(Received for publication, December 2, 1994; and in revised form, January 16, 1995)
From the
The free nucleoplasmic Ca concentration
([Ca
]
) may regulate
many nuclear events, such as gene transcription. Since the nucleus may
possess the enzymes necessary to generate the second messenger inositol
1,4,5-trisphosphate (Ins(1,4,5)P
), and because the nuclear
envelope may enclose an Ins(1,4,5)P
-releasable
Ca
store, we tested the hypothesis that nuclear
and/or cytosolic levels of Ins(1,4,5)P
can control
[Ca
]
. To assay
[Ca
]
, we measured the
fluorescence of the Ca
indicator fluo 3 in the
nucleus of Xenopus oocytes by confocal microscopy. When we
injected Ins(1,4,5)P
into the cytosol,
[Ca
]
increased. This
increase in [Ca
]
still
occurred when heparin was present in the nucleus, but was abolished
when heparin was present in the cytosol, indicating that cytosolic
Ins(1,4,5)P
levels could control
[Ca
]
. When we injected
Ins(1,4,5)P
directly into the nucleus,
[Ca
]
increased, even
when heparin was present in the cytosol, indicating that
Ins(1,4,5)P
could control
[Ca
]
from within the
nucleus. These results provide functional evidence for
Ins(1,4,5)P
receptors facing the nucleoplasm and raise the
possibility that a phosphoinositide cycle situated at the nuclear
membranes can control Ca
-dependent nuclear functions.
Despite an increasing recognition that Ca may
participate in the regulation of such key nuclear events as DNA
replication and gene transcription(1) , little is known about
how cells control their free nucleoplasmic Ca
concentration
([Ca
]
). (
)Because large pores cross the nuclear membranes, it has
long been assumed that Ca
ions can freely diffuse
from the cytosol into the nucleus, and that
[Ca
]
passively
reflects the free cytosolic Ca
concentration
([Ca
]
). However,
recent reports(2, 3, 4, 5, 6, 7, 8, 9) have
suggested that Ca
concentration gradients can exist
between the nucleoplasm and the cytosol, and that
[Ca
]
can be regulated
independently from
[Ca
]
.
The second
messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P) is a
likely candidate to regulate
[Ca
]
. In a wide
variety of cells, Ins(1,4,5)P
binds to high affinity
receptors that form Ca
channels in the membrane of
organelles that store intracellular Ca
. When
Ins(1,4,5)P
binds to its receptor, the Ca
channel opens and Ca
flows from the stores into
the cytosol(10) . If cytosolic Ca
diffused
into the nucleus, then Ins(1,4,5)P
could regulate
[Ca
]
by controlling
[Ca
]
. However,
Ins(1,4,5)P
could also regulate
[Ca
]
independently
from [Ca
]
.
The
suggestion that [Ca]
might be regulated independently from
[Ca
]
is supported both
by the structure of the nuclear envelope and by the presence therein of
proteins that regulate Ca
concentration. The nuclear
envelope consists of an inner and an outer membrane separated by the
perinuclear space. The perinuclear space is continuous with the lumen
of the endoplasmic reticulum(1) , which contains the
Ins(1,4,5)P
-sensitive Ca
stores in most
cells(10) . The nuclear membranes possess a Ca
ATPase (11, 12) , which could serve to
accumulate Ca
within the perinuclear space. The
nuclear membranes also contain Ins(1,4,5)P
receptors(13, 14, 15) . Stimulation of
these receptors might allow Ca
to be released from
the perinuclear space. If some of these Ins(1,4,5)P
receptors were located on the inner nuclear membrane, then an
increase in the nucleoplasmic concentration of Ins(1,4,5)P
would trigger the release of Ca
from the
perinuclear space directly into the nucleus. Localization to the
nucleus of the enzymes necessary to produce phosphatidylinositol
4,5-bisphosphate (16, 17) and of the enzyme that
hydrolyzes phosphatidylinositol 4,5-bisphosphate to Ins(1,4,5)P
(17, 18, 19, 20) supports the
notion that Ins(1,4,5)P
levels could indeed increase within
the nucleoplasm.
We used the Xenopus oocyte model to test
the hypothesis that Ins(1,4,5)P regulates
[Ca
]
both indirectly
by increasing [Ca
]
,
and directly by triggering the discharge of Ca
from
the perinuclear space into the nucleoplasm.
We obtained Xenopus oocytes as described previously (21) . Injection pipettes were backfilled with: fluo 3 (1
mM) (Molecular Probes); Ca green dextran (1
mM) (Molecular Probes); Ins(1,4,5)P
(10
to 10
M)
(Calbiochem); a mixture of fluo 3 (1 mM) and
Ins(1,4,5)P
; heparin (mass = 13.3-15 kDa,
0.1-30 mg/ml) (Calbiochem); or CaCl
(50
µM). Cytosolic microinjections were performed as described
before(21) . Microinjected volume was kept at 0.7 nl. We used
non-pigmented oocytes from albino frogs because their nuclei can be
microinjected under direct vision. To determine if we could accurately
target intracellular injections to the nucleus or to the cytosol, we
used the PMT2-SEAP vector (Genetics Institute, Cambridge, MA). This
plasmid directs the expression of a secretable alkaline phosphatase,
but only does so when delivered to the cell nucleus(22) . When
we injected the PMT2-SEAP plasmid into the nucleus, 91% of the oocytes
secreted alkaline phosphatase (n = 570 over 13 trials).
Conversely, when we injected the plasmid deliberately into the
perinuclear cytosol, none of the oocytes secreted alkaline phosphatase (n = 110 over 11 trials). These data indicate that our
microinjections could accurately target or avoid the nucleus.
To
measure [Ca]
, we
injected the fluorescent Ca
indicator fluo 3 into an
oocyte nucleus. Fluorescence was visualized using a laser scanning
confocal microscope (Bio-Rad, MRC-600) as described
previously(23) . Under unstimulated conditions, intranuclear
injection of fluo 3 resulted in a circular intracellular area where
fluorescence was increased above background (Fig. 1, closed
arrows). Because of the plasma membrane autofluorescence, we could
also observe the external outline of the cell (Fig. 1, open
arrows). As we focused deeper into the oocyte, the diameter of the
cellular outline increased progressively. In contrast, the diameter of
the circular fluo 3-filled area initially increased, but then
decreased, indicating that we were optically sectioning through the
spherical nucleus. For all experiments, we chose the confocal plane
showing the largest nuclear diameter (i.e. crossing the center
of the nucleus) and acquired sequential images (8-bit pixel depth, 254
169 pixels) from that plane every 2 s. Images obtained from
typical experiments are shown in Fig. 1. Because the nuclear
diameter is around 300-400 µm(24) , the
15-µm-thick confocal plane (thickness measured with the tilted
mirror technique, Bio-Rad Technical Bulletin 101) excluded the cytosol
situated either superficially or deep to the nucleus. The diameter of
the nuclear cross-section did not increase during the 5-min time course
of a typical experiment (for example, in Fig. 1, compare the
first and last image of each series) indicating that the dye did not
leak significantly into the cytosol over this short period of time.
Nevertheless, we considered the possibility that the fluorescent signal
at the edge of the nuclear cross-section could originate from
fluorescent dye that had crossed the nuclear membrane and was therefore
in the cytosol. To avoid measuring changes in
[Ca
]
, we excluded the
periphery of the nucleus from our analysis, and only measured the
fluorescence values within a 50-µm radius from the nuclear center.
We also performed some experiments with dextran-conjugated calcium
green instead of fluo 3. Because of its large size (mass = 70
kDa), the dextran conjugate should not allow the fluorescent dye to
cross the nuclear pores. Experiments performed with this dye produced
results similar to those obtained with fluo 3. Data was analyzed with
IMAGE version 1.56 software (National Institutes of Health).
Significance in the fluorescence changes was established using
Student's t test for paired data. Because neither fluo 3
nor Ca
green is a ratioing dye, the magnitude of the
fluorescence changes depends upon the dye concentration, and therefore
cannot be compared between different experiments.
Figure 1:
Confocal optical sections through the
animal pole of Xenopus oocytes; each row represents a
different experiment. For each experiment, images were acquired every 2
s, and three representative images are shown for each experiment.
Fluorescence increases from blue to magenta, to red, and to yellow. On all of the images, the nucleus (closed arrows) fluoresces because it has been
injected with fluo 3. In contrast, the cytosol (cyt)
that surrounds the nucleus does not fluoresce. The plasma membrane
autofluorescence defines the external outline of the cell (open arrows). Field width is 1.26 mm for all of the
panels. A, cytosolic injections of Ins(1,4,5)P (panels 1-9) or Ca
(panels10-12). Injection of Ins(1,4,5)P
(10
M) into the cytosol transiently
increased nuclear fluorescence (panels1-3,
times = 0, 100, and 276 s), even when heparin (10 mg/ml in
pipette) was preinjected into the nucleus (panels 4-6,
times = 0, 22, and 80 s). Cytosolic Ins(1,4,5)P
did
not increase [Ca
]
when
heparin was preinjected in the cytosol (panels 7-9,
times = 0, 36, and 66 s). An injection of CaCl
also
transiently increased nuclear fluorescence (panels
10-12, times = 0, 72, and 198 s). B, nuclear injections of Ins(1,4,5)P
. Injection of
Ins(1,4,5)P
into the nucleus increased nuclear fluorescence
despite the presence of heparin in the cytosol (panels1-3, times = 0, 60, and 170 s). Contrast
this experiment to that where Ins(1,4,5)P
and heparin were
both injected in the cytosol (Fig. 1A, panels
7-9). When heparin was injected in the nucleus, a nuclear
Ins(1,4,5)P
injection failed to increase nuclear
fluorescence (panels 4-6, times = 0, 30, and 50
s). Smallarrows point to the bath wall abutting the
oocyte.
When we injected Ins(1,4,5)P (10
M) into the cytosol of the oocyte, we found an increase
in the fluorescence of nuclear fluo 3 (3.9-fold increase over base-line
level, n = 3, see Fig. 1A, panels
1-3 for an example) or nuclear dextran-conjugated
Ca
green (1.5-fold increase over baseline, n = 3, not shown). These increases in fluorescence reflect an
increase in [Ca
]
.
Nuclear fluorescence returned to baseline within 65 ± 12 s (mean
± S.E.). This time course is similar to the time course with
which cytosolic Ca
increases following an injection
of Ins(1,4,5)P
(25, 26) . Thus, the
simplest explanation for these results is that Ins(1,4,5)P
first released Ca
into the cytosol, and that
this Ca
diffused into the nucleus. However, we also
considered the possibility that Ins(1,4,5)P
itself diffused
into the nucleus, where it triggered the release of Ca
from the perinuclear space into the nucleoplasm.
To
distinguish between those two possibilities, we used heparin, which
prevents Ins(1,4,5)P from binding to its receptor, thereby
inhibiting Ca
release(27, 28) . We
have previously used heparin to inhibit the propagation of
Ins(1,4,5)P
-induced Ca
waves in the
oocyte(23) . If the increase in
[Ca
]
resulted from an
increase in the concentration of Ins(1,4,5)P
in the
nucleus, then adding heparin to the nucleoplasm would be
expected to prevent a cytosolic Ins(1,4,5)P
injection from
increasing [Ca
]
.
Conversely, if the increase in
[Ca
]
resulted from an
increase in the concentration of Ins(1,4,5)P
in the
cytosol, then adding heparin to the cytosol should prevent a
cytosolic Ins(1,4,5)P
injection from increasing
[Ca
]
.
Fig. 1A (panels 4-6) shows results
from a cell in which heparin had been injected into the nucleus. When
we injected Ins(1,4,5)P into the cytosol of the cell, we
found that [Ca
]
increased (210 ± 53% of baseline, n =
5, p < 0.02). An example of the time course of this rise in
[Ca
]
is shown in Fig. 2A (tracinga). However, when we
injected Ins(1,4,5)P
into the cytosol of cells that had
previously been injected with heparin in the cytosol (examples are
given in Fig. 1A, panels 7-9, and Fig. 2A, tracingb),
[Ca
]
failed to
increase (96 ± 6% of base-line level, n = 6,
difference not statistically significant). These results, which are
summarized in Fig. 2B, indicate that Ins(1,4,5)P
can act in the cytosol to increase
[Ca
]
. This conclusion
also predicted that Ca
must be able to diffuse from
the cytosol into the nucleus. To test this prediction, we loaded oocyte
nuclei with fluo 3 and then injected Ca
(7 nl of 50
µM CaCl
solution) into the perinuclear
cytosol. As expected, injection of cytosolic Ca
increased [Ca
]
(187 ± 66%, n = 6, p <
0.03) (Fig. 1A, panels10-12).
Figure 2:
A,
time course of the changes in nuclear fluorescence following an
Ins(1,4,5)P injection (time = 5 s) in four different
cells (tracings a-d). Ins(1,4,5)P
was
injected either into the cytosol (solid lines) or into the
nucleus (dashed lines). Injection of Ins(1,4,5)P
into the cytosol increased nuclear fluorescence when heparin was
present in the nucleus (tracinga), but did not do so
when heparin was present in the cytosol (tracingb).
In contrast, injection of Ins(1,4,5)P
into the nucleus
increased nuclear fluorescence when heparin was present in the cytosol,
but did not do so when heparin was present in the nucleus (tracing d). B, summary of the data from experiments like
those shown in Fig. 1and in Fig. 2A. Each bar
represents the peak increase in nuclear fluorescence (mean ±
S.E., base line = 100%) resulting from the experimental
conditions indicated under the bar. Ins(1,4,5)P
or heparin
were injected either into the cytosol (C) or into the nucleus (N).
In all of the experiments described above, we injected
Ins(1,4,5)P into the cytosol. Because we do not know if
cytosolic Ins(1,4,5)P
can diffuse into the nucleus, those
experiments did not address the possibility that changes in the
concentration of Ins(1,4,5)P
in the nucleus could also
regulate [Ca
]
. To test
this hypothesis, we injected Ins(1,4,5)P
(10
M) directly into the nucleus. Before injecting
Ins(1,4,5)P
into the nucleus, we injected heparin into the
cytosol to block the action of any Ins(1,4,5)P
which might
escape into the cytosol, either through the injection pipette, through
the nuclear puncture site, or through the nuclear pores. In this way,
we wanted to exclude the possibility that an increase in
[Ca
]
was simply
secondary to an increase in
[Ca
]
. Under these
conditions, injection of Ins(1,4,5)P
into the nucleus
produced a robust increase in
[Ca
]
(395 ±
140% of base-line level, n = 6, p < 0.02).
We considered the possibility that if Ins(1,4,5)P
rapidly
exited the nucleus, the perinuclear Ins(1,4,5)P
concentration may be sufficient to competitively overcome the
inhibitory action of heparin in the cytosol. To address this
possibility, we reduced the concentration of Ins(1,4,5)P
injected into the nucleus 100-fold (10
M in the pipette) and increased the cytosolic heparin concentration
3-fold (30 mg/ml). Despite these conditions, nuclear Ins(1,4,5)P
still increased [Ca
]
in 6 out of the 8 cells tested (437 ± 148%, p < 0.03) (Fig. 1B, panels 1-3,
and Fig. 2A, tracingc). To rule out
the possibility that a nuclear Ins(1,4,5)P
injection
disrupted the nuclear envelope or increased
[Ca
]
nonspecifically,
we preinjected heparin (0.1 mg/ml) into the oocyte nucleus. Subsequent
injection of Ins(1,4,5)P
(10
M)
into the nucleus did not increase
[Ca
]
(102 ± 6%
of base-line levels) in 6 out of the 8 cells tested (Fig. 1B, panels 4-6, and Fig. 2A, tracingd). The increase in
[Ca
]
observed in the
remaining 2 cells may have occurred because some Ins(1,4,5)P
leaked in the cytosol at the injection site. These results, which
are summarized in Fig. 2B, indicate that an increase in
the concentration of Ins(1,4,5)P
in the nucleus can
increase [Ca
]
.
Our
results indicate that changes in either the cytosolic or the nuclear
Ins(1,4,5)P concentration can control
[Ca
]
. At present, the
relative physiological importance of these two mechanisms is unknown,
and we can only speculate that their co-existence increases the
flexibility with which a cell can regulate its
[Ca
]
. The presence of
both of these mechanisms does not imply that
[Ca
]
will increase
with any stimulus which increases intracellular Ins(1,4,5)P
concentration. Production of Ins(1,4,5)P
at the
plasma membrane may only yield a localized, submembranous increase in
[Ca
]
, but may not
increase in [Ca
]
.
Conversely, production of Ins(1,4,5)P
in the nucleoplasm
may increase [Ca
]
without significantly increasing
[Ca
]
. In the oocyte
for example, the high cytosolic Ca
-buffering capacity (23, 29) would prevent Ca
which
escapes from the nucleus from traveling beyond the immediate
perinuclear cytosol. Moreover, in the absence of cytosolic
Ins(1,4,5)P
, an increase in
[Ca
] in the perinuclear cytosol would fail
to extend to the rest of the cell as an actively propagating
Ca
wave(23, 29) .
Since our data
indicate that Ins(1,4,5)P receptors face the nucleoplasm,
the failure of cytosolic Ins(1,4,5)P
to increase
[Ca
]
in the presence
of cytosolic heparin suggests that Ins(1,4,5)P
does not
readily cross the nuclear envelope. Thus, a nuclear phosphoinositide
cycle would be required to increase nuclear Ins(1,4,5)P
concentration. Little is known about how this cycle may be
regulated. Some evidence points toward regulation via a plasma membrane
growth factor receptor (18) , while other work suggests
regulation by events occurring within the nucleus(16) . In the
context of our data, this last possibility is particularly intriguing,
since it would imply that the nucleus can completely self-regulate its
Ca
signal.