Reconstitution of Calcium-triggered Membrane Fusion Using “Reserve” Granules*

Calcium-gated secretion of proteins involves the transfer of “reserve” granules, exocytotic vesicles that are cytoplasmic and, hence, plasma membrane-naive, from the cell interior to the surface membrane where they dock prior to fusion. Docking and subsequent priming steps are thought to require cytoplasmic factors. These steps are believed to induce fusion competence. We have tested this hypothesis by isolating reserve granules from sea urchin eggs and determining under which conditions these granules will fuse. We find that isolated reserve granules, lacking soluble cofactors, support calcium-dependent membrane fusion in vitro. Preincubation with adenosine 5′-3-O-(thio)triphosphate and guanosine 5′-3-O-(thio)triphosphate did not prevent fusion. Thus, isolated reserve granules have all the necessary components required for calcium-gated fusion prior to docking.

The ontogeny of granules destined for calcium-triggered exocytosis involves many steps, including the biosynthesis of granule components, transport of granules to the plasma membrane, and docking with the plasma membrane. As in most cells, mature secretory granules from sea urchin eggs are derived from vesicles which bud from the Golgi apparatus (1). It is known that secretory granules isolated from a cell surface complex consisting of sea urchin egg plasma membrane sheets and tightly associated cortical granules can be reconstituted to fuse with the plasma membrane (2,3), with other cortical granules (4), and even with liposomes in response to elevated calcium in vitro (5). It is believed that cortical granules have multiple copies of the machinery required for calcium-triggered fusion (6). It is clear that docked granules arrested at a stage just prior to being triggered can support membrane fusion in vitro. In contrast, the point during the development of secretory granules where fusion complexes are assembled and capable of supporting both homotypic and heterotypic membrane fusion is not known. It has been proposed that the assembly of a complex of proteins that are thought to mediate membrane fusion occurs during and subsequent to granule docking (7)(8)(9). This assembly is thought to require cytoplasmic factors. Furthermore, other "activation" steps subsequent to granule docking are thought to be required for a fusion complex to support triggered membrane fusion (10,11).
Sea urchin eggs undergo a massive calcium-triggered exocytotic event called the cortical reaction immediately following fertilization. Approximately 18,000 docked cortical granules fuse with the plasma membrane during the first few minutes (12). Secreted granule contents are integral to the construction of the fertilization envelope, a barrier to polyspermy (13). Sea urchin eggs also contain "reserve" secretory granules, distinct from cortical granules, which are localized in the cytoplasm of the unfertilized egg. These granules translocate from the cytoplasm to the inner surface of the plasma membrane, dock, and fuse with the plasma membrane at various times, ranging from minutes to days, after the cortical reaction (14 -23). One example of reserve secretory granules in sea urchin are the yolk granules. Despite their name, it appears that sea urchin yolk granules do not serve as a food source during early embryonic development, and their abundance is stable between fertilization and day 3 of development (24). Between day 3 and 7 of development, yolk granules disappear from the cytoplasm (24). During this same late period, a protein with an apparent molecular mass of 160 kDa, the major protein marker observed in the SDS-PAGE 1 profile of yolk granules, is deposited between the cells of the developing embryo, presumably via a secretory process (20). A 160-kDa protein is the main constituent of a secreted protein complex, called toposome (17,18), responsible in part for cell-cell adhesion during sea urchin embryonic development (20). The toposome complex has been isolated from sea urchin egg yolk granules (17), and immunohistochemical study has localized it in these same granules (20). Indeed, electron microscopic analysis of yolk granules in the cells of sea urchin gastrula indicates that yolk granules can fuse with the plasma membrane in a calcium-dependent manner (25).
In this study homogenates of both unfertilized and fertilized eggs were fractionated by free flow electrophoresis, a rapid separation technique that has been extensively used to purify intracellular vesicles (26 -31). A fraction that contains reserve secretory granules was identified, and the requirements for fusion were determined using reconstitution.

EXPERIMENTAL PROCEDURES
Obtaining and Handling Gametes-Eggs and sperm of the sea urchin Lytechinus pictus were obtained by intracelomic injection of 0.5 M KCl. Eggs were collected in sea water and sperm were collected dry. Before homogenization, eggs were dejellied by several passes through 110-m Nitex mesh and were washed three times in sea water. Fertilization * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  was achieved by mixing eggs and diluted sperm (20:1, v/v in sea water) at a volume ratio of 50:1.
Free Flow Electrophoresis-A free flow electrophoresis apparatus (Octopus, Dr. Weber GmbH, Kirchheim-Heimstetten, Germany) providing marginal flows with high conductivity (31), modified to a chamber size of 500 ϫ 100 ϫ 0.3 mm, was used for homogenate separation. GEHB buffer was used as chamber buffer and GEHB supplemented with 50 mM HEPES and 50 mM Bis-Tris were used as electrode and margin buffer. The optimal current for the electrophoretic separation of sea urchin egg granules was 80 mA, which was reached at a voltage across the chamber of 1000 V at 17°C. The homogenate was injected into the separation chamber close to the cathode and separated at a flow rate of 700 ml/h. 96 fractions were collected after electrophoretic separation and the turbidity of each fraction was measured by absorbance at 405 nm in a Spectramax 250 microtiter dish reader to detect fractions containing granules.
Reconstitution of Calcium-triggered Fusion-Free flow electrophoresis fractions containing granules were centrifuged onto glass coverslips as described previously (4,5). Coverslips were placed in a perfusion chamber (32), washed with PKME buffer (50 mM PIPES, pH 6.7, 425 mM KCl, 10 mM MgCl 2 , 5 mM EGTA, 1 mM benzamidine) and were observed by differential interference contrast and fluorescence microscopy. In some experiments PKME was supplemented with either ATP (2 mM), GTP (1 mM), ATP␥S (2 mM), or GTP␥S (1 mM). Fusion was triggered by perfusion with PKME buffer or nucleotide supplemented PKME buffer containing 316 M free calcium (5). In some experiments fusion was triggered by photo-releasing calcium from DM-nitrophen ("caged calcium") (32). To reconstitute granule-plasma membrane fusion, sea urchin egg cortices were prepared as described previously (33), but were treated with PKME containing 316 M calcium to trigger the fusion of any attached cortical granules. Next, granules from unfertilized eggs were labeled with the hydrophobic fluorescent marker octadecyl rhodamine B (1:1000 dilution of a 1 mM stock of octadecyl rhodamine B in ethanol), as described elsewhere (5), and were centrifuged onto coverslips containing plasma membrane sheets in PKME buffer.
Microscopy-Granules were imaged with a Zeiss Plan-Neofluar 63 ϫ 1.25 numerical aperture objective using both differential interference contrast optics and standard rhodamine epifluorescence optics on an upright Zeiss microscope. Reconstituted fusion of granules with sea urchin egg plasma membrane sheets was imaged using a Nikon Fluar 10 ϫ 0.5 numerical aperture objective with a rhodamine filter set. Sea urchin egg cortices were prepared as described previously (33). Electrophoretic fractions of egg homogenates containing fluorescent endosomes were made from eggs fertilized in sea water containing fluorescent dextrans (34). Caged calcium was released from DM-nitrophen in PK buffer (50 mM PIPES, 450 mM KCl, 1 mM benzamidine, 1 mM DM-nitrophen and 0.5 mM CaCl 2 , pH 6.7) as described previously (32).
Electron Microscopy-Fraction C granules were fixed in 0.3% glutaraldehyde in PKME buffer for 30 min at room temperature followed by a 1-h incubation in 3.0% glutaraldehyde. Granules were washed in Hendry's phosphate buffer and post-fixed in 1% OsO 4 in the same buffer. The granules were then washed in distilled water and stained with saturated uranyl acetate for 15 min. Samples were dehydrated in a graded ethanol and acetone series. Dehydrated samples were embedded in Epon-Araldite and cured at 60°C for 48 h. Cured blocks were ultrathin sectioned using a Leica Ultracut-UCT. Sections were stained with saturated uranyl acetate and Reynold's lead citrate and were observed in a Jeol 100CX transmission electron microscope. L. pictus eggs were fixed and stained as described previously (34).

RESULTS
Homogenates of unfertilized and fertilized eggs were fractionated by free flow electrophoresis (Fig. 1). Two major fractions were isolated from homogenates of unfertilized eggs. The first fraction, A, contained small 0.77 Ϯ 0.19-m diameter (mean Ϯ SD, n ϭ 45) granules. The second fraction, C, contained larger granules (1.48 Ϯ 0.25 m diameter, n ϭ 50). Upon fertilization, peaks A and C decreased in magnitude, and a new fraction, B, appeared between peak A and C. The exact migration distance of fraction B relative to fractions A and C was variable between preparations. Microscopic analysis of the fraction from unfertilized eggs corresponding in position to fraction B of fertilized eggs revealed a mixture of granules similar in size to those comprising fractions A and C. Occasionally, plasma membrane fragments with associated cortical granules were observed in fraction B. In contrast, aggregates of larger granules and occasionally plasma membrane sheets devoid of cortical granules were observed in fraction B isolated from homogenates prepared from fertilized eggs. Microscopic analysis of isolated sea urchin cortices from unfertilized eggs (data not shown) allowed us to measure the diameter of the plasma membrane docked cortical granules in L. pictus, 0.77 Ϯ 0.21 m (mean Ϯ S.D., n ϭ 50). These were similar in size to the granules in fraction A, but were distinct from those in fraction C.
To determine the origin of the components migrating in Top panel, homogenates of unfertilized and fertilized sea urchin eggs were fractionated by free flow electrophoresis. 96 fractions were collected, and their turbidity (absorbance at 405 nm) was measured in a microtiter dish spectrophotometer to detect fractions by the light scattering activity of granules. A low fraction number corresponds to components with a low charge to surface area ratio, while high fraction numbers correspond to a higher mobility and thus a high charge to surface area ratio. Two major peaks, A and C, were resolved in unfertilized eggs (solid line). Homogenates of fertilized eggs (broken line) showed a dramatic change in the migration pattern with the appearance of a new peak, B migrating between the positions of peaks A and C. Bottom panels, equal volumes (1 ml) of the isolated fractions (indicated by the arrows in the upper panel) from unfertilized (upper set) and fertilized (lower set) eggs were centrifuged onto glass coverslips and observed by video-enhanced differential interference contrast microscopy. Size bar is 10 m. fraction B obtained from fertilized eggs we examined fractions of both fertilized and unfertilized eggs by SDS-PAGE (Fig. 2). The electrophoretic migration patterns of polypeptides from fractions A and C isolated from unfertilized eggs reveal that these fractions are composed of unique assemblies of proteins. Furthermore, fractions A and C are well resolved by free flow electrophoresis. While the SDS-PAGE pattern observed from egg homogenate is identical between unfertilized and fertilized eggs, following fertilization many of the proteins detected in fractions A and C are now found in fraction B.
Reserve granules are by definition undocked granules resident in the cytoplasm but capable of being mobilized to migrate to the plasma membrane where they will ultimately fuse. The observation that some components resident in fraction C of unfertilized eggs migrate with fraction B of fertilized eggs suggests that some of these granules have been recruited into the large complexes observed in fraction B. We tested for calcium triggered fusion activity in fraction C by reconstitution in vitro. Granules were brought into close proximity by centrifugation onto glass coverslips. Next they were placed into PKME buffer which has a high concentration of Mg 2ϩ (10 mM) but less than 1 M free calcium. We exposed fraction C granules isolated from either unfertilized or fertilized eggs to PKME buffer containing 316 M free calcium. Upon perfusion with calcium, we observed the disappearance of high contrast edges between individual granules and the concomitant formation of larger refractile structures with high contrast edges (Fig. 3). These changes are consistent with the fusion of the membranes of these granules and the coalescence of their contents. When exocytotic granules fuse with each other they form larger structures called "fusosomes" (4). The fusosomes formed by the fusion of fraction C granules were stable structures which were observed for several minutes after the addition of calcium.
To verify that the reaction observed by differential interference contrast microscopy was actual membrane fusion, we labeled fraction C granules with octadecyl rhodamine B, a hydrophobic fluorescent membrane marker. Next, labeled granules were mixed with unlabeled granules and brought into close proximity by centrifugation onto glass coverslips. In calcium free buffer (prior to uncaging), dye transfer was not observed between labeled and unlabeled granules. Membrane fusion was triggered by the release of caged calcium and ob-served by differential interference contrast and fluorescent microscopy. Fig. 4 shows a group of granules before and after the release of caged calcium. Differential interference contrast microscopy shows a group of granules coalescing to form a large fusosome upon exposure to calcium. Fluorescent microscopy reveals that the hydrophobic membrane marker resident in one of the granules has spread throughout the membrane of the newly formed fusosome and suggests that their membranes have merged.
Reconstituted membrane fusion with fraction C granules did not require added ATP or GTP. There was no apparent change in the amount of fusion activity observed when ATP (2 mM) or GTP (1 mM) was added (data not shown). If nucleotide hydrolysis is not required for reconstitution, then incubation in nonhydrolyzable analogs of these nucleotides should not interfere with calcium triggered fusion. In Fig. 5 we see that incubation in either ATP␥S (2 mM), GTP␥S (1 mM), or PKME buffer alone did not result in any significant inhibition of calcium triggered granule-granule fusion. Using video-enhanced microscopy, we counted the number of membrane delineated bodies observed to fuse after perfusion with calcium. We observed 50 Ϯ 10% (mean Ϯ S.E., n ϭ 12) fusion with calcium in PKME buffer, 44 Ϯ 18% (n ϭ 3) fusion with calcium in the presence of ATP␥S, and 43 Ϯ 22% (n ϭ 4) fusion with calcium in the presence of GTP␥S. Fusion was never observed in the absence of calcium; exposure to mM concentrations of Mg 2ϩ in PKME buffer did not trigger fusion. Experiments using granules from any single preparation gave comparable amounts of fusion regardless of nucleotide treatment. We did however observe a significant amount of variability between preparations of granules in the amount of fusion observed between preparations of granules. To test if this variability was a function of the fraction of granules contacting other granules we plotted our PKME buffer fusion data as a function of the number of granules per m 2 (Fig. 6). We observed a strong correlation between the density of granules and the fraction which fused in response to calcium. Above a density of 0.3 granules/m 2 we observed that 78.9 Ϯ 4.5% (n ϭ 7) of fraction C granules fused upon exposure to calcium. Light microscopic and gel electrophoretic analysis of fraction C components support the view that fraction C is comprised of a homogeneous population of granules. If, however, fraction C was contaminated with a subpopulation of nonfusogenic components we might expect less than maximal  3. Calcium-triggered homotypic fusion of fraction C granules. Fraction C granules isolated from either unfertilized or fertilized eggs (1 ml) were pelleted onto glass coverslips and transferred to a microscope perfusion chamber (32) where they were imaged by videoenhanced differential interference contrast microscopy. Slides were perfused with calcium-free PKME buffer. After a 5-min preincubation, the slides were perfused with PKME buffer containing 316 M free calcium to trigger fusion. Each perfusion step involved 500 l of buffer, approximately 3.5 times the chamber volume. Size bar is 10 m. membrane fusion. To test its homogeneity, we examined fraction C by thin section electron microscopy. Fig. 7 (bottom panel) reveals a population of granules which are homogeneous with regard to size. Granules fell into three general categories based on the density of staining. 9.2% were densely stained, 3.5% were lightly stained, and 87.3% were moderately stained. In comparison, thin section electron microscopy of L. pictus eggs revealed four classes of organelles that fell into the size range of 0.5-2.0 m in diameter, mitochondria, yolk granules, acidic granules, and the cortical granules, which are found adjacent to the plasma membrane (Fig. 7, top panel). Yolk granules appeared identical to the fraction C granules and were also found to fall into roughly three categories based on the density of staining. Together these observations support the hypothesis that nucleotides are not required to reconstitute calcium trig-gered fusion and indicate that Ͼ85% of isolated fraction C granules are morphologically similar and at least 80% are fusion-competent.
Biological fusion reactions are thought to have a short lived reaction intermediate called a lipidic stalk (35). The formation of these highly curved lipidic stalks can be inhibited by the inclusion of lipids with an intrinsic curvature opposite to that which facilitates stalk formation. One such class of lipid, lysolipids, when introduced into the contacting monolayers of mem- FIG. 4. Differential interference contrast and fluorescent imaging of calcium-triggered fusion. Fraction C granules isolated from unfertilized eggs were mixed with granules labeled with the fluorescent membrane marker octadecyl rhodamine B (R18) and pelleted onto glass coverslips. The top two panels show the differential interference contrast (DIC) and fluorescent (R18) image of the same field before exposure to calcium (t ϭ 0). Next a UV shutter was opened to uncage calcium (ϩCa), and the granules were imaged at 42 s (R18) and 57 s (DIC). Size bar is 5 m.

FIG. 5. ATP␥S and GTP␥S do not inhibit calcium-triggered fusion.
Granules isolated from fraction C of unfertilized eggs were pelleted onto glass coverslips and imaged by video microscopy. Slides were perfused with calcium free PKME buffer, or PKME buffer containing either 2 mM ATP␥S or 1 mM GTP␥S. After a 5-min preincubation at room temperature, the slides were perfused with the same buffer containing 316 M free calcium to trigger fusion. Size bar is 10 m.
FIG. 6. Fusion efficiency is dependent of the density of granules. Granules isolated from fraction C of unfertilized eggs were pelleted onto glass coverslips and imaged by video microscopy. Slides were perfused with calcium-free PKME buffer. Slides were then perfused with PKME buffer containing 316 M free calcium to trigger fusion. The density of granules was determined by counting the total number of vesicles in the video field before perfusion with calcium, and the amount of fusion was determined by counting the number of vesicles that had fused at 2 min after calcium perfusion. brane fusion partners inhibit membrane fusion (35,36). Subsequent removal of lysolipids from these membranes lifts the block. Lysolipids are known to inhibit the exocytotic fusion of mast cells, chromaffin cells, and sea urchin egg cortical granules in vitro (35)(36)(37). Exposing docked cortical granules to a pulse of buffer containing high calcium in the presence of inhibitory concentrations of lysolipid, was found to "activate" granules (38). When lysolipid was subsequently removed, granules were observed to fuse, even in the absence of calcium in the bulk solution. In contrast, isolated cortical granules when exposed to calcium were not "activated" (3). These data suggest that for calcium "activation," granules must be tightly apposed to their target membrane during exposure to calcium. If the fusion of fraction C granules also proceeds by a "stalk mechanism," lysolipids should reversibly inhibit membrane fusion in this system. Likewise, if the fraction C granules are tightly docked to each other after reconstitution, we might expect to observe calcium activation. Fig. 8 shows four successive video fields of fraction C granules. Lysolipid (100 M) by itself had no observable effect. The subsequent addition of calcium in the presence of lysolipid failed to trigger fusion. Removal of the calcium and subsequently of the lysolipid was also without effect. Subsequent addition of calcium in the absence of lysolipid did, however, trigger fusion, demonstrating that the block of membrane fusion was reversible.
If fraction C granules are exocytotic granules, they should be capable of fusion with the plasma membrane of the egg. We labeled granules with octadecyl rhodamine B and centrifuged them onto the surface of a coverslip that contained large sheets of egg plasma membrane. Within seconds of exposure to calcium we observed an increase of fluorescence and dye transfer to these plasma membrane sheets (Fig. 9). Granules which had alighted onto the surface of the glass coverslip did not change in fluorescence intensity. These observations are consistent with the dequenching of octadecyl rhodamine B (39) and dye redistribution into the egg plasma membrane sheets, subsequent to granule-plasma membrane fusion. Granule localization on glass or plasma membrane was confirmed by fluorescent imaging after perfusion with PKME buffer containing the fluorescent membrane probe RH414 (1 M) to visualize membrane sheets (data not shown).

DISCUSSION
The main finding of this study is that 80% of isolated reserve secretory granules are fusion-competent when triggered by elevated concentrations of calcium. If this in vitro observation reflects the situation in the intact cell, then an important FIG. 8. Lysophosphatidylcholine inhibits calcium-triggered fusion. Fraction C granules from unfertilized eggs were sequentially exposed to PKME buffer containing 100 M lauroyl lysophosphatidylcholine (LPC; upper left) and LPC with 316 M calcium (upper right). Next calcium was removed by perfusion with PKME containing 100 M LPC, followed by the removal of LPC by perfusion with PKME (lower left). Finally granules were challenged by perfusion with PKME containing 316 M calcium (lower right). Size bar is 10 m.
FIG. 9. Reconstitution of calcium-triggered heterotypic fusion. Granules isolated from fraction C of unfertilized eggs were labeled with the hydrophobic fluorescent membrane marker octadecyl rhodamine B. Sea urchin egg cortices were prepared on polylysine treated glass coverslips and exposed to PKME buffer with 316 M calcium to trigger the fusion of all docked cortical granules. After washing the granule-free plasma membrane sheets with calcium-free PKME buffer, octadecyl rhodamine B labeled granules were centrifuged onto the cytoplasmic surface of the plasma membrane sheets and placed into a microscope chamber in PK buffer (A). The slide was exposed to UV light at t ϭ 0 to initiate the release of calcium from the "caged calcium" compound DM-nitrophen. The field was imaged by fluorescence microscopy at t ϭ 1.2 (B), 3.2 (C), 5.2 (D), 7.2 (E), and 9.2 s (F) after opening the UV shutter. The arrowhead in frame A indicates a granule that fuses with an underlying sheet of plasma membrane during the course of this experiment. The arrow in frame A indicates a fluorescent granule whose fluorescence does not change because it had alighted on the bare surface of the coverslip. Size bar is 50 m.
implication of this finding is that granule proteins responsible for triggered membrane fusion are already assembled and primed at a step prior to vesicle docking. Numerous yolk granule fusion complexes must be assembled at least 3 days prior to their physiological role in mediating the secretion of the toposome complex. One explanation for why reserve secretory granules would have preassembled and primed fusion complexes is that the granules may have an ancillary cellular function distinct from secretion. When the integrity of the plasma membrane is breached, cells can often repair the lesion (40 -42).
Recently the mechanism of membrane repair has been studied in sea urchin eggs (43,44). In the intact egg, the mechanism of membrane repair is thought to use rapid calcium-triggered membrane fusion. Yolk granules which can rapidly fuse with other granules and with the plasma membrane in vitro are good candidates for the source of membrane used to patch lesions in the egg in vivo. Together, these observations suggest that our reconstituted system does mirror important attributes of the intact cell.
We used free flow electrophoresis to isolate vesicular fractions of sea urchin egg homogenates. The granules observed in fraction A of unfertilized eggs are of the same size as cortical granules, disappear following fertilization, and have a similar SDS-PAGE pattern as isolated cortical granules (45). Accordingly, it is likely that fraction A contains cortical granules which were dislodged from the plasma membrane during homogenization. The observation that many of the proteins associated with fraction A of unfertilized eggs migrate in fraction B prepared from fertilized eggs, and the fact that fraction B prepared from fertilized eggs contained exocytosis-associated endosomes, is consistent with cortical granule membrane proteins being retrieved by endocytosis into the membranes of large endosomes migrating in fraction B isolated from homogenates prepared from fertilized eggs.
In contrast to fraction A, the granules in fraction C are distinct from the docked cortical granules observed on sea urchin egg plasma membrane fragments isolated from unfertilized eggs. First, these granules have a diameter of 1.48 Ϯ 0.25 m as opposed to the diameter of cortical granules, 0.77 Ϯ 0.21 m, in this species. Second, the protein composition of these granules is distinct from the composition of the cortical granules which migrate in fraction A (see Fig. 2) and from the published composition of sea urchin cortical granules (45). Finally, these granules can be isolated from homogenates prepared from fertilized eggs in which cortical granules have already fused.
Four lines of evidence suggest that the large 1.5-m diameter granules observed in fraction C are reserve secretory granules. First, the morphology of fraction C granules as observed by electron microscopic analysis is identical to the unique morphology of sea urchin egg yolk granules (compare Fig. 7, top  and bottom). Yolk granules have a characteristic homogeneous appearance with uniform dark staining (25). They also have small dark 10 -50-nm subparticles (25). These inclusions were observed in fraction C granules as well. Second, the major protein component observed in the SDS-PAGE profile of these large granules has an apparent molecular weight of 160 kDa. A 160-kDa protein is the main constituent of a secreted protein complex called toposome, which is also the most abundant protein in sea urchin yolk granules (17,18). Comparison of the SDS-PAGE profile of the large granules we have studied (see Fig. 2, lanes C 1 -C 3 ) with the published SDS-PAGE profile of isolated yolk granules (45) suggests an identical pattern. Thus, based on their morphology and SDS-PAGE profile, it is likely that the granules we have isolated in fraction C are the cytoplasmic yolk granules responsible for secreting the cell adhe-sion toposome complex binding the cells of the developing embryo between day 3 and 7 of development. Third, like predocked cortical granules (2)(3)(4)(5), these large granules can be reconstituted to fuse with other granules, or with the plasma membrane in a calcium dependent manner. Finally, some reserve granules should associate with the cytoskeleton after fertilization to allow those granules to translocate to the plasma membrane, dock, and fuse with the plasma membrane. Many of the large fraction C granules form aggregates upon fertilization consistent with association with the egg cytoskeleton upon egg activation.
Calcium triggered membrane fusion in this system was reversibly inhibited by lysolipids. Thus, it is possible that fusion proceeds via stalk structures, the proposed membrane intermediate for several forms of biological membrane fusion reactions, including sea urchin cortical granule exocytosis, mast cell degranulation (35) and calcium triggered secretion in chromaffin cells (37). While calcium dependent fusion with fraction C granules can be reconstituted, exposing these granules to calcium in the presence of inhibitory concentrations of lysolipid did not result in any activated granules; fusion was not observed when calcium and then lysolipid were removed (see Fig.  8). This is in contrast to docked cortical granules which can be activated (38), and suggests that we have not reconstituted tight docking, perhaps because fraction C granules, as reserve granules, have never been docked with the plasma membrane. It is possible that fraction C granules may be missing key components which may be required for biological docking but which are not required for fusion. This is supported by the observation that yolk granules attached to egg plasma membrane sheets with 1/7 the efficiency of isolated cortical granules in an in vitro binding assay (46). It is also possible that the components required for biological docking may be different from the components responding to the experimental manipulations used to bring granule membranes into close juxtaposition in this study. Alternatively, the lack of activation with calcium in the presence of lysolipid might indicate a difference between homotypic fusion and heterotypic membrane fusion.
It is not clear why 20% of isolated fraction C granules did not fuse under our reconstitution conditions. While it is possible that some granules fail to fuse because of the absence of limiting soluble factors, this seems unlikely because 1) addition of ATP, GTP, ATP␥S, or GTP␥S did not significantly alter the amount of fusion observed, and 2) incubation with an egg homogenate gave no significant increase in the amount of fusion observed when tested with a subsequent pulse of calcium (as compared with mock-treated granules, data not shown). Nonetheless, the majority of the reserve granule pool isolated from eggs can undergo rapid calcium-dependent membrane fusion in vitro. Thus, cellular components that respond to calcium, discriminate between calcium and magnesium, and can rapidly mediate membrane fusion must be present on these fusion-competent granules. This functional observation may have utility in identifying the molecular components required for calcium-triggered membrane fusion in this system; a candidate fusion protein should reside on these granules prior to docking.
The ability of reserve granules to fuse with membranes in a calcium-dependent, nucleotide-independent manner is consistent with the observation that the N-ethylmaleimide-sensitive factor (NSF) driven release of ␣-soluble NSF attachment protein (SNAP) can precede docking and fusion in yeast vacuoles (47), and it supports the hypothesis that the role played by the ATPase NSF may represent an early step in the exocytotic cascade (48) such as in priming granules for docking or fusion (11,49). Because yolk granules are fusion-competent, ATP-de-pendent steps in secretion required to "prime" fusion complexes (50,51) must also take place prior to granule docking. It is possible that under certain conditions primed secretory components might become deprimed in the course of experimental manipulation. In this case, ATP might act as a cofactor to reprime these components, even subsequent to docking. This might explain why under some conditions calcium triggered exocytosis in sea urchin eggs can be preserved by the addition of ATP (52,53). The fact that exocytosis can be triggered by calcium in the absence of any added ATP, in so many different cell types (48, 50, 54 -58), as well as the observation that isolated vesicles (5,59) contain components sufficient to support membrane fusion with liposomes, suggests that the assembly and activation of fusion complexes on intracellular vesicle occurs prior to docking in many systems.