Bioscience Reports, Vol. 8, No. 4, 1988
Exocytosis Reconstituted from the Sea
Urchin Egg is Unaffected by Calcium
Pretreatment of Granules and Plasma
Membrane
Tim Whalley and Michael Whitaker ~
Received March 28, 1988
Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by
recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The
isolated cortical granules are unaffected by calcium concentrations up to i raM, nor do granule
aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and
cortical granules can be pretreated with 1 mMCa before reconstitution without affecting the
subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations.
On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar
calcium provided that one of their number is in contact with the plasma membrane. If exocytosis
involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated
production of a fusogen can occur only when contiguity exists between cortical granules and plasma
membrane. They also suggest that a substance involved in exocytosis can diffuse and cause piggy-back
fusion of secretory granules that are in contact with the plasma membrane. Our results are also
consistent with a scheme in which calcium ions cause a reversible, allosteric activation of an exocytotic
protein.
KEY WORDS: calcium; exocytosis; sea urchin egg; phosphoinositide; diacylglycerol.
INTRODUCTION
E x o c y t o s i s is an e s s e n t i a l c e l l u l a r m e c h a n i s m for t h e e x p o r t o f s e c r e t o r y p r o d u c t s .
M u c h is k n o w n a b o u t t h e c o n t r o l o f e x o c y t o s i s at t h e level o f s e c o n d m e s s e n g e r s
w h e r e i n t r a c e l l u l a r c a l c i u m [Cai] (1), p o l y p h o s p h o i n o s i t i d e s ( 2 - 5 ) , a n d G T P
b i n d i n g p r o t e i n s (6) h a v e r e g u l a t o r y roles. C e r t a i n o t h e r p r o t e i n s m a y also b e
i m p o r t a n t in e x o c y t o s i s . S o m e p r o t e i n s a s s o c i a t e d w i t h s e c r e t o r y g r a n u l e s a r e
f u s o g e n i c at m i c r o m o l a r c a l c i u m c o n c e n t r a t i o n s (7). C a l c i u m b i n d i n g p r o t e i n s
such as c a l m o d u l i n m a y h a v e a r o l e in t h e c a l c i u m t r a n s d u c i n g p a t h w a y (8). It has
also b e e n s u g g e s t e d t h a t c y t o s k e l e t a l p r o t e i n s a r e i m p o r t a n t in t h e fusion
m e c h a n i s m (9). H o w e v e r , e l u c i d a t i o n o f t h e m o l e c u l a r m e c h a n i s m o f m e m b r a n e
Department of Physiology, University College London, Gower Street, London WCIE 6BT, UK.
i To whom correspondence should be addressed.
335
0144-8463/8810800-0335506.00/0(~ 1988PlenumPublishingCorporation
336
Whalley and Whitaker
fusion has proved difficult because of the problems of studying this process
in vitro.
In the sea urchin egg, cortical secretory granules are attached to the inner face
of the plasma membrane. Exocytosis occurs at fertilization in response to an
increase in cytoplasmic free calcium ion concentration (10). Plasma membrane
can be isolated with the cortical granules still attached and exocytosis will occur in
vitro when 1-3/~MCa is added (11, 12). Exocytosis is easy to follow in this
system because there is an obvious morphological change in response to calcium:
the cortical granules (1 #m diameter) can be observed in the light microscope and
disappear when they fuse with the plasma membrane (13). Hydrolysis of
membrane polyphosphoinositides (PPI) occurs at fertilization (14) and it has been
suggested that the diacylglycerol produced is a fusogen which promotes exocytosis
(15, 16). In the isolated cortex, PPI hydrolysis is stimulated at calcium ion
concentrations which cause exocytosis (16). Using this in vitro preparation it has
been shown that exocytosis is insensitive to microtubule and microfilament
inhibitors (13). Calcium appears to be the sole physiological trigger of exocytosis,
a simpler situation than in other exocytotic systems with multiple second
messenger controls (2-5). Neither protein kinase C nor GTP binding proteins
play a modulatory role since exocytosis is unaffected by TPA or DAG (15) and
the reported effects of guanine nucleotides (18) are due to Ca mobilisation rather
than a direct effect upon the exocytotic apparatus (15, 17).
The reconstitution of exocytosis in sea urchin eggs was achieved by Crabb
and Jackson (18). Reconstitution is a very useful approach that allows manipulation of individual components of the system and provides a way of investigating
the molecular mechanism of exocytosis. The aim of the present work was to
define the role of calcium ions in the fusion event by determining the effects of
treating the two isolated components (secretory granules and plasma membrane)
with calcium. We have used slightly different methods to achieve an in vitro
reconstitution and have studied the effects of calcium ion treatment of each
component of the system on the subsequent responsiveness of the reconstituted
system to calcium ions.
MATERIALS AND METHODS
Handling of Eggs
Eggs of Lytechinus pictus were obtained by intracoelomic injection of
0.5 M KCI. The jelly coat was removed by passage through Nitex mesh (27). Eggs
were maintained at 16~
Preparation of Cortical and Plasma Membrane Lawns
Eggs were attached to glass slides or coverslips pretreated with 0.050.1 mg/ml poly-L-lysine and were rinsed gently with intracellular medium (IM:
220 mM potassium glutamate, 500 mM glycine, 10 mM sodium chloride, 2.5 mM
ExocytosisReconstituted from Sea Urchin Eggs
337
magnesium chloride, 2.5 mM adenosine 5'-triphosphate, 10 mM EGTA, pH 6.7)
or PKME medium (50 mM PIPES, 425 mM KC1, 2.5 mM magnesium chloride,
2.5 mM adenosine 5'-triphosphate, 10mM EGTA, pH6.7; ref. 18). The eggs
were sheared with a jet of medium leaving isolated cortical fragments attached to
the glass. Plasma membrane lawns were made by isolating cortices as above and
then shearing with a more forceful jet of medium which removed more than 95%
of the cortical granules but left the plasma membrane intact.
Preparation of Cortical Granules
Eggs were attached to glass petri dishes pretreated with I mg/ml poly-Llysine and were gently rinsed several times with IM or PKME. Cortical lawns
were prepared as described above and were exhaustively washed until free of
cytoplasmic contaminants. The cortical granules were removed by shearing the
cortical lawns with IM or K E A (450 mM potassium chloride, 5 mM EGTA,
50mM ammonium chloride, pH9.1; ref. 18). The isolation of granules was
accompanied by a clearing of the translucent cortical lawn preparation which
proved a convenient measure of the extent of granule detachment. An examination of the granule preparation by differential interference contrast microscopy
indicated that the suspension consisted mainly of monomeric granules but
aggregates were also present (10% by number). Granules prepared in PKME
were neutralised to pH 6.7 by the addition of 1 M PIPES immediately before
reconstitution.
Preparation of Reconstituted Cortical Lawns
A perfusion chamber was constructed by pressing two strips of PTFE tape
onto the edges of a glass coverslip. The centre of the slide was coated with
0.1 mg/ml poly-L-lysine, rinsed, and a slurry of eggs was applied and allowed to
settle for 2 minutes. Plasma membrane lawns were then prepared. A No. 1
coverslip was pressed on to the PTFE supports to complete the perfusion
chamber. A suspension of isolated cortical granules was introduced into the
chamber by capillary action and these were allowed to settle for 10-20 minutes.
Unbound cortical granules were removed by perfusing the chamber with three
washes of the appropriate medium. Perfusion of the preparation was achieved by
placing filter paper strips at one end of the chamber.
Assessment of the Extent of Exocytosis
Exocytosis from cortical fragments attached to glass coverslips with poty-Llysine was assessed by measuring the decrease in intensity of scattered light from
a dark field image as previously described (19, 20). The extent of exocytosis in
reconstituted lawns was determined by counting the number of unfused and fused
cortical granules in a field containing three plasma membrane lawns before and
after the addition of a particular calcium-containing solution.
338
Whalley and Whitaker
Calcium Containing Solutions
Calcium-EGTA buffers were used in experiments on exocytosis in isolated
cortical fragments. IM contained 1 0 m M E G T A and various total calcium
concentrations. The free Ca was calculated using the constants of Martell and
Smith (21). The calcium-EGTA ratios and free Ca were 0.528, 1.78/~M; 0.715,
3.98/~M; 0.877, 11.2/~M; 0.914, 16.6/tM; 0.943, 25.1/tM. For exocytosis in
reconstituted lawns sufficient 100 mM CaCI2 in IM or PKME was added to IM or
PKME to give the desired final free Ca. Calcium concentrations were routinely
measured with a calcium sensitive electrode (WPI, Inc., New Haven, CT, USA),
and were adjusted to pH 6.7.
Calcium Pretreatment of Cortical Granules
Cortical granules used for pretreatment experiments were prepared as
described previously, except that the medium contained 2 mM EGTA. The pH
was adjusted to 6.7 and 100 mM CaC12 in PKM (PKME lacking EGTA) was
added to give a final calcium concentration of 1 mM. The granules were incubated
for 5 minutes in 1 mM calcium followed by the addition of sufficient 100 mM
E G T A in PKM to give a final E G T A concentration of 10 mM. The pH was
adjusted to 6.7 by the addition of 1 M KOH. Cortical granules were used as soon
as this treatment was completed.
RESULTS
Reconstitution of an Exocytotically Competent System
Plasma membrane lawns were prepared by dislodging the cortical granules
with a jet of buffer. When isolated cortical granules (prepared as described in
Materials and Methods) were added back to these isolated lawns, both monomeric and aggregated granules adhered. Typically, some 30-60 monomers o r
aggregates settled on each plasma membrane fragment (surface area: 4500/2m2).
Figure 1 illustrates a typical experiment. A plasma membrane fragment from
which the cortical granules have been removed is shown. Some cortical granules
remain attached to the lawn (A). A suspension of isolated cortical granules was
added. These were allowed to settle for 10 minutes and unattached granules were
removed by washing. Both monomers (&), dimers (O), and oligomers ( ~ ) are
seen attached to the plasma membrane. Granules have also attached themselves
to the coverslip ( 0 ) - Fewer cortical granules adhere to the coverslip than to the
plasma membrane (Table 1). When 100 #M Ca in IM is added to the reconstituted lawn, the granule monomers, dimers, and oligomers attached to the plasma
membrane disappear: they have undergone exocytosis (18). We have occasionally
observed that a string of granules attached to the plasma membrane by only one
of their number will all undergo exocytosis when treated with calcium. The
cortical granules attached to the coverslip have not changed in their appearance,
indicating that observed change is due to a PM/CG interaction.
Exocytosis Reconstituted from Sea Urchin Eggs
339
Fig. 1. Reconstitution of exocytosis in vitro in sea urchin eggs. Top left: Plasma membrane
fragments attached to a glass coverslip with polylysine. A few cortical granules remain attached (A).
(]entre: The same fragments after the addition of a suspension of isolated cortical granules allowed to
settle for 10 minutes. Granule aggregates are present ( ~ ) and a number of granules have settled onto
the glass (4~). Top right: After the addition of 100/zM calcium in IM. The granules attached to the
plasma membrane have undergone exocytosis; the granules attached to the glass are unaffected.
Differential interference contrast optics. Scale bar is 10/~m.
Calcium Sensitivity of Exocytosis
We investigated the differences in the Ca sensitivities of cortical lawns and
preparations reconstituted in different media. Figure 2 shows the effects of
different Ca concentrations on secretion from native cortical fragments and from
reconstituted lawns (RL) prepared in IM or PKME. Both of the reconstituted
100
75.
s
/
5o-
|
fo
./ j
0
A~
6-0
5,0
A
4-0
3.0
pCa
Fig. 2. A comparison of in vitro exocytosis in native and reconstituted preparations. Reconstituted
exocytosis in IM (11) is 10 fold less sensitive to calcium than native in vitro exocytosis (0) and 100 fold
less sensitive in PKME medium (&).
340
WhaUey and Whitaker
Table 1.
Binding of cortical Secretory granules
under various conditions
Experimental
condition
Number of CG per*
1000 #m 2
(a) PM lawns
Control
PM pretreated
with 10 mM Ca 2+
CG pretreated
with 10 mM Ca 2+
PM & CG pretreated
with 10 mM Ca 2+
7.5 • 0.82"
8.1 • 0.51
8.2 • 1.3
7.4 • 0.49
(b ) Polylysine-treated coverslip
Control
2.0•
* Mean & SEM are shown (n = 6, excepta : n = 12).
systems have lost some sensitivity towards Ca. The calcium required for half
maximal stimulation of exocytosis in cortical fragments was 6.3/~M; it was 25/~M
for IM prepared RL's and 250 #M for RL's prepared in PKME.
The Effect of Calcium Pretreatments
We investigated the effects of calcium pretreatment on the subsequent
efficacy of reconstitution. Either cortical granules, plasma membrane, or both
were pretreated for 10 min with 1 mM calcium. Calcium was by the addition of
J.
1 0 0 84
75"
,9
~
u
xoso
4,l
2 5 84
II
5.0
4.0
3"0
pCa
Fig. 3. Treating either plasma membrane, cortical granules or both with 1 mM calcium prior to
reconstitution does not affect the subsequent sensitivity to calcium. The experiment was performed in
PKME medium. Control ( 0 ) ; calcium pre-treated plasma membrane (O); calcium pre-treated cortical
granules (11); both calcium pre-treated (lk). All show half maximal exocytosis at 250/~M calcium and
100% exocytosis at 1 mM calcium.
ExocytosisReconstituted from Sea Urchin Eggs
341
E G T A and the pre-treated granules and plasma membrane were reconstituted.
The number of cortical granules bound to plasma membrane fragments was the
same as controls (Table 1). Pretreated granules and plasma membrane also
underwent calcium stimulated exocytosis. Figure 3 illustrates that none of the
calcium pretreatment procedures affected the secretory response of the reconstituted system. In these experiments, where preparation was performed in PKME
and K E A buffers, neither the Cas0~ nor the calcium required for 100%
exocytosis is affected. We also found that if cortical granules were added to the
perfusion chamber in the presence of i mM calcium, they spontaneously fused
immediately they contacted plasma membrane fragments.
DISCUSSION
Calcium-Sensitivity of the Reconstituted Exocytosis
We have found that the reconstituted system is not as sensitive to calcium in
vitro as the undissociated cortical lawn preparation, a result that has previously
been reported (18). We found that reconstitutions in IM were an order of
magnitude more sensitive to calcium than reconstitutions in PKME. We might
attribute this to the fact that IM is considerably less chaotropic than PKME which
contains 450 mM KC1. Anions such as C1- are known to cause solubilisation of
proteins (22). This result suggests that labile protein factors may be responsible
for the loss of sensitivity to calcium. Sasaki (23) has reported that if the cortical
lawns of Hemicentrotus pulcherrimus eggs are treated with KC1, there is a ten-fold
decrease in the calcium sensitivity. He showed that a heat labile protein of
approximately 100kD could restore the calcium sensitivity of KC1 treated
cortices. Loss of a similar protein may explain our observation, though we have
not succeeded in restoring the calcium sensitivity with the application of
cytoplasmic extracts.
Specificity of Calcium-Induced Granule Discharge
Secretory granules that contacted the plasma membrane underwent fusion,
whereas those that settled on glass did not undergo any morphological change.
This indicates that calcium-stimulated granule discharge can only occur when
there is a juxtaposition of the cortical granules and plasma membrane. Nor do
granule aggregates undergo any calcium-induced lysis of fusion when in suspensJ[on or attached to glass surface. This suggests to us that the isolated secretory
granules are free of contaminating plasma membrane. It is therefore unlikely that
the granules pull off tiny patches of plasma membrane with them as they
dissociate from the plasma membrane during the isolation step.
Calcium Pretreatment
Our experiments indicate that secretory granules are unaffected by calcium
concentrations two orders of magnitude greater than those required to cause
342
Whalley and Whitaker
exocytosis. There are no obvious morphological or functional differences between
calcium-treated and untreated granules in the reconstituted preparation. Nor does
calcium pretreatment of the plasma membrane before reconstitution affect the
subsequent exocytosis. If the processes underlying exocytosis include calciumdependent enzyme-substrate interactions, these results suggest that enzyme and
substrate can interact only when the secretory granule and the plasma membrane
are in contact: it seems to us highly unlikely that such a substrate could otherwise
escape depletion during calcium-pretreatment of granules or plasma membrane.
An equally plausible alternative is that calcium ions act reversibly at an allosteric
site (24) on a calcium sensitive fusion protein (25).
Generation of Lipid Fusogens
One hypothesis explaining the role of calcium in exocytosis in sea urchin eggs
is that calcium activates phosphoinositidase C (PIC), and leads to elevated levels
of diacylglycerol (DAG). DAG is fusogenic (26-28). Calcium causes polyphosphoinositide turnover in isolated cortices at concentrations which cause exocytosis
(16), and at fertilization the increase in D A G levels follows the same time course
as the Ca transient and exocytosis (14). Neomycin--an aminoglycoside which
binds to phosphatidylinositol bisphosphate (PtdlnsP2)--inhibits both exocytosis
and PtdlnsP2 hydrolysis with the same concentration dependence (15, 20). On
this hypothesis, contiguity between the plasma membrane and cortical granules is
necessary in order that PIC have access to its substrate, PtdlnsP2 being located in
the plasma membrane and PIC in the granule membrane, for example.
Our observation that all the granules of an aggregate will undergo mutual
fusion when only one of the granules comprising the aggregate is in contact with
the plasma membrane is a strong indication that a diffusible fusogen is generated
by calcium only when the secretory granule is in contact with the plasma
membrane. This sort of behaviour is not without precedent. Sea urchin eggs and
mast cells are ceils that undergo a rapid and concerted exocytosis. In both these
cell types, piggy-back exocytosis in which granule fuses with granule has been
reported (29, 30). It is entirely possible that this diffusible substance is DAG. The
reconstituted system can be used to test this idea.
ACKNOWLEDGEMENTS
This work was supported by funds from the Science and Engineering
Research Council and the Wellcome Trust. TW is an SERC Scholar.
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