©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Changes in either Cytosolic or Nucleoplasmic Inositol 1,4,5-Trisphosphate Levels Can Control Nuclear Ca Concentration (*)

(Received for publication, December 2, 1994; and in revised form, January 16, 1995)

Daniel J. Hennager (1) Michael J. Welsh (2) (4) (3) Sylvain DeLisle (1) (3)(§)

From the  (1)Veterans Administration Medical Center and (2)Howard Hughes Medical Institute, and the (3)Departments of Internal Medicine and (4)Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(3)), and because the nuclear envelope may enclose an Ins(1,4,5)P(3)-releasable Ca store, we tested the hypothesis that nuclear and/or cytosolic levels of Ins(1,4,5)P(3) 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(3) 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(3) levels could control [Ca]. When we injected Ins(1,4,5)P(3) directly into the nucleus, [Ca] increased, even when heparin was present in the cytosol, indicating that Ins(1,4,5)P(3) could control [Ca] from within the nucleus. These results provide functional evidence for Ins(1,4,5)P(3) receptors facing the nucleoplasm and raise the possibility that a phosphoinositide cycle situated at the nuclear membranes can control Ca-dependent nuclear functions.


INTRODUCTION

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]). (^1)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(3)) is a likely candidate to regulate [Ca]. In a wide variety of cells, Ins(1,4,5)P(3) binds to high affinity receptors that form Ca channels in the membrane of organelles that store intracellular Ca. When Ins(1,4,5)P(3) 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(3) could regulate [Ca]by controlling [Ca]. However, Ins(1,4,5)P(3) 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(3)-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(3) 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(3) receptors were located on the inner nuclear membrane, then an increase in the nucleoplasmic concentration of Ins(1,4,5)P(3) 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(3)(17, 18, 19, 20) supports the notion that Ins(1,4,5)P(3) levels could indeed increase within the nucleoplasm.

We used the Xenopus oocyte model to test the hypothesis that Ins(1,4,5)P(3) regulates [Ca] both indirectly by increasing [Ca], and directly by triggering the discharge of Ca from the perinuclear space into the nucleoplasm.


MATERIALS AND METHODS

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(3) (10 to 10M) (Calbiochem); a mixture of fluo 3 (1 mM) and Ins(1,4,5)P(3); heparin (mass = 13.3-15 kDa, 0.1-30 mg/ml) (Calbiochem); or CaCl(2) (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 times 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(3) (panels 1-9) or Ca (panels10-12). Injection of Ins(1,4,5)P(3) (10M) 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(3) did not increase [Ca] when heparin was preinjected in the cytosol (panels 7-9, times = 0, 36, and 66 s). An injection of CaCl(2) also transiently increased nuclear fluorescence (panels 10-12, times = 0, 72, and 198 s). B, nuclear injections of Ins(1,4,5)P(3). Injection of Ins(1,4,5)P(3) 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(3) 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(3) injection failed to increase nuclear fluorescence (panels 4-6, times = 0, 30, and 50 s). Smallarrows point to the bath wall abutting the oocyte.




RESULTS AND DISCUSSION

When we injected Ins(1,4,5)P(3) (10M) 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(3)(25, 26) . Thus, the simplest explanation for these results is that Ins(1,4,5)P(3) 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(3) 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(3) 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(3)-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(3) in the nucleus, then adding heparin to the nucleoplasm would be expected to prevent a cytosolic Ins(1,4,5)P(3) injection from increasing [Ca]. Conversely, if the increase in [Ca] resulted from an increase in the concentration of Ins(1,4,5)P(3) in the cytosol, then adding heparin to the cytosol should prevent a cytosolic Ins(1,4,5)P(3) 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(3) 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(3) 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(3) 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(2) 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(3) injection (time = 5 s) in four different cells (tracings a-d). Ins(1,4,5)P(3) was injected either into the cytosol (solid lines) or into the nucleus (dashed lines). Injection of Ins(1,4,5)P(3) 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(3) 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(3) 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(3) into the cytosol. Because we do not know if cytosolic Ins(1,4,5)P(3) can diffuse into the nucleus, those experiments did not address the possibility that changes in the concentration of Ins(1,4,5)P(3) in the nucleus could also regulate [Ca]. To test this hypothesis, we injected Ins(1,4,5)P(3) (10M) directly into the nucleus. Before injecting Ins(1,4,5)P(3) into the nucleus, we injected heparin into the cytosol to block the action of any Ins(1,4,5)P(3) 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(3) 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(3) rapidly exited the nucleus, the perinuclear Ins(1,4,5)P(3) 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(3) injected into the nucleus 100-fold (10M in the pipette) and increased the cytosolic heparin concentration 3-fold (30 mg/ml). Despite these conditions, nuclear Ins(1,4,5)P(3) 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(3) 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(3) (10M) 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(3) 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(3) in the nucleus can increase [Ca].

Our results indicate that changes in either the cytosolic or the nuclear Ins(1,4,5)P(3) 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(3) concentration. Production of Ins(1,4,5)P(3) 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(3) 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(3), 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(3) receptors face the nucleoplasm, the failure of cytosolic Ins(1,4,5)P(3) to increase [Ca] in the presence of cytosolic heparin suggests that Ins(1,4,5)P(3) does not readily cross the nuclear envelope. Thus, a nuclear phosphoinositide cycle would be required to increase nuclear Ins(1,4,5)P(3) 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.


FOOTNOTES

*
This work was supported by grants from the American Lung Association and the Department of Veterans Affairs (to S. D.) and from the Howard Hughes Medical Institute (to M. J. W). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 500 EMRB, Newton Rd., University of Iowa, College of Medicine, Iowa City, IA 52242. Fax: 319-335-7623.

(^1)
The abbreviations used are: [Ca], free nucleoplasmic Ca concentration; Ins(1,4,5)P(3), inositol 1,4,5-trisphosphate.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.