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MS 9809 Received 5 July 1999; accepted after revision 20 August 1999.
A hallmark of living systems is their organizational complexity, at the macromolecular, organelle, cellular, tissue and behavioural levels. At the cellular level, biological membrane fusion is a major mechanism for controlling the compartmentalization of macromolecular assemblies. Membrane fusion is a fundamental process involved in many biological activities including cellular secretion, enveloped virus infection, muscle formation and egg fertilization. The time, location and extent of biological membrane fusion appear to be controlled by proteins. Proteins and protein complexes thought to be involved in the pre-fusion and membrane-membrane merger processes are highly organized and possess structural motifs identifiable by both crystallography and spectroscopy (Skehel & Wiley, 1998). While the knowledge of protein structures is extremely important in our research, these structures have not yet, in and of themselves, revealed the mechanism of membrane fusion. Rather, determining how proteins regulate and drive biological membrane fusion requires synergy between biophysical, biochemical and physiological studies of the fusion process.
How biology ensures an organized, reliable and controlled response with regard to exocytotic membrane fusion, is exemplified by fertilization. Seconds after fertilization, there is an elevation of the proteinaceous vitelline coat of many eggs, including human eggs, that has captivated scientists for over a century. Upon fertilization, a large, transient increase in cytoplasmic calcium triggers a massive synchronous exocytosis of cortical vesicles that lie docked to the inner leaflet of the plasma membrane; virtually every vesicle fuses during fertilization despite the complexity of the process. In echinoderm eggs, these vesicles are 
1 µm in diameter, and are easily imaged (Fig. 1). One of the great advantages of echinoderm fertilization is that it is a physiological reaction that can be easily studied in the laboratory. In nature, both male and female gametes are released into seawater where fertilization then occurs. In the laboratory, the major differences involve removal of a protective jelly coat from the eggs, and use of artificial (defined) rather than natural seawater. Another major advantage of this preparation was discovered in 1975, when Victor Vacquier published a procedure for preparing the plasma membrane of the sea urchin egg with attached cortical vesicles (Vacquier, 1975). Dejellied eggs adhere extremely well to polycation-coated glass or plastic. When the eggs are sheared with appropriate solutions, the bulk of the egg is removed leaving large plasma membrane fragments adhering to the glass; fully functional cortical vesicles remain attached to these membrane fragments despite vigorous washing. The cortical vesicles are primed, docked, and ready for release. An elevated free calcium concentration appears to be the sole requirement for cortical vesicle exocytosis (Whitaker & Baker, 1983; Zimmerberg & Liu, 1988; Blank et al. 1998a). This preparation possesses even greater versatility because the secretory vesicles can be purified from the plasma membrane and fusion reconstituted. Vesicles brought into contact with the plasma membrane (Crabb & Jackson, 1985; Whalley & Whitaker, 1988), each other (Vogel & Zimmerberg, 1992; Vogel et al. 1992; Coorssen et al. 1998) or other membranes (Vogel et al. 1992) fuse in response to calcium. Thus, the vesicles retain all the components necessary for calcium-triggered fusion.
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A, prior to fertilization (left), the proteinaceous vitelline coat of the sea urchin egg of Lytechinus pictus is not visible in this differential interference contrast image. Upon fertilization (right), the massive synchronous exocytosis of cortical vesicles leads to the formation of a fertilization envelope bounded by the vitelline coat. The envelope appears as a ring surrounding the egg. B, cortical vesicle exocytosis is observed in the intact egg. Prior to fertilization (left), primed, docked and ready-to-release vesicles line the inner leaflet of the plasma membrane. Upon fertilization (right), an increase in calcium concentration triggers vesicle exocytosis. Vesicles disappear during exocytosis as a result of hydration and dispersal of vesicle content. See Vogel et al. 1996. Magnifications: A, × 500; B, × 2500. | ||
Vesicle fusion can be directly imaged or monitored using changes in light scattering. Light scattering is used to quantify the amount of fusion since changes in optical density are directly proportional to the loss of vesicles by fusion (Haggerty & Jackson, 1983; Zimmerberg et al. 1985). This decrease in light scattering upon vesicle fusion is due to the sudden, dramatic decrease in the refractive index of the vesicles as hydration and dispersal of contents occurs: the contents of a single vesicle can hydrate and largely disperse in 
17 ms (Zimmerberg et al. 1985; Shafi et al. 1994). Changes in light scattering can be monitored using a spectrophotometer (Haggerty & Jackson, 1983; Sasaki & Epel, 1983), a microtitre plate reader (Vogel et al. 1991; Vogel & Zimmerberg, 1992; Coorssen et al. 1998) or a microscope photometer (Zimmerberg et al. 1985; Blank et al. 1998a). A forward scattering microscope photometer is easily constructed using a darkfield condenser, a low numerical aperture objective and a photodetector. A stop-flow system was designed to allow switching of solutions using turbulent rather than laminar flow (Kaplan et al. 1996; Blank et al. 1998a). Turbulent flow reduces the diffusion-limited unstirred layer and allows a 95 % change in ionic concentration within 0·3 s at the surface of the coverslip. (Kaplan et al. 1996). Faster rates of change in the free calcium concentration are achieved using a simple photolysis system employing a mercury arc lamp and solutions containing the photocleavable calcium chelator DM-nitrophen (Vogel et al. 1991; Shafi et al. 1994). The kinetics of the system can be studied with these tools. Our goal is to relate proteins participating in exocytosis with identified, measurable steps that describe the fusion process.
Kinetic characteristics of sea urchin cortical vesicle exocytosis
Sea urchin cortical vesicle exocytosis exhibits features characteristic of calcium-triggered exocytosis observed in other secretory cell types (Fig. 2), for both Lytechinus pictus and Strongylocentrotus purpuratus. The onset of exocytosis after exposure to calcium can be detected as the onset of content dispersal which is in the order of 10 ms (Vogel et al. 1991). The entire population of vesicles can fuse within seconds (Shafi et al. 1994; Vogel et al. 1996). The rate of fusion increases, peaks, and then decreases, and these changes in rate occur in the continued presence of calcium. The kinetics vary with calcium concentration but do not scale proportionally. A graded response to calcium is observed; there are calcium concentrations that result in only a fraction of the available vesicle population fusing. The amount of fusion is independent of the sequence of calcium concentrations used to elicit the response, depending only upon the final calcium concentration. A sequence of two challenges, one to an intermediate level of calcium followed by a second higher calcium concentration, yields the same amount of fusion as a single challenge at the higher calcium concentration. What hypotheses can explain these observations?
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A, fits to the kinetic behaviour observed during calcium-triggered exocytosis in the sea urchin cortical preparations of S. purpuratus. Both percentage fusion and rate (% fusion/sec) are non-linear functions of the calcium concentration. The kinetics vary with calcium (left) but do not scale proportionally (right). B, illustration of a sequence of two challenges (left), one to an intermediate level of calcium (green line) followed by a second higher concentration (red line), yield the same amount of fusion as a single challenge at the higher concentration (dashed red line). C, the amount of fusion (black line) is independent of the sequence of calcium concentrations used to elicit the response, depending only upon the final calcium concentration (green and red circles). See Blank et al. 1998a,b. | ||
A significant body of work on exocytosis in the chromaffin cell has led to the development of an explanation for the calcium dependence of fusion (Neher & Zucker, 1993). In that system, a single linear reaction consisting of multiple binding equilibria followed by one irreversible transition to fusion is a model consistent with the data obtained by capacitance measurements (Heineman et al. 1993, 1994). However, this model is not consistent with the sea urchin data. In the absence of concurrent trafficking processes, fusion initiated in a homogeneous population of vesicles should continue until every vesicle has fused. The simplest explanation as to why this does not occur is that other limiting processes are coupled to a linear reaction scheme. Permanent inactivation of the remaining vesicles following sub-maximal stimulation was not observed; the remaining vesicles still have the ability to fuse in response to higher free calcium concentrations (Blank et al. 1998a). If the cessation of fusion was due to reversible, calcium-dependent inactivation of the fusion machinery, then returning the preparation to a lower free calcium concentration should reset the fusion process. Upon reset, a second exposure to the initial calcium concentration should result in proportional fusion of the remaining vesicles. This property was not observed (Blank et al. 1998a). Changing the rate of calcium delivery from micromolar per millisecond (µM ms-1) to micromolar per second (µM s-1) had no effect on the final level of fusion, ruling out adaptation and rate-dependent processes (Blank et al. 1998b). Thus, the activity of the system could not be attributed to a linear fusion reaction coupled to decreases in the free calcium concentration, calcium-dependent inactivation, or adaptation.
If the cessation of fusion occurred because a specific sub-population of vesicles was removed through fusion (heterogeneity), then only exposure to higher calcium concentrations should produce more fusion (Baker & Knight, 1981). The amount of fusion will be additive and depend only upon the final concentration of calcium. These properties were observed (Blank et al. 1998a,b; Coorssen et al. 1998; Tahara et al. 1998). In vivo , heterogeneity in vesicle fusion is observed during fertilization; cortical vesicles fuse in a pattern consistent with a non-uniform distribution of calcium thresholds (Terasaki, 1995; Matese & McClay, 1998). What properties of the protein machinery could account for these behaviours?
Functionally defining the fusion complex
We define the fusion complex as a functional entity, the minimal complex that can cause a vesicle to fuse. To explain the effects of calcium on a secretory system in which the vesicles are docked, primed and ready for release we use the concept of spare fusion complexes randomly activated by calcium. By definition, a vesicle requires only one active fusion complex to fuse, and vesicles fuse with an increased rate proportional to the number of active fusion complexes present. Our hypothesis is that an increase in calcium increases the average number of participating fusion complexes, and that these activated fusion complexes are randomly distributed among vesicles (Vogel et al. 1996). At free calcium concentrations that result in sub-maximal responses, some vesicles will not have an activated complex and do not fuse. Whenever a situation arises where the outcome (here fusion) is dependent upon the absence or presence of one or more entities, then the Poisson distribution should be considered. The Poisson distribution describes the random distribution of activated fusion complexes among many vesicles. For example, if 100 vesicles are created from a membrane having 100 complexes then, on average, there will be 1 complex per vesicle. However, each vesicle will not have 1 complex. Rather, some vesicles will have 0 complexes, some 2, some 3, etc. In this example the mean number of complexes per vesicle is 1. The Poisson distribution predicts that 37 % of the vesicles will have no complexes, 37 % will have 1 complex, 18 % will have 2 complexes, and 8 % will have 3 or more complexes. Remarkably, the mean number of active fusion complexes per vesicle can be uniquely determined from the number of vesicles that did not fuse (had no active fusion complexes). This property of the Poisson distribution has been used to examine the biochemical identity of the fusion complex (Vogel et al. 1996; Coorssen et al. 1998).
How large is the spare capacity of a vesicle or how many fusion complexes per fully docked vesicle can be activated with saturating concentrations of calcium? The prediction of this model is that random removal of fusion complexes should diminish the reserve such that back-extrapolation to the average number of maximally activated fusion complexes at the unperturbed level represents the maximum spare capacity. In terms of both exposure time and concentration, the alkylating agent N-ethylmaleimide (NEM) irreversibly inhibits fusion with a single exponential decay. At maximally saturating levels of free calcium, extrapolation to either zero time or concentration indicates that the spare capacity is 9 fusion complexes per vesicle at the docking site. For example, activation on average of only 1/9 (11 %) of the fusion complexes results in 67 % fusion. A Poisson average number of 9 active fusion complexes per vesicle would leave 0·01 %, or 1 vesicle per 104, inactive (fusion incompetent). This was confirmed in the planar cortex by directly counting the vesicles remaining after maximal stimulation (Vogel et al. 1996). A Poisson average of 4·5 active fusion complexes was determined for exocytosis during fertilization (Vogel et al. 1996). Random activation of fusion complexes with spare capacity can account for the reliable and controlled response observed in this calcium-triggered system. Moreover, we can link function and biochemistry whenever a loss of spare capacity is induced by modification of specific proteins; when a system has spare capacity, a 50 % loss of function does not correspond to a 50 % loss or alteration in the molecules involved in that function. Significant inhibition in the extent of fusion occurs only when the spare capacity is reduced substantially from the unperturbed level of 9 to < 1.
The SNARE complex
Are SNARE complexes components of the fusion complex? The SNARE proteins are present in the cortical membranes of S. purpuratus and L. pictus (Fig. 3) (Avery et al. 1997; Conner et al. 1997; Tahara et al. 1998). In addition to the SNARE protein monomers, detergent extracts of isolated secretory vesicles also contain an 70 kDa component recognized by antibodies to the proteins VAMP and syntaxin (Tahara et al. 1998). Analysis of this 70 kDa region from SDS-PAGE gels reveals the presence of three proteins: VAMP, syntaxin and SNAP-25. This protein complex is lost upon boiling in SDS sample buffer. In native membranes, the complexed components are protected against cleavage by clostridial toxins (Rossetto et al. 1994). These properties (Hayashi et al. 1994; Ferro-Novick & Jahn, 1994; Pelligrini et al. 1994) define the SNARE complex identified in other secretory systems; the sea urchin possesses the same proteins and protein complexes.
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The two left lanes are immunoblots of membrane extracts of cortical vesicles from the sea urchin S. purpuratus. These extracts contain proteins that are immunoreactive with antibodies to rat brain VAMP2 (VAMP lane) and syntaxin 1A (Syntaxin lane). Following protein recovery, boiling and a second separation by SDS-PAGE, a high molecular mass band present in the initial unboiled extract of vesicle proteins is shown to contain VAMP, syntaxin and SNAP-25 (right-hand immunoblots). Values to the left of the blots are molecular mass in kDa. The ~70 kDa complex is the SNARE core complex identified in other secretory systems. See Tahara et al. 1998. | ||
Surprisingly, the SDS-resistant SNARE complex immunoreactive signal is absent from extracts of native membranes that have been exposed previously to calcium (Tahara et al. 1998; Coorssen et al. 1998). This is unexpected, as the high in vitro stability of the SNARE complex presumably means that disruption could only occur via an ATPase such as the NEM-sensitive factor, NSF (Glick & Rothman, 1987; Malhotra et al. 1988). However, it is not the SNARE complex itself that is sensitive to calcium. Rather, the SNARE complex in the context of the entire membrane assembly is sensitive to calcium. That is, the isolated or reconstituted SNARE complex in detergent solution is insensitive to calcium. Only in the native membrane itself is immunoreactivity of the complex affected by calcium. Furthermore, immunoreactivity of both cis and trans SNARE complexes (Ungermann et al. 1998a,b, 1999) are disrupted upon exposure to calcium. Does the calcium-dependent disruption of the SNARE complex signal correlate with either of the SNARE hypotheses? The original SNARE hypothesis suggested that SNARE complex disruption itself was the final event causing membrane fusion (Sollner et al. 1993; Rothman, 1994). This hypothesis does not fit because strontium and barium, which substitute for calcium and support fusion, do not disrupt the complex signal, even at concentrations that yield maximal fusion (Coorssen et al. 1998). Fusion can therefore occur without a loss in the SNARE complex signal. A modified SNARE hypothesis (SNAREpins) states that the formation of the SNARE complex drives fusion (Weber et al. 1998; Chen et al. 1999). However, both in cortices and a homotypic vesicle-vesicle fusion assay, we find that the calcium concentration at which the complex signal is lost is 6 µM. Treatment with 3 µM calcium results in 30 % fusion and no loss of complex signal; 6 µM calcium yields an additional 30 % fusion and total loss of complex signal, and 100 µM calcium elicits total fusion of the vesicle population (Coorssen et al. 1998). Fusion can therefore be elicited at a range of calcium concentrations, regardless of the SNARE complex signal. No direct correlation exists between fusion and the immunoreactive SNARE complex, as there are also conditions in which fusion does not occur but calcium still disrupts the SNARE complex signal (Coorssen et al. 1998).
A regulatory role for the SNARE complex
What is the role of the SNARE complex in exocytosis? The VAMP-selective protease, tetanus toxin, when injected into the unfertilized sea urchin egg prevents wound-induced exocytosis of cortical vesicles following their osmotically driven undocking from the plasma membrane (Bi et al. 1995). Partial inhibition of exocytosis at 5·9 µM Ca2+ is observed following tetanus toxin treatment of egg cortices, despite apparently complete cleavage of VAMP by the toxin (Avery et al. 1997); this result is consistent with an alteration in the calcium sensitivity of the preparation. The imperfect correlation between protein cleavage and loss of function may therefore reflect experimental perturbations in priming and redocking and not a direct effect on the final steps of exocytosis. As in many systems, SNARE elements seem to play an important role in the pathway of cortical vesicle fusion in the sea urchin egg. Our working hypothesis is that SNARE complex formation is needed to bring the membranes destined to fuse into intimate contact, thereby defining the amount of calcium required for efficient fusion. This interpretation is supported by the following experiment. If vesicles are allowed to make contact in the presence of sufficient calcium to prevent formation of the SDS-resistant SNARE complex immunoreactive signal, there is an 30-fold loss of calcium sensitivity for fusion. Fusion occurs, but requires higher concentrations of calcium; fusion appears to proceed by the usual mechanism, but with diminished calcium sensitivity (Coorssen et al. 1998). The correlation between the loss of complex signal and diminished calcium sensitivity is supported by the response to strontium and barium observed under these assay conditions; fusion occurs without a loss of complex signal and fusion sensitivity. This interpretation is also supported by experiments using a neuronal-VAMP (synaptobrevin) knockout in Drosophila, in which fusion activity (exocytosis) could be rescued by manipulations that increased presynaptic free calcium concentrations (Yoshihara et al. 1999). Our view is that SNARE complexes aid in bringing membranes together, that this is their role in fusion, but that the degree of membrane interaction incurred by the SNARE complex is not in itself sufficient to drive membrane fusion in the sea urchin system. The SNARE complex appears to play a modulatory role in defining a pre-fusion exocytotic complex, in establishing conditions or recruiting additional components for the formation of the calcium-sensitive, activation-competent fusion complex (Coorssen et al. 1998). This hypothesis is supported in homotypic fusion of yeast vacuoles where disruption of the SNARE complex following membrane contact does not affect ensuing fusion (Ungermann et al. 1998b). Any modulatory component, i.e. SNARE proteins, coupled to the fusion complex, may therefore alter the energetics of the fusion process, but may not contribute sufficient energy for fusion. Thus, the results from this system are not consistent with the SNARE complex by itself being the universal minimal fusion machine, but leave open possible roles of individual SNARE components, other SNARE complexes, synaptotagmin, other proteins, and complexes thereof.
In summary, the sea urchin egg is an experimental system for the study of calcium-triggered exocytosis with a variety of functional, reduced preparations with which to test for and identify proteins that catalyse exocytosis. By functionally defining the fusion complex in terms of a random activation of fusion complexes, we have developed an analytical and predictive description of calcium-dependent exocytosis that can now be used to unify biophysical, biochemical and physiological studies. Our basic hypothesis is that membrane fusion is essentially a lipidic process regulated by proteins (Zimmerberg et al. 1980, 1993; Monck & Fernandez, 1992; Chanturiya et al. 1997; Chernomordik et al. 1998). By analogy with viral fusion, we would expect fusion proteins to form a supramolecular complex, contributing to a protein 'fence' which forms a ring to focally isolate membrane lipid components at a defined fusion site (Chernomordik et al. 1998). In addition to providing apposition of membranes and lipid isolation, the ultimate role for the proteinaceous machinery involved in biological fusion is most likely the lowering of the same energy barriers that must be overcome to fuse adherent phospholipid membranes.
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We would like to thank our colleagues, Drs Irina Kolosova, Peter Backland, Kim Timmers, Ludmilla Bezrukov and Masahiro Tahara for stimulating and vigorous discussions throughout the work discussed in this review.
Corresponding author
J. Zimmerberg: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA.
Email: joshz{at}helix.nih.gov
Author's present address
S. S. Vogel: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2630, USA.
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 M. G. Mozhayeva, M. F. Matos, X. Liu, and E. T. Kavalali Minimum Essential Factors Required for Vesicle Mobilization at Hippocampal Synapses J. Neurosci., February 18, 2004; 24(7): 1680 - 1688. [Abstract] [Full Text] [PDF]
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 A. J. Poustka, D. Groth, S. Hennig, S. Thamm, A. Cameron, A. Beck, R. Reinhardt, R. Herwig, G. Panopoulou, and H. Lehrach Generation, Annotation, Evolutionary Analysis, and Database Integration of 20,000 Unique Sea Urchin EST Clusters Genome Res., December 1, 2003; 13(12): 2736 - 2746. [Abstract] [Full Text] [PDF]
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 A. L. Abbott, R. A. Fissore, and T. Ducibella Identification of a Translocation Deficiency in Cortical Granule Secretion in Preovulatory Mouse Oocytes Biol Reprod, December 1, 2001; 65(6): 1640 - 1647. [Abstract] [Full Text] [PDF]
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