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Critical role of cortical vesicles in dissecting regulated exocytosis: overview of insights into fundamental molecular mechanisms.

Abstract. Regulated exocytosis is one of the defining features of eukaryotic cells, underlying many conserved and essential functions. Definitively assigning specific roles to proteins and lipids in this fundamental mechanism is most effectively accomplished using a model system in which distinct stages of exocytosis can be effectively separated. Here we discuss the establishment of sea urchin cortical vesicle fusion as a model to study regulated exocytosis--a system in which the docked, release-ready, and late [Ca.sup.2+]-triggered steps of exocytosis are isolated and can be quantitatively assessed using the rigorous coupling of functional and molecular assays. We provide an overview of the insights this has provided into conserved molecular mechanisms and how these have led to and integrate with findings from other regulated exocytotic cells.

Exocytosis

Deuterostomia is a super phylum that encompasses hemichordates (Enteropneusta), echinoderms (sea urchins), and chordates (vertebrates); along with the phylum protostomia, it represents all animals having bilateral symmetry at least early in development (Fig. 1). Many mechanisms underlying fundamental, critical cellular processes are highly conserved throughout deuterostomes (Knight and Scrutton, 1986; Knight and Baker, 1987; Pennisi, 2006; Rast et al., 2006: Sodergren et al., 2006; Morgan, 2011), Maturing oocytes, eggs, and early embryos of sea urchins have thus been used as model systems to study diverse cellular processes for over a century, providing numerous initial and key insights into the mechanisms underlying fertilization, development, calcium ([Ca.sup.2+]) signaling, the immune response, cell cycle regulation, and exocytosis (Gross et al., 1999; Burke et al., 2006; Rast et al., 2006; Whitaker, 2006; Agnello and Roccheri, 2010; Ramos and Wessel, 2013).

Like the majority of eukaryotic cells, sea urchin eggs contain secretory vesicles--membrane-bound organelles that fuse with the plasma membrane (PM) to release their cargo into the extracellular space. Regulated exocytosis (i.e., triggered or stimulus-coupled secretion) forms the basis of such diverse functions as fertilization, wound healing, and neurotransmission. The exocytotic pathway is composed of defined stages: vesicles (i) translocate toward the PM where they (ii) tether, dock, and (iii) undergo priming reactions (i.e., become release-ready or fusion competent) and upon triggering (e.g., elevation of intracellular free calcium ([[[Ca.sup.2+]].sub.free]) levels), (iv) fuse with the PM and release their contents into the extracellular space; or in the case of compound exocytosis, vesicles fuse with each other (i.e., homotypic fusion) prior to or during merger with the PM (Fig. 2). Although various cell types carry out specialized physiological functions and hence require unique modulatory factors, the fundamental mechanism by which membrane fusion proceeds is highly conserved from protists through to mammals (Whitaker, 1994; Burgoyne and Morgan, 2003; Plattner and Kissmehl, 2003; Barclay et 2012). We thus define the minimal components essential for membrane merger as belonging to the fundamental fusion mechanism (FFM), and when coupled with accessory factors that enhance or regulate priming, docking, [Ca.sup.2+] sensing (and perhaps even fusion pore opening and stability), the physiological fusion machine (PFM) (Churchward and Coorssen, 2009; Rogasevskaia et al., 2012).

Regulated exocytosis has thus been intensely investigated in a range of cell types including endocrine and exocrine cells, neurons, and eggs. Some cell types provide unique experimental advantages that enable the study of a particular characteristic; for example, electrophysiological and imaging studies of neuroendocrine cells have provided key insights into vesicle maturation states (Neher, 1998; S[empty set]-rensen et al., 2003; Rizzoli and Betz, 2005; Becherer and Rettig, 2006; Stevens et al., 2011), and genetic studies utilizing model organisms such as Caenorhabditis elegans and yeast have identified several components of the exocytotic pathway (Brenner, 1974; Broadie and Richmond, 2002; Wickner, 2002; Barclay et al., 2012). Therefore, multiple stages leading to content release are recognized, and several components critical to the exocytotic process are known. The basic aim of exocytosis research is now to understand the exact sequence or stage at which these components act, how they interact to effect release and to identify the remaining machinery needed. The sea urchin cortical vesicle (CV) is an optimal system with which to address these questions. The coupled biochemical and physiological study of sea urchin egg CV release has enabled the examination of protein and lipid components at a strictly defined stage of the exocytotic pathway (i.e., post docking and priming) (Vogel et al., 1991; Zimmerberg et al., 1999, 2000; Szule and Coorssen, 2003; Churchward and Coorssen, 2009; Chasserot-Golaz et al., 2010).

Cortical Vesicle Fusion

Polyspermy, the fertilization of an egg with more than one sperm, usually results in an unviable zygote. In the sea urchin oocyte, there are two mechanisms that prevent polyspermy. Sperm-egg contact causes a transient electrical depolarization, shifting the resting membrane potential to near positive values (i.e., the fast block [Jaffe, 1976]); and subsequent fusion of CV with the egg PM releases glycoprotein-rich material, resulting in elevation of the fertilization envelope that acts as a mechanical barrier to sperm entry and protects the developing egg (Rothschild and Swann. 1952; Schuel, 1984). Since fertilization occurs externally and sea urchin oocytes encounter numerous sperm at once, the formation of the fertilization envelope must be rapid. To facilitate this, urchin CV are stably docked to the PM (~15, 000 or more per egg), having translocated there during oogenesis (Berg and Wessel. 1997; Morgan, 2011). With all CV in this fully primed, fusion-ready state, there are no upstream reserve pools of CV in the cytoplasm and thus no further vesicle translocation, docking, and priming after the triggering elevation of intracellular [[[Ca.sup.2+]].sub.free], contrary to what happens in many other regulated exocytotic events (Smith et al., 1998; Nagy et al., 2002; Rizzoli and Betz, 2005; Becherer and Rettig, 2006; Stevens et al., 2011). Cortex preparations isolated from the unfertilized egg have thus been likened to giant synaptic active zones because all CV constitute a single release-ready pool (Zimmerberg et al., 2000). Notably, in contrast to mammalian secretory vesicles, CV retain this fusion-competent or readily releasable state in vitro, even after isolation from the PM; CV thus serve as a unique tool with which to rigorously and quantitatively assess the fundamental molecular mechanisms underlying the docked or primed state and fast [Ca.sup.2+]-triggered fusion (Vogel et al., 1996; Coorssen et al., 1998, 2003; Tahara et al., 1998; Blank et al., 2001; Szule et al., 2003; Whalley et al., 2004; Churchward et al., 2005; Hibbert et al., 2006; Rogasevskaia and Coorssen, 2011; Rogasevskaia et al., 2012).

[Ca.sup.2+] is the trigger

In the early 1970s, several quantitative studies utilizing the sea urchin egg demonstrated an increase in intracellular [[[Ca.sup.2+]].sup.free] immediately after fertilization (i.e., sperm entry), supporting the [[Ca.sup.2+]] theory of egg activation (Clothier and Timourian, 1972; Chambers et al., 1974; Nakamura and Yasumasu, 1974; Steinhardt and Epel, 1974). Soon after, Vacquier (1975) described a procedure to isolate sheets of PM with docked vesicles by shearing eggs adhered to a support surface. This so-called isolated planar cortex can be rinsed free of all cytosolic factors, and fusion can subsequently be triggered only by the addition of micromolar [[[Ca.sup.2+]].sub.free]. During this time, research from other laboratories showed that fusion could be induced by incubating eggs with [Ca.sup.2+] ionophore and inhibited by [Ca.sup.2+] chelating agents (Vacquier, 1975; Zucker and Steinhardt, 1978; Baker et al., 1980; Terasaki, 1995). It was this work in the cortex that first demonstrated that fast, [Ca.sup.2+]-triggered membrane fusion occurs in the absence of ATP and other cytosolic factors (Vacquier, 1975; Baker and Whitaker, 1978); [Ca.sup.2+] is necessary and sufficient, and thus is the final trigger for the membrane-fusion step that defines regulated exocytosis (Katz and Miledi, 1965; Schneggenburger and Neher, 2005). Well over a decade after these groundbreaking studies using urchin oocyte preparations. ATP was confirmed to be an upstream priming factor in neuroendocrine cells (Holz et al., 1989), and in subsequent years it was discovered that docked vesicles undergo further priming reactions before attaining fusion competence (Parsons et al., 1995; Plattner et al., 1997; Klenchin and Martin, 2000; Liu et al., 2010). It was not until 2002 that the ATP-independence of synaptic vesicle release was also confirmed (Heidelberger et al., 2002). Notably, a somewhat related preparation of PM sheets with attached vesicles was developed using neuroendocrine cells (Avery et al., 2000). Unlike the isolated urchin cortex, these neuroendocrine cells undergo rapid depriming and require cytosol to enable exocytosis, resulting in a heterogeneous vesicle population that does not allow the rigorous biochemical analysis of the late stages of regulated release that is facilitated by the urchin egg preparations. Thus, as a result of pioneering work on urchin cortex preparations, even later fusion models that still postulated an ATP requirement for the final process of membrane merger would be reassessed and shown not to fully account for the native release reaction (see the following; Soliner et al., 1993a, b; Rothman, 1994; Coorssen et al., 1998; Tahara et al., 1998; Szule and Coorssen, 2003).

CV can also be detached from the isolated cortex and added back onto sheets of PM, where they stably dock and undergo [[Ca.sup.2+]]-triggered fusion (Crabb and Jackson, 1985; Whalley and Whitaker, 1988). Though this reconstituted system is slightly less efficient (i.e., only a few CV fully dock and there is a higher half-maximal [Ca.sup.2+] threshold relative to the native cortex), the results of these studies indicated that some of the molecular machinery required to carry out docking (and perhaps priming reactions) resides on the PM. Notably, this work on the urchin cortex was the first successful reconstitution of regulated exocytosis (Avery et al., 1999). Thus, in terms of a highly accessible model of a fundamental cellular mechanism, sea urchin CV are stably docked to the PM, having undergone all necessary priming reactions, and require only an elevation in [[[Ca.sup.2+]].sub.free] to trigger fusion. Furthermore, biochemical analyses of membrane fusion are substantially more direct and rigorous because the removal of cytosol further reduces the background arising from protein and lipid components not required for the triggered membrane-merger steps. The ability to clearly separate the fully docked or primed state from the subsequent [Ca.sup.2+]-triggered stages of exocytosis has enabled researchers to ascribe roles for specific proteins and lipids at a well-defined late stage of regulated exocytosis (Whitaker and Baker, 1983; Zimmerberg et al., 1999, 2000: Coorssen et al., 2003; Whalley et al., 2004; Churchward et al., 2005; Leguia et al., 2006; Rogasevskaia and Coorssen, 2006, 2011; Rogasevskaia et al., 2012).

Further development yielded isolated cell-surface complexes (CSC; PM fragments with docked CV) via homogenization--essentially planar cortices in suspension, perhaps best compared to mammalian cell ghosts (Decker and Lennarz, 1979; Crabb and Jackson, 1985; Jackson et al., 1985). Because a gravid urchin sheds gram quantities of eggs, CSC can be obtained at a very high yield; not only are such large quantities of stage-specific preparations unique to this system, but they are essential for most effectively combining physiological and biochemical analyses of exocytosk in the same experiment. This simplified preparation provides better access to the fully docked state and active sites, enabling large-scale and higher-throughput screening of pharmacological compounds that may influence the process of docking, priming, and membrane fusion (Haggerty and Jackson, 1983; Whitaker and Baker, 1983; Whalley and Sokoloff, 1994; Vogel et al., 1995. 1996; Coorssen et al., 2003; Whalley et al., 2004; Hibbert et al., 2006; Furber et al., 2009a, b, 2010; Rogasevskaia and Coorssen, 2011).

CV can also undergo homotypic fusion (Pickett and Edwardson, 2006). Such compound exocytosis is a conserved and prominent feature of many regulated secretory cells including eosinophils (Scepek and Lindau, 1993), pancreatic acinar cells (Nemoto et al.. 2001; Thorn and Parker, 2005), mast cells (Alvarez de Toledo and Fernandez, 1990), neutrophils (Lollike et al., 2002), and ribbon synapses (Matthews and Sterling, 2008; Cho and von Gersdorff, 2012), as well as sea urchin oocytes (Chandler, 1984; Zimmerberg et al., 1985). Compound exocytosis can be separated into two general pathways: (i) vesicles undergoing homotypic fusion prior to fusion with the PM, as in ribbon synapses and mast cells (Alvarez de Toledo and Fernandez, 1990; Matthews and Sterling, 2008; Graydon et al., 2011); or (ii) vesicles fusing to a "primary" vesicle that first fuses stably with the PM, as in acinar cells (Nemoto et al., 2001). Compound exocytosis maintains efficient function in these systems as (i) vesicles can forgo translocation to the PM and hence minimize reaction time; (ii) in certain cell types. limited available PM contact space is optimally utilized (Pickett and Edwardson. 2006); and (iii) the need for large or fast exo-cytotic responses can be fully accommodated.

CV-PM fusion in the egg and other secretory cells also shares many other evolutionarily conserved features with CV-CV fusion. (i) The [Ca.sup.2+] sensitivity of CV-CV and CV-PM fusion are comparable, with submaximal responses to [Ca.sup.2+] (i.e., there is a distribution of [Ca.sup.2+] sensitivities across the population of CV) that follow a sigmoidal pattern (Knight and Scrutton, 1986; Knight and Baker, 1987; Vogel et al., 1996; Blank et al., 1998a, b, 2001; Coorssen et al., 1998); these responses are comparable to and translationally invariant relative to those of other secretory cells (Knight and Scrutton, 1986), and indeed similar responses are seen in mammalian CNS neurons (Wolfel et al.. 2007). (ii) CV fusion is inhibited by certain protein-modifying agents such as N-ethylmaleimide, trypsin, and the positive curvature lipid lysophosphatidylcholine (Haggerty and Jackson, 1983; Jackson et al., 1985; Vogel and Zimmerberg, 1992; Chernomordik et al., 1993; Vogel et al., 1993; Coorssen et al., 1998; Churchward et al., 2005; Furber et al., 2009a); comparable inhibition has been documented in other secretory cell types including pancreatic (Eliasson et al., 1997), chromaffin (Frye and Holz, 1985), and pituitary cells (Kaye et al., 1992). Furthermore, CV are capable of fusing with pure lipid membranes (i.e., liposomes or phospholipid bilayer systems) and other CV that have been proteolytically inactivated (Vogel et al., 1992; Vogel and Zimmerberg, 1992; Chanturiya et al., 1999). Thus, the minimal essential machinery sufficient to support docking, priming, triggering, and membrane fusion resides on the CV membrane (Vogel et al., 1992; Vogel and Zimmerberg, 1992); this does not imply that there are not critical components on the PM, but could indicate that they tend to be involved in targeting or are more modulatory in nature and thus contribute to the tuning of physiological responses.

Monitoring Exocytosis

Initially, CV-PM fusion was assessed microscopically by counting the number of CV present in cortex preparations before and after the addition of [Ca.sup.2+]. Though this approach provided early evidence of the relationship between [Ca.sup.2+] and membrane fusion (Vacquier, 1975; Steinhardt et al., 1977), it is labor-intensive and highly susceptible to sampling bias; a high-throughput assay was needed to enable large-scale biochemical analyses (e.g., screening for compounds that affect exocytosis). Measuring release of CV-specific enzymes such as ovoperoxidise or [beta]-glucanase provided a more quantitative and reliable approach (Foerder and Shapiro, 1977; Moy et al., 1983). In the mid-1980s, several laboratories documented a decrease in optical density proportional to CV content dispersal after membrane fusion and devised assays to measure this change and thereby measure the extent and kinetics of CV fusion (Haggerty and Jackson, 1983: Sasaki and Epel, 1983: Shafi et al., 1994). Parallel studies established that content dispersal resulted from CV membrane fusion rather than from lysis (Haggerty and Jackson. 1983: Crabb and Jackson, 1985).

Light-scattering assays were then adapted and further developed to monitor homotypic CV fusion (Vogel and Zimmerberg, 1992; Coorssen et al., 1998, 2003; Tahara et al., 1998; Szule et al., 2003). CV-CV fusion results in the formation of fusosomes, highly unstable structures that burst and disperse their combined content immediately after fusion; since CV content hydrates "explosively," this results in a very rapid decrease in light scattering (Vogel and Zimmerberg, 1992). Packed CV layers, or lawns, are easily produced on a large scale via centrifugation of CV suspensions in multiwall plates, and the extent of fusion at varying [[[Ca.sup.2+]].sub.free] is quickly measured in a microplate reader by assessing changes in optical density. The centrifugation step mimics the docking step, but does impact on (i.e., contribute energy to) the fusion process itself (Coorssen et al., 1998). Alternatively, CV are allowed to settle into contact because endogenous docking machinery on their membranes is sufficient to support CV-CV contact and engagement; comparison of results from settle and standard CV fusion assays thus enables identification of factors acting in the docking and priming reactions and fusion, respectively (Coorssen et al., 1998, 2003; Zimmerberg et al., 1999, 2000; Churchward et al., 2005; Furber et al., 2009a; Rogasevskaia and Coorssen, 2011; Rogasevskaia et al., 2012).

As noted previously, CV and other secretory vesicles show a heterogeneous response to [Ca.sup.2+]; that is, there are [[[Ca.sup.2+]].sub.free] at which only a fraction of vesicles fuse, and this submaximal response follows a sigmoidal pattern (Knight and Scrutton, 1986; Knight and Baker, 1987; Hide et al., 1993). Two possible models to explain this phenomenon have been tested using urchin egg preparations: (i) within a population of CV there is a homogenous distribution of [Ca.sup.2+]-sensitive fusion complexes that are selectively disabled at varying [Ca.sup.2+] concentrations; or (ii) CV have inherently varying [Ca.sup.2+] sensitivities since each has a different number of fusion complexes. CV can be held at a [[[Ca.sup.2+]].sub.free] sufficient to cause a fraction of them to fuse, and subsequent challenge with a maximal [[[Ca.sup.2+]].sub.free] results in 100% fusion. Hence, the first model was ruled out as [Ca.sup.2+] clearly did not inactive fusion complexes (Blank et al., 1998a). Instead, there appear to be subpopulations of vesicles with inherently varying [Ca.sup.2+] sensitivities. The current working hypothesis thus posits the existence of multiple fusion complexes distributed on CV membranes in a Poisson-like manner (Vogel et al., 1996). One activated fusion complex is sufficient to cause fusion, and increasing [[[Ca.sup.2+]].sub.free] increases the average number of activated fusion complexes per CV (Blank et al., 1998a, b). In cortex, CSC, and CV preparations from the eggs of Strongylocentrotus purpuratus, progressive inactivation of fusion complexes by proteolysis, thiol reactive regents, or cholesterol (CHOL) removal indicated an average of 6-9 fusion complexes per CV (Vogel et al., 1996: Blank et al., 1998a; Churchward et al., 2005).

Proteins in Exocytosis

In general, due to their stage-specific (i.e., fusion-ready) nature, CSC and CV preparations enable direct and quantitative testing for the roles of specific proteins in the fully docked state and the fast, [Ca.sup.2+]-triggered steps of regulated release. At its simplest, this testing identifies only those components critical to the minimal essential docking. triggering, and fusion mechanisms that are likely the conserved basis upon which different modulatory and accessory components act in other secretory cell types to regulate and tune their responses to specific physiological needs and states. Indeed, working with purified CV further concentrates the fusion complexes (i.e., the large background contributed by the PM is removed) and enables the most rigorous coupling of quantitative functional and molecular analyses, as necessary to effectively dissect molecular mechanisms (Coorssen et al., 1998, 2003; Tahara et al., 1998; Szule et al., 2003; Whalley et al., 2004; Hibbert et al., 2006; Furber et al., 2009a. b. 2010). Furthermore, as urchins largely diverged before the gene duplication characteristic of later evolution in other species (Fig. 1), they tend to have only a single version of most proteins, making this an ideal system with which to identify minimal essential components of the FFM.

SNARE proteins

The SNARE superfamily of proteins is characterized by an evolutionarily conserved sequence of 60-70 amino acids (the SNARE motif) that facilitate their characteristic inter-membrane interactions. Although often labeled as vesicle (v-) or target (t-)-SNAREs to indicate their originally described subcellular localization, this notation can be misleading as both classes are localized to vesicles and the PM. Structurally, they are designated Q-SNAREs if they contain a central glutamine at

the SNARE motif and R-SNAREs if they contain an arginine residue (Fasshauer et al., 1998). Syntaxin (syt), synaptobrevin (syb), and the so-called synaptosomal associated protein of 25 kDa (SNAP-25) together form an SDS-resistant heterotrimeric complex that spans the vesicle and PM, bringing the two into close proximity (i.e., ~2 nm; Sutton et al., 1998). Syb and syt are membrane-anchored via a transmembrane domain present at the C-terminus and contain cytosolic SNARE domains that facilitate characteristic coiled-coil interactions (Hanson et al., 1997; Poirier et al., 1998). SNAP-25 contains two SNARE motifs, one each at the N--and C-termini, and is membrane-anchored via palmitoylated cysteine residues in the central domain. The SNARE complex is said to be in a trans-configuration prior to fusion, with the proteins residing on opposing membranes, and in cis-configuration after fusion, with the proteins residing on the single merged membrane: nonetheless, the latter also exists on isolated vesicles. Upon fusion, the complex is disassembled by an ATPase, the N-ethylmaleimide-sensitive factor (NSF), and its receptor, the soluble NSF attachment protein (SNAP).

Since the discovery of SNARE proteins (Novick et al., 1981; Trimble et al., 1988; Baumert et al., 1989; Kaiser and Schekman, 1990; Bennett et al., 1992; Sollner et al., 1993b) and proposal of the initial SNARE hypothesis (Sollner et al., 1993a; Rothman, 1994), a large body of research in the field has been aimed at understanding their exact role in exocytosis (Coorssen et al., 1998, 2003; Tahara et al., 1998: Weber et al., 1998: Schoch et al., 2001: Sorensen et al., 2003; Szule and Coorssen, 2003; Szule et al., 2003; Borisovska et al., 2005; Jahn and Scheller, 2006; Sorensen, 2009; Sudhof and Rothman, 2009; Bacaj et al., 2010; Rizo and Sudhof, 2012). Syb, SNAP-25, and syt are present on CV membrane at densities comparable to synaptic vesicles (Coorssen et al, 1998, 2002; Tahara et al., 1998; Takamori et al., 2006); indeed mammalian central nervous system SNAREs share an identity of about 73% (~83% similarity) with their urchin progenitors and orthologs (Table 1). Thus, the CV-CV fusion system has proven to be invaluable in testing several aspects of SNARE hypotheses. First, the native SNARE complex was shown to be [Ca.sup.2+] sensitive: low [[[Ca.sup.2+]].sub.free], could trigger disassembly of the complex, and fusion could nonetheless subsequently be triggered by applying a higher [[[Ca.sup.2+]].sub.free] (Tahara et al., 1998). In addition, as in other secretory cells, fusion could be triggered by alternate divalent cations (i.e., [Sr.sup.2+] or [Ba.sup.2+]), but these did not result in disruption of the SNARE complex, indicating that the final process of membrane merger could occur irrespective of the presence or absence of SNARE complexes (Coorssen et al., 1998; Zimmerberg et al., 1999). Using both CSC and isolated CV, membrane fusion has also been shown to occur in the absence of NSF (Whalley et al., 2004), again consistent with the original observations that cytosolic components are not critical to the fundamental fusion mechanism, and that the minimally essential components are on the CV membrane. During this time, homotypic fusion of yeast vacuoles was also shown to proceed after SNARE complex disassembly (via treatment with excess yeast homologs of NSF and [alpha]-SNAP), indicating the need for a downstream fusogen (Ungermann et al., 1998). Together, these were the first studies to critically evaluate the initial SNARE hypothesis, which posited membrane fusion to be catalyzed by the ATP/NSF-mediated disassembly of an inter-membrane SNARE complex, establishing that this did not effectively describe the native mechanism of regulated fusion.

Table 1

Conservation of proteins functioning in the exocytotic cycle;
extent of conservation between Strongylocentrotus purpuratus
and Homo sapiens

Protein        Percent identity *  Percent similarity *

SNAP-25           62.3% (127/204)       77.0% (157/204)
Syntaxin          75.3% (189/251)       86.5% (217/251)
Synaptobrevin       81.7% (76/93)         86.0% (80/93)
Synaptotagmin     65.4% (268/410)       78.0% (320/410)
Rab3A             83.4% (161/193)       94.3% (182/193)
NSF               64.8% (480/741)       80.4% (596/741)
Tubulin           84.5% (383/453)       93.8% (425/453)
Actin             92.6% (349/377)       96.0% (362/377)
Calmodulin        96.1% (146/152)       97.4% (148/152)

* Calculated using EMBOSS matcher (Rice et al., 2000) with
protein sequences from the National Center for Biotechnology
Information (NCBI) protein database.


A revised SNARE hypothesis (i.e., SNAREpin) proposed that assembly and the rapid "zippering-up" of the complex drives membrane fusion, the critical inter-membrane complexes thus being transient. The SNAREs were initially shown to induce lipid mixing when reconstituted into liposomes (Weber et al., 1998), and a later study demonstrated some degree of content mixing (Nickel et al., 1999); however, these reactions occurred at a very slow rate (i.e., not resembling physiological fusion), required high protein densities, and seemed to work only with specific liposome preparations or protein reconstitution methods (Chen et al., 2006; Dennison et al., 2006; reviewed in Churchward and Coorssen, 2009). Quantitative removal of CV SNARE cytosolic domains did not result in a loss of membrane fusion; instead, there was a loss in [Ca.sup.2+] sensitivity and a decrease in the rate of fusion (Coorssen et al., 2003). Conversely, by using an alternate protease, fusion could be abolished while leaving the majority of SNAREs intact (Szule et al., 2003). Moreover, disassembly of CV SNARE complexes by treatment with NSF and [alpha]-SNAP reduced the [Ca.sup.2+] sensitivity of fusion (but only marginally relative to the shift seen after SNAREs were ablated with proteases; Fig. 3), but not the ability to fuse (Zimmerberg et al., 2000). On the whole, it is evident that SNARE cytosolic domain interactions (i.e.. those responsible for inter-membrane contact) do not drive or necessarily define the ability to fuse, but the question remains: what then caused the varying changes in [Ca.sup.2+] sensitivity seen in these studies (Fig. 3) (Zimmerberg et al., 2000; Coorssen et al., 2003; Szule and Coorssen, 2003; Szule et al., 2003)? The proteases used were not selective for the SNAREs, and other proteins necessary for vesicle docking, priming, and [Ca.sup.2+] sensing are also likely to have been differentially proteolysed, and to different extents. In particular, synaptotagmins, a family of [Ca.sup.2+]-and phospholipid-binding proteins involved in exocytosis--including postulated roles in fusion (Tucker et al., 2004; Zimmerberg et al., 2006; Martens et al., 2007; Kochubey et al., 2011; Vennekate et al., 2012)--have a large number of papain cleavage sites and would have undergone extensive proteolysis in the SNARE ablation study (Table 2); this and the proteolysis of other proteins could thus well have contributed to the extensive loss in [Ca.sup.2+] sensitivity (Coorssen et al., 2003). Numerous lipids (i.e., CHOL, arachidonic acid, sphingosine, and phosphatidylserine) and proteins (including synaptotagmin, complexin, snapin, and munc18) are also known to bind SNAREs (Hata et al., 1993; Ilardi et al., 1999; Chen et al., 2002; Mitter et al., 2003; Rickman and Davletov, 2003; Stace and Ktistakis, 2006; Dai et al., 2007; Latham et al., 2007; Darios et al., 2009), and both SNARE ablation and targeted disassembly of complexes by NSF and [alpha]-SNAP would almost certainly have hindered at least several of these selective interactions as well.

Table 2
Summary of proteolytic cleavage sites in SNARE proteins
and synaptotagmin

                              Total Number of Protease Cleavage Sites

                             Trypsin       Papain       Clostripain

Protein              Number      Ave  Min     Ave  Min      Ave  Min
                         of
                   Isoforms
                   Analyzed

Syntaxin                 94       42   29      81   53        20    6

SNAP25                   25       12    8      25   20         6    5
(N[H.sub.2]-term)

SNAP25 (C-term)          25       17   10      37   17         8    5

Synaptobrevin            73       19   12      42   23         9    4

Synaptotagmin            59       63   42     134   77        22    4

                    Number of Amino Acids From Transmernbrane
                                Region to First Cleavage Site

Protein            Trypsin       Papain       Clostripain

                       Ave  Min     Ave  Max          Ave  Max

Syntaxin                 3    4       3    4            6   14

SNAP25                   4    6       3    6           21   29
(N[H.sub.2]-term)

SNAP25 (C-term)          3    6       3    6            9   14

Synaptobrevin            9   11       9   11           11   63

Synaptotagmin            5    9       4    6           35  161

Amino acids are counted from the N[H.sub.2] and C--terminal
sides of the palmiloylated cysteine residues in the center of
SNAP25. Adapted from Coorssen et al. (2003).


As the techniques used would not have affected the SNARE transmembrane domains, it thus remains to be seen how these may contribute to the late stages of regulated exocytosis (Langosch et al., 2007). Nonetheless, testing of fusion models using the CV system pointed to a critical modulatory role for the SNAREs--contributing to the efficiency of fusion, possibly by contributing to tuning the [Ca.sup.2+] sensitivity of the reaction to the known physiological range; thus, a key contribution of the SNAREs may be in priming, rather than in fusion per se (Coorssen et al., 1998, 2003; Tahara et al., 1998; Zimmerberg et al., 2000; Szule and Coorssen, 2003; Szule et al., 2003). At the time of this work, approximately a decade of research internationally, using a plethora of cell types, had clearly established a critical role for the SNAREs in the exocytotic pathway; a critical function in priming and promotion of docking It well with the literature if not with the more prevalent interpretations.

Indeed, the critical role of SNAREs in docking and priming has now been well established in neuroendocrine cells. Unlike CV in the urchin egg, secretory vesicles of chromaffin cells are concurrently present at various stages of secretory readiness. As a result, the exocytotic response, monitored via the whole-cell patch clamp technique or total internal reflection fluorescence (TIRF) microscopy, can be separated into two distinct kinetic phases: (i) the initial exocytotic burst from the readily releasable pool and the slowly releasable pool that together represent the differentially primed pool of vesicles that respond immediately upon stimulus; and (ii) a second sustained component representing vesicles that were unprimed at the beginning of stimulus but matured to a primed state during the stimulus (Parsons et al., 1995; Smith et al., 1998; Nagy et al., 2002; Rizzoli and Betz, 2005; Becherer and Rettig, 2006; Stevens et al., 2011). Notably, neither mutation of select amino acid residues in the SNAP-25 SNARE motif, nor deletion of amino acid residues corresponding to the botulinum neurotoxin cleavage site abolished fusion in chromaffin cells; however, a higher [Ca.sup.2+] concentration (>100 [micro]mol [1.sup.-1]) was needed to elicit a response (Sorensen et al., 2003). Similarly, chromaffin cells from syb II knockout mice showed a diminished response from the primed pool but with sustained release unaffected (Borisovska et al., 2005); a long-lasting high [Ca.sup.2+] stimulus did not rescue a response from the primed pool. Interestingly, urchin CV fuse with an [EC.sub.50] of about 250 [micro]mol [1.sup.-1] [[[Ca.sup.2+]].sub.free] in the absence of SNARE cytosolic domains (Coorssen et al., 2003), and thus perhaps an even higher [[[Ca.sup.2+]].sub.free] would have elicited a response from the syb-deficient cells. Therefore, with insufficient SNARE contributions, an impairment of an attached, primed state prevents a physiological stimulus from eliciting an exocytotic response (Szule and Coorssen, 2003). However, as clearly demonstrated in the urchin CV model and a number of other fast, [Ca.sup.2+]-triggered exocytotic systems, the process of membrane merger itself is unaffected (Szule and Coorssen, 2003; Becherer and Rettig. 2006; Churchward and Coorssen, 2009; Stevens et al., 2011; Rizo and Suclhof, 2012). Thus, the ability to fuse--the capacity for native secretory membranes to undergo regulated merger--is not defined by the SNAREs per se. Nonetheless, without the SNAREs modulating some aspect of attachment and priming, the efficiency of the fusion reaction would seem unlikely to satisfy the physiological needs of the systems under analysis.

Importantly, SNAREs contain other evolutionarily conserved domains, potentially affecting the process of membrane merger. Recently, the role of conserved tryptophan residues within the syb II juxtamembrane domain was tested (Borisovska et al., 2012). In syb II knockout mice, expression of syb II protein carrying tryptophan mutations failed to completely rescue the fast component (primed pool) but was able to fully rescue the sustained phase of release, and the amount of transmitter released from these cells remained unchanged; therefore, these conserved residues are components of the machinery needed for the maintenance of a primed state.

Actin

Cytoskeletal components play an active role in vesicle mobility and aid in their translocation and attachment to the PM (Vitale et al., 1995). Among the numerous cytoskeletal components that modulate exocytosis, the role of actin has been extensively studied (Eitzen et al, 2002; Eitzen, 2003; Hibbert et al., 2006; Malacombe et al., 2006). In a number of cells that undergo regulated exocytosis, pharmacological agents that stabilize actin polymerization have been shown to inhibit secretion by hindering vesicle translocation to the PM, and agents that destabilize actin filaments increase the secretory response due to an increase in the number of docked vesicles (Burgoyne and Cheek, 1987: Aunis and Bader, 1988; Vitale et al., 1995; Omata et al., 2000; Jog et al., 2007; Porat-Shliom et al., 2012). In addition to these established roles, actin has also been proposed to play an active role in the fusion of yeast vacuoles: F-actin assembly (i.e., actin polymerization) was posited to drive membrane fusion (Eitzen et al., 2002; Eitzen, 2003). As ATP is required for the isolated yeast vacoules to achieve fusion competence, it is difficult to assess the effects of pharmacological agents on membrane fusion itself. In contrast, because CV are locked in a fully docked, primed, and fusion-ready stage, they have enabled direct testing of postulated late roles of cytoskeletal elements in the fast [Ca.sup.2+]-triggered steps of exocytosis.

Early research utilizing urchin cortex preparations found no adverse effects on membrane fusion after treatment with agents that stabilize or destabilize the actin network (Whitaker and Baker, 1983). To achieve a more quantitative analysis and ensure full access of reagents to all the fusion machines. a high-sensitivity immunoblotting protocol was used to measure CV membrane actin density, and the effects of latrunculin B (inhibits actin polymerization). jasplakino-lide (induces actin polymerization, and stabilizes actin filaments), coffin (actin-binding protein that disassembles F-actin), and exogenous actin were tested (Coorssen et al., 2002; Hibbert et al., 2006). At concentrations sufficient to affect actin polymerization states, none of these reagents had an effect on membrane fusion; 1000-fold molar excess of exogenous actin also was without effect. As previously stated, in sea urchin oocytes, CV translocate to the PM early during oogenesis. This step has been demonstrated to require the formation of actin filaments (Berg and Wessel, 1997; Wessel et al., 2002); therefore, in isolated release-ready CV, this role of actin in exocytosis has already been carried out. These results do not exclude potential modulatory influences of actin on the triggered fusion process; indeed. F-actin and other cytoskeletal components have been implicated in the modulation of the fusion pore (Larina et al., 2007; Berberian et al., 2009; Bhat and Thorn, 2009; Bonn et al., 2013). However, the findings do indicate that actin is not a minimal essential component in the fast, [Ca.sup.2+]-triggered steps of exocytosis (Hibbert et al., 2006).

Calmodulin

Calmodulin (CaM) is a [Ca.sup.2+]--binding protein (Babu et al., 1988; Chin and Means, 2000) that has been proposed to act as a [Ca.sup.2+] sensor in regulated membrane fusion (Peters and Mayer, 1998; Burgoyne and Weiss, 2001; Quetglas et al., 2002; Burgoyne, 2007; Weiss et al., 2010). A pharmacological approach was used to test the role of CaM in yeast homotypic vacuolar fusion (Peters and Mayer, 1998). Treatment with W-7 (a CaM antagonist) (Hidaka et al., 1981) or CaM antibody inhibited yeast vacuole fusion (Peters and Mayer, 1998); however, the concentration of W-7 needed was well above the reported [IC.sub.50] (Hidaka et al., 1981). Moreover, as noted above, yeast vacuole fusion requires ATP because isolated vacuoles are not in a fully primed state, making correlation of the effects with membrane fusion difficult. Quantitative analysis of the postulated role of [Ca.sup.2+] in the late, [Ca.sup.2+] -triggered steps of CV exocytosis revealed that this [Ca.sup.2+] -binding protein is not required in the membrane-fusion reaction, and does not confer [Ca.sup.2+] sensitivity (Table 3). Here we used quantitative Western blotting to establish that there are about 6500 copies of CaM per isolated CV (i. e., density of ~2070 copies of CaM/[mu][m.sub.2]) (Coorssen et al, 2002, 2003; Hibbert et al., 2006), enabling a more targeted use of inhibitors and thus reduced potential for nonspecific effects. Even at 300 [micro][moll.sup.-1], a molar excess of about 105-fold over the total CV CaM in an aliquot of CV suspension for assay, W-7 did not have an effect on the extent, [Ca.sup.2+] sensitivity, or initial kinetics of homotypic CV fusion; comparable results were obtained with J-8 and fluphenazine-N-mustard (FNM when tested with both CSC and isolated CV (Hibbert, 2005). Similarly, incubation with excess urchin CaM or the inhibitory peptide derived from CaM Kinase (Table 3) did not affect CV fusion, nor did excess CaM antibody or other blocking peptides (not shown; Hibbert, 2005). Interestingly, 100 [micro][moll.sup.-1]' of W-7 had an effect on CV fusion in the settle assay, suggesting an effect on CV-CV docking; however, this dose was 3 times the reported [IC.sub.50] (Hidaka et al., 1981) and such spurious, non-dose-related effects on the docking assay were also seen with J-8 and FNM (not shown; Hibbert, 2005), suggesting that artifacts likely arising from the membrane intercalation of these compounds could not be excluded.

Table 3

Effects of a calmodulin inhibitor, exogenous, and I inhibitory
peptide hometypic CV fusion (n = 2-7)

Pharmacological   [DELTA][ED.sup.50]([mu]M)  [DELTA]extent(%
Agent                                                Fusion)
W-7: 300 [mu]mol           8.4 [+ or -] 3.9     1.3 [+ or -]
[1.sup.-1]                                               2.9

100 [mu]mol                1.8 [+ or -] 2.2     5.3 [+ or -]
[1.sup.-1]                                               4.5

30 [mu]mol                 0.2 [+ or -] 1.5  1.1[+ or -] 2.8
[1.sup.-1]

CaM: 3 [mu]mol             1.3 [+ or -] 1.5     0.7 [+ or -]
[1.sup.-1]                                               2.2

CaMK I:10[mu]mol            1.3 [+ or -]1.4     0.7 [+ or -]
[1.sup.-1]                                               3.3

Pharmacological   [DELTA]rate(%/s)  [DELTA][EC.sub.5]([mu]M)
Agent

W-7: 300 [mu]mol                 -          3.8 [+ or -] 2.6
[1.sup.-1]

100 [mu]mol       2.7 [+ or -] 7.7          6.1 [+ or -] 5.9
[1.sup.-1]

30 [mu]mol                       -          6.5 [+ or -] 4.7
[1.sup.-1]

CaM: 3 [mu]mol    3.0 [+ or -] 4.6          1.7 [+ or -] 3.3
[1.sup.-1]

CaMK I:10[mu]mol                 -                         -
[1.sup.-1]

Pharmacological   [DELTA]extent(%
Agent                     Fusion)

W-7: 300 [mu]mol     3.6 [+ or -]
[1.sup.-1]                    7.0

100 [mu]mol          4.4 [+ or -]
[1.sup.-1]                  7.9 *

30 [mu]mol           8.2 [+ or -]
[1.sup.-1]                    6.4

CaM: 3 [mu]mol       0.5 [+ or -]
[1.sup.-1]                    5.2

CaMK I:10[mu]mol               -
[1.sup.-1]

* Indicates statistical significance
Student's t-Test, P < 0.05).


Consistent with the data above, other evidence also indicates that CaM is not an essential [Ca.sup.2+] sensor late in the pathway of regulated exocytosis, nor are CaM-dependent effector proteins components of the FFM. As other divalent cations (i. e., [Sr.sup.2+] and [Br.sup.2+]) can trigger fusion in different secretory cell types (Coorssen et al.. 1998; Neves et al., 2001), a critical [Ca.sup.2+] sensor for regulated exocytosis is expected to bind all these cations with some efficacy. Syn-aptotagmin, a putative [Ca.sup.2+] sensor for exocytosis, satisfies this criterion (Li et al., 1995; Shin et al., 2003; Cheng et al., 2004), but CaM does not (Roufogalis et al., 1983; Neves et al., 2001). CaM is, however. the [Ca.sup.2+] sensor for fast endocytosis, a process that is not triggered by [Sr.sup.2+] or [Br.sup.2+] (Artalejo et al.. 1995).

Lipids in Exocytosis

Cholesterol

The role of lipids in the exocytotic pathway is receiving more widespread attention; in particular, the effects of CHOL on numerous fusion parameters are beginning to be understood (Churchward and Coorssen, 2009; Chasserot-Golaz et al., 2010; Rituper et al., 2010, 2012). In secretory vesicles, membrane CHOL is present at a 2-fold higher concentration relative to the PM (Decker and Kinsey, 1983), and fluorescent labeling has shown CHOL to be concentrated at the CV-PM interface (Churchward et al., 2008b).

Reducing the CHOL concentration of CV membranes via chemical extraction and sequestration (i. e., binding to meth-yl-beta-cyclodextrin [m[beta]cd] or polyene antibiotics, respectively), enzymatic reduction, or inhibition of CHOL biosynthesis adversely affects all fusion parameters (i.e., reductions in the extent, [Ca.sup.2+] sensitivity, and rate) (Church-ward et al.. 2005, 2008; Rogasevskaia and Coorssen, 2011). Such a range of methods need to be employed to ensure that effects are related directly to CHOL; for example, although m[beta]cd has been widely used to extract CHOL, selectivity varies depending on the target membrane, and other components including phospholipids, sphingolipids, and membrane proteins can also be removed (Monnaert et al., 2004; Hatzi et al., 2007; Zidovetzki and Levitan, 2007: Besenicar et al., 2008; Ormerod et al., 2012). Selectivity for CHOL cannot simply be assumed. Similarly, although statins are used to downregulate CHOL via inhibition of HMG-CoA reductase, a rate-limiting enzyme in the biosynthetic pathway, this also interferes with the production of ubiquinones, other sterols. dolichols, and prenylated proteins (Goldstein and Brown, 1990; Beltowski et al., 2009). Thus, as no commonly used "CHOL reagents" are entirely specific, it is important to use multiple different strategies in testing the role of CHOL, and to monitor membrane composition (i. e., the lipidome, and even the proteome) after treatment; thus the function of CHOL in the late, [Ca.sup.2+]-triggered steps of exocytosis have perhaps best been established for CV (Churchward et al.. 2005, 2008b; Rogasevskaia and Coorssen, 2011).

Primarily in conjunction with sphingomyelin, CHOL forms microdomains in which critical exocytotic components are concentrated, including priming factors such as the SNAREs (Lang et al., 2001), synaptotagmin, and synaptophysin (Gil et al.. 2005, 2006). Disruption of these microdo-mains, by targeting CHOL or sphingomyelin, impairs the [Ca.sup.2+] sensitivity or kinetics of fusion but not the fundamental ability of CV to fuse (Churchward et al., 2005, 2008b; Rogasevskaia and Coorssen, 2006; Churchward and Coors-sen, 2009). Thus, CHOL-protein and -lipid interactions are postulated to be critical priming factors required for efficient fusion. In addition, CHOL has also been shown to play a central role in the fusion mechanism by virtue of its intrinsic negative curvature; it is a critical component initiating formation of a hemifusion diaphragm (one of the lipidic structural intermediates proposed to enable membrane merger) at inter-membrane docking or contact sites (Chernomordik and Kozlov, 2008). In the absence of CHOL, lipids of similar curvature (e. g., phosphatidylethanolamine (PE) or diacylglycerol (DAG)) are able to selectively rescue the ability of CV to fuse; only the delivery of exogenous CHOL to the CHOL-depleted CV membrane can fully recover both the ability to fuse and the efficiency of the reaction, the latter by re-establishing the necessary microdo-mains and thus interactions between critical components (Churchward et al., 2008b).

Evidence from several secretory cell types now indicates the role of CHOL to be highly conserved. Removing platelet-membrane CHOL decreased the extent and rate of release (Ge et al., 2010). Furthermore, epicholesterol (an epimeric analog of CHOL) rescued all parameters of fusion after CHOL depletion; the authors note, though, that as only about 34% of the endogenous CHOL was depleted and an equal amount of epicholesterol introduced, the data do not definitively indicate epicholesterol and CHOL to be analogous substances in regard to their facilitation of exocytotic fusion. CHOL depletion in neuroendocrine cells (Wang et al., 2010), neuromuscular junctions (Ormerod et al., 2012), brain synaptosomes (Waseem et al., 2006), and synaptic vesicles (Linetti et al., 2010) impairs exocytosis. Considering the documented effects on membrane microdomains in these systems (Gil et al.. 2005, 2006), membrane microdo-main disruption also impairs other critical upstream components of exocytosis, including ion channels (Taverna et al., 2004, 2007; Xia et al., 2008), it is clear that CHOL is a central conserved component of exocytosis and of the final triggered fusion process, but that exact functions will be difficult to fully dissect outside of the CV. Recent work on chemical synapses certainly confirms roles for CHOL in neurotransmitter release, but further emphasizes the complicated nature of such analyses in intact systems (Smith et al., 2010; Ormerod et al., 2012). The thorough and quantitative dissection of CHOL contributions to fusion was only possible because of the direct assessments of function and membrane composition fully enabled by working with urchin CV; in the absence of this level of experimental rigor, it seems unlikely that such quantitative assessments will be possible in other secretory cells. This is particularly true in terms of assessing curvature contributions--unless this can be directly assessed, the postulated curvature contributions of other components remain purely speculative. Nonetheless, native membrane fusion is known to correlate positively with the presence of PE and DAG (Rhee et al., 2002; Uchiyama et al., 2007; Churchward et al., 2008b; Rogas-evskaia and Coorssen, 2011). Although curvature contributions have also been ascribed to phosphatidic acid (PA) in chromaffin cells (Zeniou-Meyer et al.. 2007b; Bader and Vitale, 2009), these have not been quantitatively established, and upstream priming roles related to protein binding would also be consistent with effects seen.

Phospholipids

Phospholipids are major components of cellular membranes, constituting the bulk of the bilayer and thus forming the fundamental matrix of native membranes. Of these, phosphatidylinositol (PI) contains an inositol ring that can be phosphorylated to generate a series of polyphospho-inositides (PIP) that play numerous critical functions in signal transduction pathways and as protein-binding sites (Whitaker and Aitchison, 1985; Eberhard et al., 1990; Osborne et al., 2007; Shin et al., 2010). The roles of PI, PIP, phosphatidylserine (PS), PE, and DAG in regulated exocytosis have been quantitatively investigated using supplementation or selective pharmacological manipulations of urchin CV (Churchward et al., 2008b; Rogasevskaia and Coorssen, 2011; Rogasevskaia et al., 2012). As with proteins, the goal is to distinguish modulatory components from those essential to the fast, [Ca.sup.2+] -triggered release steps. As there is no evidence of lipid metabolism in isolated CV, this is an additional attractive feature for such analyses, because the function of the particular lipid in question can be more directly addressed; this is a serious issue in analysis of intact cells given that metabolism can alter the species of interest and the localization of lipids to release sites is difficult to quantify.

Direct incorporation of anionic lipids into CV membrane results in an inhibition of fusion parameters (Churchward et al., 2008b; Rogasevskaia and Coorssen, 2011; Rogas-evskaia et al., 2012); however, evidence indicates that the effects seen after such "bulk" supplementation of anionic phospholipids are nonspecific (Rogasevskaia et al., 2012). For example, such supplementation with PI inhibits all fusion parameters, yet selectively blocking PI using neomycin does not alter the extent or [Ca.sup.2+] sensitivity of fusion; rather, there is only a selective decrease in the rate of fusion (Rogasevskaia et al., 2012). The effects seen upon direct incorporation of anionic lipids are likely the result of the random distribution of these charged species in the membrane rather than at sites of docking and fusion. Experiments with CHOL-sulfate support this hypothesis: simple delivery of exogenous CHOL-sulfate results in a decrease in the extent of CV fusion, but not in [Ca.sup.2+] sensitivity. However, supplementation with CHOL-sulfate after the removal of CV CHOL (i. e., that results in reduced fusion extent and [Ca.sup.2+] sensitivity; Churchward et al. [2005, 2008b]) rescues both [Ca.sup.2+] sensitivity and fusion extent (Rogasevskaia et al., 2012); it seems that CHOL-sulfate tends to localize to the dockding and fusion site to replace the missing CHOL. Understanding the potential artifacts associated with direct lipid incorporation allows for better controls and thus more selective analyses concerning the roles of anionic and other lipid species. Using a combination of selective blocking reagents and enzyme inhibitors, the CV studies indicate that PI/PIP do not have a direct role in [Ca.sup.2+] sensing or fusion, but function upstream, likely serving as specific binding sites for proteins promoting docking and priming. In contrast, evidence now suggests that polyphosphoinositides may act as a [Ca.sup.2+] sensor, a role first proposed decades ago on the basis of studies of liposome fusion but not directly tested in native vesicles until now (Rogasevskaia et al., 2012).

Like CHOL, PE has been shown to modulate the rate and extent of CV fusion (Rogasevskaia and Coorssen, 2011). As PE also has an intrinsic curvature comparable to that of CHOL, the reduced extent of fusion seen with lower total amounts in the CV membrane correlates with its negative curvature contribution to the fusion machine (Churchward et al., 20081b; Rogasevskaia and Coorssen, 2011). Interestingly, diminished levels of CV membrane PE do not impair [Ca.sup.2+] sensitivity, an efficiency parameter linked to CHOL-enriched microdomains and thus thought to be regulated by specific protein-protein and protein-lipid interactions (Churchward et al., 2005, 2008b; Rogasevskaia and Coors-sen, 2006). PA has also been proposed to promote formation of the hemifusion diaphragm via its negative curvature (Zeniou-Meyer et al., 2007a; Bader and Vitale, 2009). Indeed, expressing inactive phospholipase D1 inhibits exocytosis at cholinergic synapses (Humeau et al., 2001). However, there is no evidence to suggest this was solely due to a decrease in PA at the docking and fusion site: though the presence and localization of PLD was tested, changes in PA concentration (i. e., at the fusion site or at a cellular level) were not monitored (Humeau et al., 2001). As PA is not able to rescue fusion in CHOL-depleted CV (Church ward et al., 2008b), it would again seem that this anionic lipid is more likely to function upstream of fusion to aid in localizing necessary proteins contributing to the physiological fusion machine (Stace and Ktistakis, 2006; Mendonsa and Engebrecht, 2009). Indeed, an earlier study of [Ca.sup.2+] -triggered exocytosis in platelets clearly demonstrated that no major phocpholipase activities are directly associated with triggered release but could function upstream to promote a fusion-ready state (Coorssen. 1996).

Future Directions

By virtue of their fully docked, primed, and release-ready state, sea urchin cortical vesicles (CVs) have enabled rigorous, quantitative assessment of the roles of specific proteins and lipids in these conserved, fundamental cellular mechanisms that enable exocytosis. As outlined above, many components critical to the late steps of regulated exocytosis have yet to be linked to specific functional stages, and it is likely that some components remain to be identified (or at least linked to the mechanisms). Several small molecules are known to affect the process of [Ca.sup.2+] -triggered membrane merger; however, the protein or lipid targets of these agents remain unknown (Haggerty and Jackson, 1983; Whalley and Sokoloff, 1994; Vogel et al., 1995; Coorssen et al., 2003; Szule et al., 2003; Furber et al., 2009a, b, 2010). For example, N-ethylmaleimide (NEM) is an alkylating agent that potently and irreversibly inhibits membrane fusion; indeed, work with this reagent provided some of the first evidence that triggered release was at least in part mediated by proteins (Haggerty and Jackson, 1983: Vogel et al., 1992; Whalley and Sokoloff, 1994). Although NSF is so named due to its interaction with NEM, this protein is not required for membrane fusion, and the fusion-critical proteins affected by NEM remain unknown (Whalley et al., 2004; Furber et al., 2009a). In contrast, another thiol-modifying reagent, iodoacetamide, has been shown to have a biphasic effect, enhancing the [Ca.sup.2+]sensitivity and rate of CV *fusion at lower concentrations but inhibiting them at higher levels (Furber et al., 2009a). These studies indicate the existence of two distinct thiol sites that affect the [Ca.sup.2+] -triggered fusion mechanism; iodoacetamide alters one that modulates the efficiency of fusion (likely at the level of a [Ca.sup.2+] sensor), and NEM and a host of others interfere with protein sites required to enable fusion. Thiol-reactive agents can therefore be used to identify [Ca.sup.2+] sensors (or related regulatory proteins) and proteins affecting membrane merger. Because certain fluorescent thiol reagents cause the same effect as their parent compounds (Furber et al., 2010), utilizing these with stage-specific CV holds the promise of an unbiased approach to labeling and identifying proteins critical to the late. [Ca.sup.2+] -triggered steps of regulated exocytosis. Furthermore, certain proteases could also be used to identify critical proteins. For example, treatment with chymotrypsin has only a limited effect on CV SNARE cytosolic domains, yet it irreversibly inhibits CV fusion. Therefore, identifying the targets of this protease will allow the list of candidate proteins that mediate fast. [Ca.sup.2+] -triggered membrane fusion to be further refined.

The role of SNAREs in exocytosis has been a subject of intense debate (Zimmerberg et al., 2000; Duman and Forte, 2003; Rizo, 2003; Szule and Coorssen, 2003; Churchward and Coorssen, 2009; Rizo and Sudhof, 2012). As we have discussed herein, it is evident that SNARE cytosolic domains are not necessary for membrane merger and therefore are not part of the FFM. The general conclusion based on findings from SNARE complex disruption studies in the cell-surface complex and CV is that SNAREs and SNARE-protein or -lipid interactions mediate aspects of the [Ca.sup.2+] sensitivity and kinetics of fusion, likely indicative of roles in priming; however, the specific interactions within the PFM that confers these modulatory functions are unclear. Papain quantitatively ablated SNARE cytosolic domains (and thus zippering-up and inter-membrane complex formation) without disrupting the fundamental ability of CV to fuse; however, the fusion mechanism was no longer efficient, with the EC50 shifting to about 250 umol [1.sup.-1] [[C[a.sup.2+]].sub.free] (Coorssen et al., 2003). However, the protease is not selective for the SNAREs, and proteins necessary for the [Ca.sup.2+] sensing, priming, and docking of vesicles are also likely to have been proteolysed. In particular, synaptotagmin. a [Ca.sup.2+] - and phospholipid-binding protein having on the order of 134 papain cleavage sites in its cytosolic domain, would have undergone extensive proteolysis (Table 2); if the extent of CV synaptotagmin proteolysis could be quantitatively correlated with the documented shift in [Ca.sup.2+] sensitivity, this would provide direct evidence for a central role of synap-totagmin in [Ca.sup.2+] sensing and fusion. Treatment of CV with NSF + a-SNAP or low [[ [Ca.sup.2+]].sub.free] also resulted in a loss of [Ca.sup.2+] sensitivity (i.e., E[C.sub.50] shifting to ~90 umol [1.sup.-1] [[C[a.sup.2+]].sub.free] : Fig. 3), and this shift can be ascribed to a loss of SNARE protein interactions (Coorssen et al., 1998; Tahara et al., 1998; Zimmerberg et al., 2000). However, treatment with NSF disassembles cis-SNARE complexes for subsequent recycling of the component SNAREs, but is not known to disassemble trans-complexes, as these are said to be functionally resistant to such disassembly (Weber et al., 2000); thus treatment of CV with NSF + a-SNAP would not definitively indicate that trans-SNARE inter-membrane interactions are not required for membrane fusion. In addition to disassembling the cis-SNARE trimer, a-SNAP is known to bind monomeric syt and SNAP-25, although with lower affinity (Hanson et al., 1995; McMahon and Sudhof, 1995), and the interactions of the SNAP-25/syt dimer with other components including synaptotagmin have been postulated to confer [Ca.sup.2+] sensitivity (Rickman et al., 2004; de Wit et al., 2009). However. [Ca.sup.2+] treatment of CV resulted in the disassembly of both trans- and cis-SNARE complexes, demonstrating (like the proteolytic ablation studies) that inter-membrane SNARE interactions are not essential for membrane fusion (Coorssen et al.. 1998: Tahara et al., 1998). Nonetheless, as the shift in [EC.sub.50] was comparable after treatment of CV with either low [[[Ca.sup.2+]].sub.free] or NSF + a-SNAP, together these data suggest that it is cis-SNARE interactions in the vesicle membrane that contribute to the [Ca.sup.2+] sensitivity of the fusion reaction; indeed [Sr.sup.2+] and [Ba.sup.2] triggered fusion but did not disrupt SNARE complexes (Coorssen et al., 1998: Tahara et al., 1998). Thus, when SNARE interactions were blocked by exposure of CV to excess exogenous binding partners (Szule et al., 2003), it is likely that SNAP-25/syt dimers (or even cis-SNARE trimers) remained intact and could interact with other components required to mediate [Ca.sup.2+] sensitivity: perhaps this explains why no fusion parameters were affected by the treatments with exogenous proteins. This suggests an interpretation perhaps not previously apparent from the SNARE interaction models widely proposed in the literature. Simply, as there are conditions in which [Ca.sup.2+] sensitivity and efficient kinetics remain in the absence of trans-SNARE interactions, it is possible that specific cis-SNARE interactions contribute after SNARE coiled-coil interactions (or perhaps even despite them). Fusion-ready CV are thus at a state of "maturation" in which trans-SNARE functions have already been carried out, and cis-SNARE interactions (i.e., potentially including interactions of SNARE-dimers, -trimers. or monomers with other critical proteins and lipids in and on the vesicle membrane) are needed for efficient fusion to occur. Is this, by definition, the functional PFM? Are the postulated interactions themselves contributing to function, or do the SNAREs help to localize other critical components (i.e., much like CHOL-enriched microdomains (Church-ward and Coorssen, 2009)) to ensure fast, efficient fusion under physiological conditions? In considering that the fundamental molecular mechanisms enabling regulated exocytosis are conserved, then in certain secretory processes such as neurotransmission, the [Ca.sup.2+] -triggered fusion steps may occur on a time scale that cannot currently be effectively resolved in situ. That is, current techniques do not provide the temporal resolution needed to distinguish between the formation of trans-SNARE complexes and the intra-membrane, cis interactions leading to membrane merger. If these cis interactions are indeed critical, particularly between SNAREs and other protein- and lipid-binding partners, this might well explain why reconstituted proteoliposome models do not broadly and effectively recapitulate the characteristics of native fusion (Chen et al., 2006; Dennison et al., 2006; Churchward and Coorssen, 2009); perhaps we just do not yet have an effective understanding of the local native "mix" of components and their interactions. Thus, based on data garnered over the last decade using sea urchin cell-surface complex and CV preparations in tightly coupled functional--molecular assays, the suggestion is that SNARE and other protein or lipid interactions in and on the vesicle (and target?) membranes are critical aspects of regulated exocytosis (i.e., contributing to or defining the PFM); this certainly best fits with the findings that CV can fuse with pure lipid membranes, albeit with lower efficiency (Vogel et al., 1992; Chanturiya et al., 1999). It is the physiological nature of the egg and the docked, release-ready state of CV that enables the dissection of the mechanisms contributing to fast, [Ca.sup.2+] -triggered steps of exocytosis (i.e., both the FFM and the PFM). It would seem that these preparations from the unfertilized urchin egg may still have much to teach us into the 21st century.

Acknowledgments

The authors dedicate this paper to Victor Vacquier for his groundbreaking contributions to exocytosis research. By initiating the use of these native membrane preparations in a "reductionist" approach, he set the stage for many critical research contributions over the last 40 or so years.

The authors thank Dr. Andrew Braun (University of Calgary) for generously providing CaM inhibitory peptides [BK.sub.a], MLCK, and CaM Kinase I. PSA would like to thank the UWS School of Medicine for scholarship funding. JRC acknowledges the support of the University of Western Sydney, the UWS School of Medicine, and an anonymous private Australian foundation, as well as past support from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the Alberta Heritage Foundation for Medical Research that supported much of the original research from his group that is referred to herein.

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Received 19 January 2013; accepted 13 June 2013.

* To whom correspondence should be addressed. E-mail: j.coorssen@ uws.edu.au

[dagger] Current address: School of Paediatrics and Child Health. University of Western Australia, Perth, Western Australia 6006.

Abbreviations: CaM, calmodulin; CHOL, cholesterol; CSC, cell-surface complex; CV, cortical vesicle; DAG, diacylglycerol; FFM, fundamental fusion mechanism; NSF. the N-ethylmaleimide-sensitive factor: PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP, polyphosphoinositides; PM, plasma membrane; SNAP, soluble NSF attachment protein.

PRABHODH S. ABBINENI (1), JULIE E. HIBBERT (2), ([dagger]), AND JENS R. COORSSEN (1), (*)

(1) Department of Molecular Physiology, and Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia; and (2) Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, AB, T2N 4N1, Canada
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