout but not synaptobrevin-2/vamp2 knockout blocks synaptic vesicle fusion (from Verhage et al., 2000, and Schoch et al., 2001). A, Recordings of spontaneous synaptic fusion events at the neuromuscular junction from littermate control embryos (left traces) and Munc18-1 knockout embryos (right traces, both at embryonic day 18.5). At the bottom of the spontaneous release traces, responses elicited by α-latrotoxin are shown. Note that not a single synaptic fusion event is observed in Munc18-1 knockout synapses. B, Recordings of spontaneous synaptic events in neurons cultured from littermate control mice and synaptobrevin-2 knockout mice (syb 2 -/-). At the bottom, summary graphs of the frequency of spontaneous events, their amplitudes, and their rise times are depicted. Although synaptobrevin-deficient neurons exhibit only 10% of wildtype spontaneous synaptic fusion, the individual events are similar to those observed in wild-type controls. 30

31 Figure 6 Origianl identification of a novel binding mode of SM proteins to SNARE complexes via the N-terminal sequence of syntaxins (from Yamaguchi et al., 2002, and Dulubova et al., 2002). The top images show an alignment of the N-terminal sequences of syntaxins involved in endoplascmic reticulum, endosomal, and Golgi membrane fusion reactions; sequences are from a variety of species ranging from yeast to man (similar motifs are also present in other syntaxins not shown). Below the alignment is a GST-pulldown assay to demonstrate that the N-terminal sequences of Tlg2 and its mammalian homolog syntaxin-16 bind the SM protein Vps45p but not the SM protein Sly1p, whereas the N-terminal sequence of the syntaxins Sed50 and syntaxin-5 bind Sly1p but not Vps45p. At the bottom, a model of the binding of the SM protein Vps45 to an assembled SNARE complex containing Tlg2p is shown. 31

32 Figure 7 Discovery of synaptotagmins and of the Ca 2+ -binding properties of C 2 -domains. A, Cloning of Syt1 revealed the canonical domain structure of synaptotagmins that includes a single transmembrane region and two C 2 -domains (Perin et al., 1990). Note that only a subset of synaptotagmins are glycosylated in their intravesicular sequence as shown, and some synaptotagmins either bind Ca 2+ in a different stoichiometry, or not at all. B, Ca 2+ -dependent phospholipid binding by the Syt1 C 2 A-domain (from Davletov and Südhof, 1993). Data shown measure the binding of radioactive phospholipids to immobilized control GST-fusion protein, or to a GST-fusion protein of the Syt1 C 2 A-domain. Note the steep Ca 2+ -cooperativity of binding and micromolar Ca 2+ -affinity. C, Atomic structures of the Syt1 C 2 A- and C 2 B-domains with three and two bound Ca 2+ -ions, respectively (from Sutton et al., 1995; Ubach et al., 1998, and Fernandez et al., 2001). Images show ribbon diagrams with cyan colored β-strands, orange α- helices, and orange balls for the Ca 2+ -ions. 32

33 Figure 8 Syt1 knockout completely ablates fast synchronous but not asynchronous release (from Geppert et al., 1994). Traces depict synaptic responses to isolated action potentials; standard views are shown under (i) and expanded views under (ii) to illustrate that the Syt1 knockout completely ablates fast synchronous response, but enhances slow asynchronous responses. 33

34 Figure 9 Proof that Syt1 acts as Ca 2+ -sensor in synaptic membrane fusion using knockin mice with a mutation that changes the Syt1 Ca 2+ -affinity (from Fernandez-Chacon et al., 2001). A, Diagram of the Ca 2+ -binding site architecture of the Syt1 C 2 A-domain, and location of the two neutralizing amino-acid substitutions, R233Q and K236Q, that were introduced into knockin mice. Note that the K236Q mutation has no detectable effect on Syt1 properties, and was used as a negative control. B, Demonstration that the R233Q mutation decreases the apparent Ca 2+ - affinity of Syt1 during phospholipid binding, measured using a Syt1 fragment containing both C 2 - domains that was isolated from the brains of wild-type and R233Q mutant knockin mice. Data show binding of the Syt1 fragment to liposomes as a function of the free Ca 2+ -concentration. C. Data showing that the R233Q mutation decreases the apparent Ca 2+ -affinity of synaptic exocytosis similar to its effect on the Syt1 Ca 2+ -binding affinity. Figure depicts amplitudes of synaptic responses induced at different concentrations of extracellular Ca

35 Figure 10 Different synaptotagmins confer distinct properties onto synaptic vesicle fusion (from Xu et al., 2008). A, Representative traces of isolated inhibitory synaptic responses from rescue experiments of Syt1 knockout (Syt1 KO) neurons. Traces depict a comparison of synaptic responses from wild-type neurons (WT), Syt1 knockout neurons without rescue, or Syt1 knockout neurons after lentiviral rescue with the eight synaptotagmin isoforms capable of Ca 2+ - binding. B, Expansion of synaptic responses from Syt1 knockout neurons expressing Syt1, Syt2, or Syt9 to illustrate their distinct kinetics. Synaptic responses were normalized in amplitude to illustrate that Syt2 expressing neurons exhibit the fastest onset and decline of synaptic transmission, whereas Syt9 expressing neurons exhibit the slowest. C, Quantitation of key kinetic parameters of synaptic responses in Syt1 knockout neurons expressing Syt1, Syt2, or Syt9 (left, rise times; right, decay times; *, **, and ***=p<0.05, 0.01, and p.001, respectively, by Student's t-test; numbers in bars show numbers of neurons analyzed). 35

36 Figure 11 Model of the pas-de-deux of synaptotagmin and complexin in Ca 2+ -triggered synaptic exocytosis (from Chen et al., 2002; Tang et al., 2007; and Maximov et al., 2009). A schematic model of the priming, superpriming and fusion-pore opening stages of synaptic vesicle fusion is shown on top, with the associated SNARE-complex assembly states. Priming is thought to involve partial trans-snare complex assembly with Munc18-1 bound. During superpriming, complexin binding simultaneously energizes assembly of trans-snare complexes, and prevents completion of trans-snare complex assembly (i.e., complexin simultaneously activates and clamps fusion). Ca 2+ -binding to synaptotagmin displaces the complexin clamp, allowing completion of trans-snare complex assembly and fusion-pore opening. The diagram below the model of superpriming shows an expansion of the SNARE/SM protein assembly with bound complexin; note that the accessory α-helix is thought to clamp trans-snare complex assembly by interfering with full assembly, whereas the very N-terminal complexin sequences energize the SNARE complex, possibly by modulating the membrane insertion points of SNARE proteins. On the bottom right we show a depiction of the modular domain structure of complexin with the currently known functional assignments, and a spacefilling model of the atomic structure of the SNARE complex containing bound complexin (diagram was modified from Maximov et al., 2009). 36