Reconstitution of Calcium-triggered Membrane Fusion Using
"Reserve" Granules*
Valery V.
Chestkov
§,
Sergey P.
Radko§,
Myoung-Soon
Cho§,
Andreas
Chrambach§, and
Steven S.
Vogel§¶
**
From the
Medical Genetics Center, Russian Academy of
Medical Sciences, Moscow, Russia, § Laboratory of
Cellular and Molecular Biophysics, NICHD and ¶ Synaptic Mechanisms
Section, NINDS, National Institutes of Health, Bethesda, Maryland
20892, and
Medical College of Georgia, Institute for Molecular
Medicine and Genetics, Augusta, Georgia 30912-2630
 |
ABSTRACT |
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.
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INTRODUCTION |
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 (7-9). 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-PAGE1 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.
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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-µm Nitex mesh and were washed
three times in sea water. Fertilization was achieved by mixing eggs and
diluted sperm (20:1, v/v in sea water) at a volume ratio of 50:1.
Homogenate Preparation--
Eggs were washed three times after
isolation with GEHB buffer (1 M glycine, 5 mM
EGTA, 5 mM HEPES titrated with Bis-Tris to pH 6.7)
supplemented with 1 mM benzamidine and 1 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid. Eggs were homogenized by six strokes of a Dounce homogenizer using the B pestle and kept at 4 °C prior to electrophoretic
separation.
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 × 100 × 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
MgCl2, 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 µM 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 µM
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 × 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 × 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 CaCl2, 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%
OsO4 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).
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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 ± 0.19-µm diameter (mean ± SD, n = 45) granules.
The second fraction, C, contained larger granules (1.48 ± 0.25 µm diameter, n = 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 ± 0.21 µm (mean ± S.D.,
n = 50). These were similar in size to the granules in
fraction A, but were distinct from those in fraction C.

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Fig. 1.
Fractionation of sea urchin egg
homogenates. 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 µm.
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To determine the origin of the components migrating in 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.

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Fig. 2.
SDS-PAGE analysis of free flow
electrophoresis fractions. Total egg homogenates and 6 ml of each
fraction from unfertilized and fertilized eggs were centrifuged at
3,000 × g for 30 min at 4 °C. Pellets were
resuspended in 100 µl of SDS-PAGE sample buffer, boiled for 2 min,
and stored at 20 °C until used. Samples (50 µl/lane) were loaded
onto SDS-PAGE gels prepared with 10% acrylamide and 0.27%
N,N -bisacrylamide as described previously (60).
Individual lanes for unfertilized (left panel) or fertilized
(right panel) eggs are: M, marker proteins;
H, egg homogenate; A, fraction constituting peak
A; B1-B3, three consecutive
fractions containing peak B;
C1-C3, three consecutive
fractions constituting peak C.
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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
Mg2+ (10 mM) but less than 1 µM
free calcium. We exposed fraction C granules isolated from either
unfertilized or fertilized eggs to PKME buffer containing 316 µM 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.

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Fig. 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 video-enhanced 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 µM free calcium to trigger fusion. Each perfusion step involved 500 µl of buffer, approximately 3.5 times the
chamber volume. Size bar is 10 µm.
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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 observed 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.

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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 = 0). Next a UV shutter was
opened to uncage calcium (+Ca), and the granules were imaged at 42 s (R18) and 57 s (DIC). Size bar is 5 µm.
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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 ± 10% (mean ± S.E.,
n = 12) fusion with calcium in PKME buffer, 44 ± 18% (n = 3) fusion with calcium in the presence of
ATP
S, and 43 ± 22% (n = 4) fusion with
calcium in the presence of GTP
S. Fusion was never observed in the
absence of calcium; exposure to mM concentrations of
Mg2+ 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 µm2 (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/µm2
we observed that 78.9 ± 4.5% (n = 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 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 µm 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 triggered fusion and indicate that >85% of
isolated fraction C granules are morphologically similar and at least
80% are fusion-competent.

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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 µM free calcium to trigger fusion. Size
bar is 10 µm.
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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 µM
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.
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Fig. 7.
Thin section electron microscopy of fraction
C granules. Top panel, thin section of an intact
unfertilized sea urchin egg. Abbreviations are: Y, yolk
granule; A, acidic vesicle. Black arrows indicate
cortical granules; white arrows indicate mitochondria. Bottom panel, thin section of fraction C granules isolated
from unfertilized eggs. Size bar is 2 µm.
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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 membrane 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 (35-37). 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 µM) 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.

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Fig. 8.
Lysophosphatidylcholine inhibits
calcium-triggered fusion. Fraction C granules from unfertilized
eggs were sequentially exposed to PKME buffer containing 100 µM lauroyl lysophosphatidylcholine (LPC; upper
left) and LPC with 316 µM calcium (upper
right). Next calcium was removed by perfusion with PKME containing
100 µM LPC, followed by the removal of LPC by perfusion
with PKME (lower left). Finally granules were challenged by
perfusion with PKME containing 316 µM calcium
(lower right). Size bar is 10 µm.
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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 µM) to visualize membrane sheets
(data not shown).

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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 µM 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 = 0 to initiate the release of calcium from the
"caged calcium" compound DM-nitrophen. The field was imaged by
fluorescence microscopy at t = 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 µm.
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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
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 ± 0.25 µm as opposed to the diameter of
cortical granules, 0.77 ± 0.21 µm, 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-µm 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 C1-C3) 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 adhesion
toposome complex binding the cells of the developing embryo between day
3 and 7 of development. Third, like predocked cortical granules (2-5),
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-dependent 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.
 |
ACKNOWLEDGEMENTS |
We thank P. Gallant, V. Matranga, M. Terasaki, K. Timmers and J. Zimmerberg for stimulating discussions;
Sven Beushausen, Paul Blank, Jens Coorssen, Elis Stanley and Jennifer
Lippincott-Schwartz for critically reading the manuscript.
 |
Note Added in Proof |
After our manuscript was submitted, a
paper was published arguing that a critical element for
calcium-dependent resealing of membrane disruptions in sea urchin eggs
is the homo- and heterotypic fusion of yolk granules (Terasaki, M.,
Miyake, K., and McNeal, P. L. (1997) J. Cell Biol. 139, 63-74).
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom all correspondence should be addressed: Medical College of
Georgia, Institute for Molecular Medicine and Genetics, 1120 15th St.
CB 2803, Augusta, GA 30912-2630. Tel.: 706-721-0713; Fax: 706-721-7915;
E-mail: stevev{at}immag.mcg.edu.
1
The abbreviations used are: PAGE, polyacrylamide
gel electrophoresis; ATP
S, adenosine
5
-3-O-(thio)triphosphate; GTP
S, guanosine 5
-3-O-(thio)triphosphate; PIPES,
piperazine-N,N
-bis(2-ethanesulfonic acid);
Bis-Tris,
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; NSF,
N-ethylmaleimide-sensitive factor.
 |
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