1 Department of Physiology, University College London, Gower Street, London WC1E
6BT, UK
2 Department of Anatomy and Developmental Biology, University College London,
Gower Street, London WC1E 6BT, UK
3 Endocrinology and Reproduction Research Branch, National Institutes of Health,
Bethesda, MD 20892, USA
* Author for correspondence (e-mail: j.carroll{at}ucl.ac.uk )
Accepted 5 March 2002
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Summary |
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Key words: Phosphatidylinositol 4,5-bisphosphate, Oocyte, GFP
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Introduction |
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Ins(1,4,5)P3 is generated via phospholipase C
(PLC)-mediated hydrolysis of PtdIns(4,5)P2
(Berridge, 1993). The mechanism
of PLC activation at fertilization is an active area of research and two main
models are emerging in which either sperm-derived PLCs (mammals) or
egg-derived PLCs (sea urchins/starfish) are activated (for reviews, see
Stricker, 1999
;
Jaffe et al., 2001
;
Carroll, 2001
). In mammals, PLC
activation and the resultant PtdIns(4,5)P2 hydrolysis and
Ins(1,4,5)P3 production must persist for several hours to
support the generation of long-lasting Ca2+ oscillations. Studies
in somatic cells suggest that the dynamics of
PtdIns(4,5)P2 hydrolysis and
Ins(1,4,5)P3 production that are required to bring about
Ca2+ oscillations involve a pulsatile generation of
Ins(1,4,5)P3 that initiates each Ca2+ spike
(Meyer and Stryer, 1988
;
Harootunian et al., 1991
;
Hirose et al., 1999
;
Nash et al., 2001
) or a
persistent low level of activation that sensitizes the
Ins(1,4,5)P3R to Ca2+-induced Ca2+
release (Wakui et al., 1989
;
Missiaen et al., 1991
;
Berridge, 1993
). In mouse eggs,
the finding that non-hydrolysable Ins(1,4,5)P3 analogues
(Sato et al., 1998
) and low
persistent levels of Ins(1,4,5)P3
(Jones and Nixon, 2000
) can
cause repetitive Ca2+ oscillations provides some support for the
latter of these two models.
To date, Ins(1,4,5)P3 and
PtdIns(4,5)P2 have been measured at fertilization in large
populations of sea urchin and Xenopus eggs using biochemical mass
assays. These studies have revealed that both Ins(1,4,5)P3
and its precursor PtdIns(4,5)P2 increase at the time of
fertilization (Turner et al.,
1984; Ciapa et al.,
1992
; Stith et al.,
1993
; Stith et al.,
1994
; Snow et al.,
1996
). This apparently counterintuitive result is explained by the
increase in PtdIns(4,5)P2 being a small difference in a
very large increase in the turnover of polyphosphoinositides (PPI)
(Ciapa et al., 1992
). Although
these biochemical studies provide insightful snapshots into the metabolism of
PPI at fertilization in sea urchins, it is not known precisely when
PtdIns(4,5)P2 changes start in relation to the
Ca2+ wave or whether a similar large increase in PPI turnover is
necessary to drive long-lasting Ca2+ oscillations in mammals.
In addition to PtdIns(4,5)P2 providing a source of
Ins(1,4,5)P3 for the release of Ca2+, recent
evidence suggests that PtdIns(4,5)P2 is also required
during exocytosis. In this role PtdIns(4,5)P2 acts as a
signaling molecule, rather than as a substrate for an enzyme, that can
interact directly with target proteins containing
PtdIns(4,5)P2-binding motifs. Acting in this mode, it has
been proposed that PtdIns(4,5)P2 is involved in a number
of steps in Ca2+-dependent exocytosis, including vesicle docking,
priming, fusion, actin remodeling and subsequent endocytosis
(Martin, 1998;
Martin, 2001
;
Cremona and De Camilli, 2001
).
Presumably, PtdIns(4,5)P2 also plays a role in the
Ca2+-dependent exocytosis of cortical granules that is stimulated
at fertilization (Xu et al.,
1994
; Abbott and Ducibella,
2001
). As such, PtdIns(4,5)P2 is required to
play multiple roles at fertilization. Frist, it is required for hydrolysis in
order to supply Ins(1,4,5)P3 and, as a consequence of the
Ins(1,4,5)P3-induced Ca2+ release, it is
required in a signaling role to participate in the exocytosis of cortical
granules. Clearly a tight regulation of PtdIns(4,5)P2 is
required at fertilization if it is to perform these dual, and competing
roles.
Recently, it has become possible to monitor
PtdIns(4,5)P2 in living cells using a GFP-fusion protein
(Stauffer et al., 1998;
Varnai and Balla, 1998
). The
indicator consists of the pleckstrin homology (PH) domain of PLC
1
coupled to GFP (PH-GFP). The PH domain has a high affinity for
PtdIns(4,5)P2 and localises to the plasma membrane,
consistent with the known distribution of PtdIns(4,5)P2 in
mammalian cells (Lemmon et al.,
1995
; Kavran et al.,
1998
; Balla et al.,
2000
). The fusion protein provides a dynamic measure of
PtdIns(4,5)P2 since activation of PLC and hydrolysis of
PtdIns(4,5)P2 leads to a redistribution of PH-GFP from the
plasma membrane to the cytosol (Stauffer
et al., 1998
; Varnai and
Balla, 1998
; van der Wal et
al., 2001
). In some cell types, high levels of
Ins(1,4,5)P3, which also has a high affinity for PH-GFP,
can compete with PtdIns(4,5)P2 for PH-GFP
(Hirose et al., 1999
;
Nash et al., 2001
). As such
there is some controversy as to whether PH-GFP reports
PtdIns(4,5)P2 or Ins(1,4,5)P3
(van der Wal et al., 2001
). In
this study, we use PH-GFP to report plasma membrane
PtdIns(4,5)P2 in mouse eggs at the time of fertilization.
We found no evidence for significant loss of plasma membrane
PtdIns(4,5)P2, rather a net increase that is dependent on
exocytosis of cortical granules.
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Materials and Methods |
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DNA construct and cRNA microinjection
The chimera encoding the PH domain of PLC1 fused to enhanced green
fluorescent protein (PH-GFP) was constructed as described previously
(Varnai and Balla, 1998
), and
cloned into pcDNA3.1, which contains a T7 primer enabling cRNA synthesis.
Plasmids were linearised after the 3'UTR with SmaI and in vitro
transcription was performed with T7 polymerase (Promega) according to the
manufacturer's protocol. The cRNA was purified by RNeasy column (Qiagen) and
polyadenylated as described previously
(Subramanian and Meyer, 1997
).
The polyadenylated cRNA was recovered by RNeasy column in Rnase-free water,
aliquoted and stored at -80°C. cRNA was diluted in injection buffer (120
mM KCl, 20 mM Hepes, pH 7.4) to a final concentration of 0.1-0.2 µg/µl,
and microinjected (estimated 2-5% of egg volume) using pressure-injection.
Imaging was started 2-3 hours after cRNA injection. Some oocytes were injected
with a cRNA encoding the same fusion-protein, carrying a point mutation (R40L)
in the PH domain on one critical basic residue involved in
PtdIns(4,5)P2 binding
(Varnai and Balla, 1998
).
Manipulation and treatment of oocytes
For [Ca2+]i measurements, oocytes were incubated in
M2 containing 10 µM fura-red-AM (Molecular Probes) at 37°C for 15
minutes. In wortmannin experiments, oocytes were preincubated with 30 µM
wortmannin (Calbiochem) for 15 minutes. This rather high concentration was
used in order to fully block type III phosphatidylinositol 4-kinase (PtdIns
4-kinase) activity in vivo using a short incubation time, and considering that
oocytes have a relatively low surface area/volume ratio compared with somatic
cells. For BAPTA experiments, oocytes were preincubated with 1 or 10 µM
BAPTA-AM (Molecular Probes) at 37°C for 15 minutes. Since 10 µM
BAPTA-AM abolished Ca2+ oscillations, fertilization was confirmed
at the end of the experiments by staining chromatin with Hoechst 33342 (see
below). For jasplakinolide experiments, oocytes were preincubated with 100 nM
Jasplakinolide (Molecular Probes) for 30-60 minutes. During the recordings,
Jasplakinolide was continuously present in the experimental chamber at the
optimal final concentration of 100 nM
(Terada et al., 2000). For
caged-Ins(1,4,5)P3 experiments, oocytes expressing PH-GFP
were further injected with NPE-caged Ins(1,4,5)P3
(Molecular Probes; 1 mM pipette concentration) 30 minutes prior to the imaging
(see below). Injections were 2-5% of egg volume (based on cytoplasmic
displacement) giving an estimated final concentration in the range of 20-50
µM. Some oocytes expressing PH-GFP were also injected with the
catalytically active light chain of Botulinum neurotoxin A (BoNT/A-LC, 5 µM
in the pipette). The final concentration of BoNT/A in the eggs is 100-250 nM,
based on the injection volume described above. BoNT/A-LC was kindly provided
by G. Schiavo (Imperial Cancer Research Fund, London, UK).
Confocal imaging and Ins(1,4,5)P3 uncaging
Zona-free oocytes were transferred to an experimental chamber seated in a
heating stage and were observed with a Zeiss LSM-510 laser-scanning microscope
(Carl Zeiss Inc.) with a 20x (0.75 NA) objective. In fertilization
experiments, confocal images were taken at 7 or 10 second intervals, as soon
as one sperm remained bound to the oocyte. Transmitted light images, GFP and
fura-red fluorescence images were acquired through the equator of the oocyte,
using the 488 nm line of an argon laser. GFP fluorescence from the PH-GFP
fusion protein was recorded through a BP505-530 emission filter. Fura-red
fluorescence and GFP were monitored in the same confocal slice, using a LP650
emission filter. For caged-Ins(1,4,5)P3 experiments,
oocytes were scanned simultaneously with the 488 nm line of the Argon laser
and the 364 nm line of a UV laser (1.5 mW). The intensity of UV illumination
was adjusted by setting the acusto-optical tunable filter (AOTF) to 0.5 or 5%
excitation, in order to photolyse low and high amounts of
caged-Ins(1,4,5)P3, respectively. For all experiments, the
488 nm laser power and pin-hole size were kept the same (confocal slices were
3.5 µm thick). For DNA staining, oocytes were incubated with 1 µg/ml
Hoechst 33342 for 15 minutes and images of the chromosomes were acquired with
UV laser scanning.
Confocal data analysis
Confocal images were analyzed using Metamorph (Universal Imaging). Plasma
membrane and cytoplasmic GFP fluorescence were measured by drawing regions
around the egg's perimeter and in the cytoplasm, respectively, and expressed
as arbitrary units. Fura-red fluorescence was measured in a cytoplasmic
circular region. The same regions were used for each individual frame of an
image series. Calculation of the ratio of membrane to cytosolic GFP
fluorescence and graphs displaying GFP or Fura-red fluorescence along time,
were obtained with Microsoft Excel 2000. Since Fura-red fluorescence decreases
when [Ca2+]i rises, changes in
[Ca2+]i were expressed as the negative value of the
change in Fura-red fluorescence relative to baseline fluorescence
(F/F), to obtain positive values for measurements of amplitude and rate
of rise.
Assay for exocytosis of cortical granules
Cortical granule exocytosis was detected on living eggs by the fluorescent
lectin-staining method (Lee et al.,
1988). Zona-free oocytes were fertilized in M2 by incubation with
sperm for 15 minutes at 37°C, then washed and left in M2 for 20 minutes.
Fertilized eggs were incubated with FITC- or TRITC-conjugated Lens
culinaris agglutinin (FITC/TRITC-LCA) for 3 minutes. Eggs were then
washed three times in M2 with gentle pipetting, and the pattern of
FITC/TRITC-LCA staining was observed by fluorescence microscopy using a
fluorescein or rhodamine filter set and a 40x objective. Images were
acquired with a CCD camera (Princeton) and analyzed with Metafluor (Universal
Imaging). Eggs displaying the typical punctate staining pattern on their
surface were considered to have released their cortical granules.
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Results |
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We next sought to verify that PH-GFP did not interfere with the early events of fertilization. Monitoring [Ca2+]i in PH-GFP-expressing eggs revealed that the fusion protein had no effect on the ability of the sperm to generate the typical series of Ca2+ transients (Fig. 2). In addition, exocytosis, as monitored by lectin staining, was similar in eggs expressing PH-GFP [87% labeled (13/15)] and in non-expressing controls [85% labeled (23/27)]. Thus, Ca2+ release and exocytosis proceeds normally in the presence of PH-GFP.
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To examine the spatial organization of the increase in PtdIns(4,5)P2, confocal scans along the animal-vegetal axis were collected during fertilization and analyzed to determine the relative changes in PtdIns(4,5)P2 in the plasma membrane of both poles. The fluorescence was lower in the animal pole at rest but there was no detectable increase at fertilization (Fig. 3A). By contrast, in the vegetal pole, a reliable increase in PtdIns(4,5)P2 was stimulated (Fig. 3A,B). The increase was reasonably uniform throughout the confocal slice of the vegetal hemisphere suggesting that it was not due to a highly localized increase at the site of sperm-egg fusion (Fig. 3B). Thus, the increase in PtdIns(4,5)P2 at fertilization is spatially and temporally organized.
|
An increase in [Ca2+]i is necessary and
sufficient for the increase in PtdIns(4,5)P2
The close association between the increases in
[Ca2+]i and PtdIns(4,5)P2 raises the
question whether Ca2+ is necessary and sufficient for the
stimulation of PtdIns(4,5)P2 synthesis. To investigate
whether an increase in [Ca2+]i is necessary, we used
BAPTA to buffer changes in [Ca2+]i. In eggs preincubated
with 10 µM BAPTA-AM for 15 minutes, fertilization-induced increases in
[Ca2+]i and PtdIns(4,5)P2 were
completely abolished (not shown; n=10). Further, slowing the kinetics
of Ca2+ release by using lower concentrations of BAPTA-AM (1 µM,
15 minutes) strongly attenuated the increase in
PtdIns(4,5)P2 (Fig.
4A; Table 1). These
experiments suggest a requirement for rapid changes in
[Ca2+]i to induce the increase in
PtdIns(4,5)P2 at fertilization.
|
To examine whether the Ca2+ increase is sufficient to stimulate PtdIns(4,5)P2 synthesis, we generated Ca2+ transients in unfertilized MII oocytes by UV-uncaging of Ins(1,4,5)P3. Uncaging low levels of Ins(1,4,5)P3 continuously by using a UV laser attenuated to 0.5% of maximum power, generated Ca2+ transients similar to those seen at fertilization (Fig. 4B, upper panel). During the first Ins(1,4,5)P3-induced Ca2+ transient, translocation of PH-GFP to the plasma membrane was observed, although at a significantly slower rate than seen at fertilization (Fig. 4B, upper panel; Table 1). The ability to drive an increase in PtdIns(4,5)P2 by triggering Ins(1,4,5)P3-induced Ca2+ release suggests a Ca2+-dependent process is controlling the increase in PtdIns(4,5)P2 at fertilization. The slower rate of rise indicates that fertilization provides a more effective stimulus for PtdIns(4,5)P2 synthesis, perhaps by invoking additional signaling pathways.
PH-GFP reports PtdIns(4,5)P2 rather than
Ins(1,4,5)P3 in mouse eggs
PH-GFP has a high affinity for Ins(1,4,5)P3 and has
previously been used as an Ins(1,4,5)P3 reporter
(Lemmon et al., 1995;
Hirose et al., 1999
;
Nash et al., 2001
). However,
the increase in plasma membrane PH-GFP fluorescence detected at fertilization
suggests that PH-GFP is reporting PtdIns(4,5)P2 and not
being influenced by fertilization-induced increases in
Ins(1,4,5)P3. To determine whether high levels of
Ins(1,4,5)P3 may compete with
PtdIns(4,5)P2 for binding PH-GFP, we released excess
Ins(1,4,5)P3 using a tenfold higher UV laser intensity
(attenuated to 5% of maximum power). The results show that excess
Ins(1,4,5)P3 causes reversible translocation of PH-GFP
from the plasma membrane to the cytoplasm
(Fig. 4B, lower panel). This
suggests that PH-GFP can report changes in Ins(1,4,5)P3
but, in mouse eggs, with a relatively low surface area-to-volume ratio, the
concentration of Ins(1,4,5)P3 generated at fertilization
is not sufficient to displace PH-GFP from the plasma membrane. This data,
consistent with a previous report (van der
Wal et al., 2001
), further validates our use of this probe as an
indicator of plasma membrane PtdIns(4,5)P2.
The increase in PtdIns(4,5)P2 is a result of
exocytosis
We have demonstrated that Ca2+ is necessary and sufficient for
the increase in PtdIns(4,5)P2, but how Ca2+
stimulates this increase is not known. A number of observations point to an
association with cortical granule exocytosis (CGE). First, the Ca2+
requirement for both processes is similar; second, cortical granules are
located in the vegetal pole, which is consistent with polarized
PtdIns(4,5)P2 synthesis; and third, the transient nature
of the PtdIns(4,5)P2 increase is consistent with the
kinetics of exocytosis in mammalian eggs
(Kline and Stewart-Savage,
1994). The relationship between PtdIns(4,5)P2
synthesis and exocytosis was investigated using actin filament-targeting drugs
known to inhibit CGE (Terada et al.,
2000
; Abbott and Ducibella,
2001
). In preliminary experiments, we noticed that the
fertilization-associated rise in PtdIns(4,5)P2 was
inhibited by cytochalasin B (n=5; not shown). The possibility that
actin filament disassembly by cytochalasin B may interfere with localization
of some elements of the PtdIns(4,5)P2 synthesis pathway
prompted us to investigate the effects of the actin filaments-stabilizing
reagent jasplakinolide. Jasplakinolide has recently been shown to inhibit
exocytosis in fertilized mouse eggs
(Terada et al., 2000
), a
finding we confirmed by monitoring CGE with lectin staining
(Fig. 5D). Exocytosis was found
to occur in only 15% (6/39) of fertilized eggs treated with jasplakinolide
(100 nM), compared with 85% of fertilized control eggs. In eggs treated with
jasplakinolide (100 nM) normal Ca2+ oscillations were generated;
however, the duration of the rise in PtdIns(4,5)P2 was
reduced to a small short-lived transient that finished well before the first
Ca2+ transient returned to baseline
(Table 1;
Fig. 5A). Changes in PH-GFP
localization were never observed for the subsequent Ca2+
transients, and one egg displayed no change in PH-GFP (not shown).
|
To interfere more specifically with exocytosis, eggs were injected with the
catalytically active light chain of Botulinum neurotoxin A (BoNT/A-LC; 5
µM). This toxin cleaves SNAP-25, which is a plasma membrane protein
essential for Ca2+-dependent exocytosis
(Xu et al., 1998) (for a
review, see Schiavo et al.,
2000
). In secretory cells, cleavage of SNAP-25 by BoNT/A slows the
kinetics of the Ca2+-triggered exocytic burst
(Xu et al., 1998
;
Schiavo et al., 2000
). BoNT/A
also cleaves SNAP-25 and inhibits exocytosis in mouse eggs
(Ikebuchi et al., 1998
). In
our hands exocytosis was detected in 47% (16/34) of fertilized eggs previously
injected with BoNT/A-LC compared with 85% of controls, which indicates a
partial inhibition of exocytosis. To examine the effect of this inhibition on
PtdIns(4,5)P2 synthesis, BoNT/A-LC was injected into eggs
expressing PH-GFP. At fertilization, Ca2+ transients were similar
to controls but the rate of rise of PtdIns(4,5)P2 was
threefold slower than controls and no obvious peak was detectable
(Table 1; Fig. 5B). Together, these data
show that interfering with exocytosis prevents the full development of the
rise in plasma membrane PtdIns(4,5)P2 (c.f.
Fig. 5C).
The increase in PtdIns(4,5)P2 is not stimulated
at fertilization of immature oocytes
At fertilization of immature oocytes it is known that Ca2+
oscillations are attenuated (Fujiwara et
al., 1993; Mehlmann and Kline,
1994
) (for a review, see
Carroll et al., 1996
) and that
they do not have the capacity to undergo Ca2+-activated exocytosis
(Abbott and Ducibella, 2001
).
Therefore, it was of interest to examine the PtdIns(4,5)P2
dynamics in immature oocytes. PH-GFP fluorescence was distributed evenly
around the plasma membrane (Fig.
6A). At fertilization, Ca2+ transients in immature
oocytes were smaller and did not persist for as long as those seen in mature
eggs (Fig. 6B). During these
increases in [Ca2+]i, there were no detectable changes
in the distribution of PH-GFP (Fig.
6B). This finding is consistent with exocytosis being required for
the increase in PtdIns(4,5)P2, although other
developmental events may also be required.
|
Micromolar wortmannin inhibits the PtdIns(4,5)P2
increase but not exocytosis
The increase in PH-GFP staining at fertilization may be a result
PtdIns(4,5)P2 synthesis or an increase in
PtdIns(4,5)P2 available for binding to PH-GFP. To
investigate the possibility that exocytosis increases
PtdIns(4,5)P2 synthesis we have pretreated the oocytes
with micromolar wortmannin (30 µM, 15 minutes), which inhibits the type III
PtdIns 4-kinase (Downing et al.,
1996). Treatment with wortmannin abolished the translocation of
PH-GFP to the plasma membrane in all eggs examined
(Fig. 7A). In four out of eight
eggs, small downward inflections in the PM/C ratio were evident, suggesting
that a limited hydrolysis of PtdIns(4,5)P2 was revealed
when synthesis was inhibited (Fig.
7A; Table 1). These
effects were not due to inhibition of PtdIns 3-kinase, as a low concentration
of wortmannin (100 nM) had no effect on PtdIns(4,5)P2
synthesis (n=3, not shown).
|
Finally, since PtdIns(4,5)P2 has been suggested to be
necessary for exocytosis (Martin,
1998; Martin,
2001
; Holz et al.,
2000
; Cremona and De Camilli,
2001
), we inhibited the PtdIns(4,5)P2 increase
with wortmannin and examined the effect on exocytosis using lectin staining.
The data show that wortmannin-treated eggs and controls fertilized in the
absence of wortmannin display a similar staining pattern. Thus wortmannin had
no effect on the exocytosis of cortical granules [80% eggs labeled (37/46);
Fig. 7B] suggesting that the
increase in PtdIns(4,5)P2 seen at fertilization is not
necessary for the fusion of cortical granules.
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Discussion |
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PH-GFP monitors plasma membrane PtdIns(4,5)P2 in
mouse eggs
The PH-GFP chimera selectively labeled the cortex of the eggs, suggesting
that it bound to the plasma membrane-associated pool of
PtdIns(4,5)P2, in agreement with previous studies
(Stauffer et al., 1998;
Varnai and Balla, 1998
;
Holz et al., 2000
;
Micheva et al., 2001
). In
fertilized eggs expressing PH-GFP, Ca2+ oscillations and CGE were
similar to that seen in control eggs, which suggests that expression of the
exogenous protein did not interfere with the physiological process of egg
activation or CGE. This is consistent with the finding that PH-GFP binding to
plasma membrane PtdIns(4,5)P2 is a dynamic process with
on-off rates in the order of seconds, allowing
PtdIns(4,5)P2 to remain available for binding other
proteins, such as PLC (Van der Wal et al.,
2001
). Together, these data demonstrate that PH-GFP can be used to
monitor PtdIns(4,5)P2 dynamics in the plasma membrane of
mouse eggs.
PH-GFP provided a reliable marker for the polarity of the egg, with a
reduced staining in the animal pole. This appears to reflect the absence of
microvilli in this region, which effectively reduces the amount of plasma
membrane available for PH-GFP labeling. This conclusion is supported by
observations that a plasma membrane marker FM 1-43 also shows a decreased
staining in the animal pole (G.H. and J.C., unpublished). In addition,
immature oocytes that are not polarized and have microvilli over the entire
plasma membrane stain evenly with PH-GFP. Our observations of PH-GFP
distribution provide no evidence for PtdIns(4,5)P2 on
intracellular membranes, although it is possible that intracellular pools of
PtdIns(4,5)P2 are not accessible to PH-GFP
(Balla et al., 2000).
Interestingly, eggs from Xenopus and sea urchin have
PtdIns(4,5)P2 in abundance in intracellular membranes
(Snow et al., 1996
;
Rice et al., 2000
). It remains
to be determined whether mammalian oocytes also have stores of
PtdIns(4,5)P2 in intracellular membranes.
The dynamics of PtdIns(4,5)P2 at fertilization:
the implications for signal transduction at fertilization
Imaging PH-GFP in single living oocytes during the latent period and the
first few Ca2+ waves did not reveal any loss of plasma membrane
PtdIns(4,5)P2. In fact the only detectable change in
plasma membrane PtdIns(4,5)P2 was an increase that follows
the first Ca2+ transient. This may seem surprising given the
requirement for PtdIns(4,5)P2 hydrolysis for
Ins(1,4,5)P3 production but it is consistent with
biochemical assays on populations of sea urchin and Xenopus eggs
(Turner et al., 1984;
Ciapa et al., 1992
;
Snow et al., 1996
). In sea
urchins, kinetic studies on PPI turnover have shown that the net increase in
PtdIns(4,5)P2 represents a small difference in a very
large increase in PPI turnover at fertilization
(Ciapa et al., 1992
). This
suggests that hydrolysis and synthesis are tightly coupled such that
hydrolysis is compensated for, or independently stimulated, at fertilization
of sea urchin eggs. Our experiments cannot inform on the rate of PPI turnover
but do provide the advantages that PtdIns(4,5)P2 dynamics
can be monitored simultaneously with Ca2+ in single eggs at a
relatively high temporal and spatial resolution. Thus we can be confident that
any fast changes just prior to or during the Ca2+ transient will
not go undetected due to the averaging effect of slight differences in timing
in a population of eggs.
The lack of any detectable loss of plasma membrane
PtdIns(4,5)P2 suggests two main models for
Ins(1,4,5)P3 production at fertilization in mammals. The
first involves plasma membrane PtdIns(4,5)P2 hydrolysis
that is rapidly compensated for by synthesis, as described above. Our data
also suggests that the hydrolysis of plasma membrane
PtdIns(4,5)P2 may be small, compared with the large PPI
turnover seen in sea urchins (Ciapa et
al., 1992). Inhibition of type III PtdIns 4-kinase with wortmannin
revealed only a limited and transient loss of PH-GFP from the plasma membrane
in about half the eggs examined. Thus, shifting the balance from synthesis to
hydrolysis, failed to reveal any significant loss of
PtdIns(4,5)P2 from the plasma membrane, which suggests
limited PtdIns(4,5)P2 hydrolysis. However, it is not known
to what extent the wortmannin-sensitive PtdIns 4-kinase contribute and it is
likely that PtdIns(4,5)P2 is also produced via
wortmannin-insensitive pathways. A limited hydrolysis of
PtdIns(4,5)P2 is also supported by the finding that low
tonic levels of Ins(1,4,5)P3, rather than larger
repetitive spikes of Ins(1,4,5)P3, provide the best mimic
for fertilization-induced Ca2+ transients in mouse eggs
[(Jones and Nixon, 2000
) and
this study]. In addition, we have shown that photoreleasing 20-50 µM
Ins(1,4,5)P3 can lead to the loss of PH-GFP from the
plasma membrane. This is consistent with the 10-100 µM
Ins(1,4,5)P3 required to displace PH-GFP in somatic cells
(van der Wal et al., 2001
) and
provides an upper limit on the amount of Ins(1,4,5)P3
generated at fertilization in mammals. Species differences in the mechanism of
sperm-induced Ins(1,4,5)P3 production may explain the
proposed difference in the extent of PPI turnover in mammals and sea urchins.
In mammals, a sperm-derived PLC is thought to be responsible for hydrolysing
PtdIns(4,5)P2 (Jones
et al., 2000
) (for a review, see
Carroll, 2001
) while in sea
urchins it is thought that the sperm activates egg PLCs
(Carroll et al., 1997
;
Shearer et al., 1999
;
Jaffe et al., 2001
). This
additional amplification step in the signaling pathway may lead to a high rate
of PtdIns(4,5)P2 hydrolysis in sea urchin eggs compared
with that provided by PLCs derived from a single sperm in mammals.
The second model suggests that plasma membrane
PtdIns(4,5)P2 is not the major source of
PtdIns(4,5)P2 used for Ins(1,4,5)P3
production at fertilization. In our studies we are addressing only plasma
membrane PtdIns(4,5)P2 (as discussed above), whereas in
sea urchins the experiments measure total cellular
PtdIns(4,5)P2. PtdIns(4,5)P2 on
intracellular membranes has been reported in Xenopus and sea urchin
eggs (Snow et al., 1996;
Rice et al., 2000
). It has not
been demonstrated that such intracellular pools of
PtdIns(4,5)P2 participate in Ca2+ signaling but
intracellular pools of PtdIns(4,5)P2 may be a
specialization required for large eggs where a Ca2+ wave propagates
through a large cytoplasmic volume
(Fontanilla and Nuccitelli,
1998
; Carroll et al.,
1994
; Deguchi et al.,
2000
). Hydrolysis of PtdIns(4,5)P2 on
intracellular membranes by Ca2+-dependent PLC
(Swann and Whitaker, 1986
;
Meyer and Stryer, 1988
), which
has been suggested to be provided by the fertilizing sperm
(Rice et al., 2000
), would
provide Ins(1,4,5)P3 at the wave-front to sustain
Ca2+ release tens or hundreds of microns from the plasma membrane.
Such a role for intracellular PtdIns(4,5)P2 remains to be
demonstrated but, at fertilization, where PLC is not thought to be activated
at the plasma membrane by a conventional receptor-coupled mechanism, it
remains a good possibility.
The increase in PtdIns(4,5)P2 is
Ca2+-dependent and related to cortical granule exocytosis
Our data show that increases in plasma membrane
PtdIns(4,5)P2 always outstrip any hydrolysis at
fertilization. This increase is strictly Ca2+ dependent such that a
Ca2+ increase is both necessary and sufficient for the increase in
PtdIns(4,5)P2. The more rapid kinetics of the increase at
fertilization compared with that of Ins(1,4,5)P3 suggests
that the fertilization Ca2+ transient is more effective or that the
sperm activates additional signaling pathways; protein kinase C is one obvious
possibility. Whatever the sperm does that stimulates the increase it is
short-lived, as the majority of the synthesis takes place during the first
Ca2+ transient.
One short-lived Ca2+-dependent event at fertilization is the
exocytosis of cortical granules (Kline and
Kline, 1992; Kline and
Stewart-Savage, 1994
). A strong connection between
PtdIns(4,5)P2 synthesis at fertilization and
Ca2+-triggered CGE is suggested using agents that inhibit
exocytosis. Three different agents that inhibit CGE in different ways, inhibit
the increase in PtdIns(4,5)P2 at fertilization while
having no effects on Ca2+ signalling. Jasplakinolide and
cytochalasin B prevent depolymerization and polymerization of actin,
respectively, both of which inhibit exocytosis in a number of systems,
including eggs (Terada et al.,
2000
; Abbott and Ducibella,
2001
). The third agent, BoNT/A, cleaves SNAP-25, a plasma membrane
protein that is important for Ca2+-activated exocytosis. BoNT/A
strongly decreased the rate of rise of PtdIns(4,5)P2,
which is consistent with its ability to slow down and partially inhibit the
exocytotic burst (Xu et al.,
1998
) (for a review, see
Schiavo et al., 2000
). The
connection between exocytosis and the increase in
PtdIns(4,5)P2 is also supported by a number of indirect
observations. First, both events have a similar Ca2+ dependence;
Ins(1,4,5)P3 stimulates both events
(Lee et al., 1988
;
Xu et al., 1994
;
Abbott and Ducibella, 2001
;
present study) while 1 µM BAPTA-AM is inhibitory
[(Kline and Kline, 1992
)
(present study)]. Second, the main burst of cortical granule exocytosis is
stimulated by the first Ca2+ transient
(Kline and Stewart-Savage,
1994
), which is also responsible for the increase in
PtdIns(4,5)P2 (present study). Third, in situations where
there is no Ca2+-stimulated exocytosis (GV stage oocytes and the
animal pole of MII eggs) there is no increase in
PtdIns(4,5)P2. Together, the Ca2+-dependency,
timing, pattern, duration and cortical location of
PtdIns(4,5)P2 increases are consistent with an involvement
of CGE in the PtdIns(4,5)P2 increase.
The mechanism by which CGE leads to an apparent increase in plasma membrane
PtdIns(4,5)P2 is not known. Inhibition of the increase by
wortmannin suggests that de novo PtdIns(4,5)P2 synthesis
is responsible. This is strengthened by the fact that CGE was not inhibited by
wortmannin treatment, suggesting that the PtdIns(4,5)P2
increase was not simply due to an increase in plasma membrane. An alternative
explanation is that exocytosis leads to the addition of
PtdIns(4,5)P2 to the plasma membrane that was previously
unavailable to PH-GFP (Balla et al.,
2000). However, the unmasking of such
PtdIns(4,5)P2 binding sites would need to be inhibited by
wortmannin. Taken together our data suggest that CGE stimulates an increase in
plasma membrane PtdIns(4,5)P2, perhaps by supplying
substrate or enzymes that promote PtdIns(4,5)P2
synthesis.
A role for the transient increase in plasma membrane
PtdIns(4,5)P2 at CGE?
Preventing the increase in PtdIns(4,5)P2 with
inhibitors of exocytosis did not inhibit the generation of Ca2+
transients at fertilization. This suggests that the increase is not necessary
for sustaining PtdIns(4,5)P2 levels for
Ins(1,4,5)P3 production. The increase in plasma membrane
PtdIns(4,5)P2 is also not apparently necessary for
exocytosis, since wortmannin inhibited the increase in
PtdIns(4,5)P2 without any obvious effect on exocytosis. A
role for PtdIns(4,5)P2 in the early,
Ca2+-independent, priming and docking steps of exocytosis has been
suggested by studies showing that exocytosis can be recovered in permeabilised
cells by providing PtdIns(4)P 5-kinase during the priming step
(Martin, 1998). By monitoring
Ca2+ and PtdIns(4,5)P2 simultaneously, it is
clear from our studies that the increase in PtdIns(4,5)P2
is after the Ca2+ rise. This suggests that the
PtdIns(4,5)P2 increase is not necessary for priming,
although basal PtdIns(4,5)P2 may be. We suggest that the
increase in PtdIns(4,5)P2 is a consequence, rather than a
cause of, exocytosis and that the role of the increase is downstream of
membrane fusion. Remodelling the actin cytoskeleton
(Sechi and Wehland, 2000
) and
endocytosis for membrane retrieval after exocytosis are known to occur after
fertilization (Kline and Stewart-Savage,
1994
; Bement et al.,
2000
; Smith et al.,
2000
; Lee et al.,
2001
) and it is here that PtdIns(4,5)P2
synthesis may play a role.
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Acknowledgments |
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