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Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos.

Parry H, McDougall A, Whitaker M - J. Cell Biol. (2005)

Bottom Line: Cell. 92:193-204).Constructs that chelate InsP3 also prevent nuclear division.An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne Medical School, Newcastle upon Tyne NE2 4HH, England, UK.

ABSTRACT
Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)-mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875-878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193-204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types.

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Functional InsP3 receptors in Drosophila embryos. (A) To determine whether Drosophila embryos had functional InsP3 calcium release channels, InsP3 was injected into cycle 8 embryos. The site of injection is indicated by an asterisk. (i) Microinjection of InsP3 (pipette concentration of 100 μM; final concentration of 200 nM) at 67 s increases [Ca]i and results in a cortical contraction in CaGr-injected embryos. The outline of the embryo is shown in white, and the contraction is seen as a retraction of the plasma membrane beneath the perivitelline envelope. (ii) InsP3 was coinjected with TMR to verify the site of injection. The top row is comprised of CaGr confocal images, whereas the bottom row displays TMR confocal images. To verify that there was no spillover from the TMR signal into the CaGr recording channel, the perivitelline space was first loaded with TMR (first image: 59 s). InsP3 microinjection causes a large calcium increase, indicating that functional InsP3 receptors are present. The cortical contraction is visible in these images by 8–9 s after the injection of InsP3. (iii) The time course of CaGr fluorescence increase in the embryo, corresponding to an increase in [Cai]. The embryo was in cycle 8. (B) Effects of wild-type and control (inactive) InsP3 sponge. (i) Nuclear cycle progression fails after microinjection of the InsP3 sponge during interphase of cycle 10 (20 mg/ml in pipette; 40 μg/ml in the embryo if uniformly distributed) but is unaffected by the mutated control sponge (20 mg/ml in pipette). (ii) Nuclear morphology visualized with fluorescein histones during mitosis of cycle 11 using the same microinjection protocol. With wild-type sponge, NEB, chromatin condensation, and metaphase plate formation occur normally, but anaphase onset is delayed, and chromosomes fail to separate. In contrast, embryos that were injected with the control sponge in parallel experiments have reformed normal nuclei in cycle 11; chromatin decondensation after mitosis occurs but is abnormal. (C) Effects of p130 InsP3-binding protein that was injected into cycle 11 embryos as they entered interphase to produce a gradient of inhibitor. (i) Imaging microinjected GFP::p130 fluorescence is associated with cytoplasm and plasma membrane and with ER at interphase and enters the nucleus as NEB occurs. At the highest concentrations, nuclei arrest in interphase of the following cycle, cycle 12. At intermediate concentrations, nuclei arrest with condensed chromosomes in metaphase of cycle 12. At lowest concentrations, nuclei arrest in interphase of cycle 13. From the fluorescence distribution, note the concentration gradient of the inhibitor (injected at a pipette concentration of 30 mg/ml = 200 μM at anaphase of cycle 10, 32 min before the time of the image shown) from the bottom to top of the field. (ii) Calibration of concentration gradient (see Materials and methods). The experimental image and model show the distribution 15 min after microinjection. Lines across the images indicate the pixels that were sampled. FL, fluorescence. Numbers on x axis represent distance in pixels. (iii) Higher magnification of a separate experiment using an identical experimental protocol to show eventual chromatin decondensation after failed anaphase onset. Temperature is 18°C. In these experiments, the time of pole bud formation was not recorded. Bars, 30 μm.
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fig3: Functional InsP3 receptors in Drosophila embryos. (A) To determine whether Drosophila embryos had functional InsP3 calcium release channels, InsP3 was injected into cycle 8 embryos. The site of injection is indicated by an asterisk. (i) Microinjection of InsP3 (pipette concentration of 100 μM; final concentration of 200 nM) at 67 s increases [Ca]i and results in a cortical contraction in CaGr-injected embryos. The outline of the embryo is shown in white, and the contraction is seen as a retraction of the plasma membrane beneath the perivitelline envelope. (ii) InsP3 was coinjected with TMR to verify the site of injection. The top row is comprised of CaGr confocal images, whereas the bottom row displays TMR confocal images. To verify that there was no spillover from the TMR signal into the CaGr recording channel, the perivitelline space was first loaded with TMR (first image: 59 s). InsP3 microinjection causes a large calcium increase, indicating that functional InsP3 receptors are present. The cortical contraction is visible in these images by 8–9 s after the injection of InsP3. (iii) The time course of CaGr fluorescence increase in the embryo, corresponding to an increase in [Cai]. The embryo was in cycle 8. (B) Effects of wild-type and control (inactive) InsP3 sponge. (i) Nuclear cycle progression fails after microinjection of the InsP3 sponge during interphase of cycle 10 (20 mg/ml in pipette; 40 μg/ml in the embryo if uniformly distributed) but is unaffected by the mutated control sponge (20 mg/ml in pipette). (ii) Nuclear morphology visualized with fluorescein histones during mitosis of cycle 11 using the same microinjection protocol. With wild-type sponge, NEB, chromatin condensation, and metaphase plate formation occur normally, but anaphase onset is delayed, and chromosomes fail to separate. In contrast, embryos that were injected with the control sponge in parallel experiments have reformed normal nuclei in cycle 11; chromatin decondensation after mitosis occurs but is abnormal. (C) Effects of p130 InsP3-binding protein that was injected into cycle 11 embryos as they entered interphase to produce a gradient of inhibitor. (i) Imaging microinjected GFP::p130 fluorescence is associated with cytoplasm and plasma membrane and with ER at interphase and enters the nucleus as NEB occurs. At the highest concentrations, nuclei arrest in interphase of the following cycle, cycle 12. At intermediate concentrations, nuclei arrest with condensed chromosomes in metaphase of cycle 12. At lowest concentrations, nuclei arrest in interphase of cycle 13. From the fluorescence distribution, note the concentration gradient of the inhibitor (injected at a pipette concentration of 30 mg/ml = 200 μM at anaphase of cycle 10, 32 min before the time of the image shown) from the bottom to top of the field. (ii) Calibration of concentration gradient (see Materials and methods). The experimental image and model show the distribution 15 min after microinjection. Lines across the images indicate the pixels that were sampled. FL, fluorescence. Numbers on x axis represent distance in pixels. (iii) Higher magnification of a separate experiment using an identical experimental protocol to show eventual chromatin decondensation after failed anaphase onset. Temperature is 18°C. In these experiments, the time of pole bud formation was not recorded. Bars, 30 μm.

Mentions: Cell cycle calcium signals in other early embryos are triggered by inositol trisphosphate (InsP3; Ciapa et al., 1994; Muto et al., 1996; Groigno and Whitaker, 1998). Drosophila possesses a single insect-specific InsP3 receptor isoform (Hasan and Rosbash, 1992; Yoshikawa et al., 1992). Deletion of the InsP3 receptor arrests larval development at second instar, and embryos show defects in cell division and endoreplication (Acharya et al., 1997). Embryonic development to second instar also requires the InsP3 receptor because no viable eggs or embryos were generated from germ line clones lacking the receptor (Acharya et al., 1997). Fig. 3 A shows that InsP3 receptors are functional in early embryos; the microinjection of InsP3 leads to calcium release. [Cai] was measured using CaGr, and localization of the injected InsP3 was determined by coinjection of rhodamine dextran with InsP3. Although InsP3 was injected into the body of the embryo, [Cai] rose at the cortex predominantly, and there is also a cortical contraction response. This experiment shows that InsP3-induced calcium release causes cortical contraction.


Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos.

Parry H, McDougall A, Whitaker M - J. Cell Biol. (2005)

Functional InsP3 receptors in Drosophila embryos. (A) To determine whether Drosophila embryos had functional InsP3 calcium release channels, InsP3 was injected into cycle 8 embryos. The site of injection is indicated by an asterisk. (i) Microinjection of InsP3 (pipette concentration of 100 μM; final concentration of 200 nM) at 67 s increases [Ca]i and results in a cortical contraction in CaGr-injected embryos. The outline of the embryo is shown in white, and the contraction is seen as a retraction of the plasma membrane beneath the perivitelline envelope. (ii) InsP3 was coinjected with TMR to verify the site of injection. The top row is comprised of CaGr confocal images, whereas the bottom row displays TMR confocal images. To verify that there was no spillover from the TMR signal into the CaGr recording channel, the perivitelline space was first loaded with TMR (first image: 59 s). InsP3 microinjection causes a large calcium increase, indicating that functional InsP3 receptors are present. The cortical contraction is visible in these images by 8–9 s after the injection of InsP3. (iii) The time course of CaGr fluorescence increase in the embryo, corresponding to an increase in [Cai]. The embryo was in cycle 8. (B) Effects of wild-type and control (inactive) InsP3 sponge. (i) Nuclear cycle progression fails after microinjection of the InsP3 sponge during interphase of cycle 10 (20 mg/ml in pipette; 40 μg/ml in the embryo if uniformly distributed) but is unaffected by the mutated control sponge (20 mg/ml in pipette). (ii) Nuclear morphology visualized with fluorescein histones during mitosis of cycle 11 using the same microinjection protocol. With wild-type sponge, NEB, chromatin condensation, and metaphase plate formation occur normally, but anaphase onset is delayed, and chromosomes fail to separate. In contrast, embryos that were injected with the control sponge in parallel experiments have reformed normal nuclei in cycle 11; chromatin decondensation after mitosis occurs but is abnormal. (C) Effects of p130 InsP3-binding protein that was injected into cycle 11 embryos as they entered interphase to produce a gradient of inhibitor. (i) Imaging microinjected GFP::p130 fluorescence is associated with cytoplasm and plasma membrane and with ER at interphase and enters the nucleus as NEB occurs. At the highest concentrations, nuclei arrest in interphase of the following cycle, cycle 12. At intermediate concentrations, nuclei arrest with condensed chromosomes in metaphase of cycle 12. At lowest concentrations, nuclei arrest in interphase of cycle 13. From the fluorescence distribution, note the concentration gradient of the inhibitor (injected at a pipette concentration of 30 mg/ml = 200 μM at anaphase of cycle 10, 32 min before the time of the image shown) from the bottom to top of the field. (ii) Calibration of concentration gradient (see Materials and methods). The experimental image and model show the distribution 15 min after microinjection. Lines across the images indicate the pixels that were sampled. FL, fluorescence. Numbers on x axis represent distance in pixels. (iii) Higher magnification of a separate experiment using an identical experimental protocol to show eventual chromatin decondensation after failed anaphase onset. Temperature is 18°C. In these experiments, the time of pole bud formation was not recorded. Bars, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171230&req=5

fig3: Functional InsP3 receptors in Drosophila embryos. (A) To determine whether Drosophila embryos had functional InsP3 calcium release channels, InsP3 was injected into cycle 8 embryos. The site of injection is indicated by an asterisk. (i) Microinjection of InsP3 (pipette concentration of 100 μM; final concentration of 200 nM) at 67 s increases [Ca]i and results in a cortical contraction in CaGr-injected embryos. The outline of the embryo is shown in white, and the contraction is seen as a retraction of the plasma membrane beneath the perivitelline envelope. (ii) InsP3 was coinjected with TMR to verify the site of injection. The top row is comprised of CaGr confocal images, whereas the bottom row displays TMR confocal images. To verify that there was no spillover from the TMR signal into the CaGr recording channel, the perivitelline space was first loaded with TMR (first image: 59 s). InsP3 microinjection causes a large calcium increase, indicating that functional InsP3 receptors are present. The cortical contraction is visible in these images by 8–9 s after the injection of InsP3. (iii) The time course of CaGr fluorescence increase in the embryo, corresponding to an increase in [Cai]. The embryo was in cycle 8. (B) Effects of wild-type and control (inactive) InsP3 sponge. (i) Nuclear cycle progression fails after microinjection of the InsP3 sponge during interphase of cycle 10 (20 mg/ml in pipette; 40 μg/ml in the embryo if uniformly distributed) but is unaffected by the mutated control sponge (20 mg/ml in pipette). (ii) Nuclear morphology visualized with fluorescein histones during mitosis of cycle 11 using the same microinjection protocol. With wild-type sponge, NEB, chromatin condensation, and metaphase plate formation occur normally, but anaphase onset is delayed, and chromosomes fail to separate. In contrast, embryos that were injected with the control sponge in parallel experiments have reformed normal nuclei in cycle 11; chromatin decondensation after mitosis occurs but is abnormal. (C) Effects of p130 InsP3-binding protein that was injected into cycle 11 embryos as they entered interphase to produce a gradient of inhibitor. (i) Imaging microinjected GFP::p130 fluorescence is associated with cytoplasm and plasma membrane and with ER at interphase and enters the nucleus as NEB occurs. At the highest concentrations, nuclei arrest in interphase of the following cycle, cycle 12. At intermediate concentrations, nuclei arrest with condensed chromosomes in metaphase of cycle 12. At lowest concentrations, nuclei arrest in interphase of cycle 13. From the fluorescence distribution, note the concentration gradient of the inhibitor (injected at a pipette concentration of 30 mg/ml = 200 μM at anaphase of cycle 10, 32 min before the time of the image shown) from the bottom to top of the field. (ii) Calibration of concentration gradient (see Materials and methods). The experimental image and model show the distribution 15 min after microinjection. Lines across the images indicate the pixels that were sampled. FL, fluorescence. Numbers on x axis represent distance in pixels. (iii) Higher magnification of a separate experiment using an identical experimental protocol to show eventual chromatin decondensation after failed anaphase onset. Temperature is 18°C. In these experiments, the time of pole bud formation was not recorded. Bars, 30 μm.
Mentions: Cell cycle calcium signals in other early embryos are triggered by inositol trisphosphate (InsP3; Ciapa et al., 1994; Muto et al., 1996; Groigno and Whitaker, 1998). Drosophila possesses a single insect-specific InsP3 receptor isoform (Hasan and Rosbash, 1992; Yoshikawa et al., 1992). Deletion of the InsP3 receptor arrests larval development at second instar, and embryos show defects in cell division and endoreplication (Acharya et al., 1997). Embryonic development to second instar also requires the InsP3 receptor because no viable eggs or embryos were generated from germ line clones lacking the receptor (Acharya et al., 1997). Fig. 3 A shows that InsP3 receptors are functional in early embryos; the microinjection of InsP3 leads to calcium release. [Cai] was measured using CaGr, and localization of the injected InsP3 was determined by coinjection of rhodamine dextran with InsP3. Although InsP3 was injected into the body of the embryo, [Cai] rose at the cortex predominantly, and there is also a cortical contraction response. This experiment shows that InsP3-induced calcium release causes cortical contraction.

Bottom Line: Cell. 92:193-204).Constructs that chelate InsP3 also prevent nuclear division.An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne Medical School, Newcastle upon Tyne NE2 4HH, England, UK.

ABSTRACT
Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)-mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875-878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193-204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types.

Show MeSH
Related in: MedlinePlus