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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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Spatial correlation of cortical calcium signals, the cortical cytoskeleton, and ER after the arrival of nuclei at the cortex. (A) Embryo coinjected with CaGr and rhodamine tubulin. (i) CaGr confocal images from metaphase of cycle 10 to metaphase of cycle 11. Increasing detector sensitivity in order to better detect the nuclear CaGr signal, which leads to saturation of the signal in the cortex. The [Cai] increase during interphase is highest in the region surrounding the nuclei. (ii) Simultaneous rhodamine tubulin confocal images from the same sections that display the microtubule configuration and permit determination of the phase of the cell cycle during mitosis. Embryo is in cycle 9. (B) Confocal images revealing the distribution of the ER during mitosis in a different embryo. Embryos were injected with the lipophilic dye DiIC18 to label the ER. During mitosis, the ER is concentrated around the mitotic spindle, at the poles during metaphase and anaphase, and at the midbody in telophase. Also note the ER-free interstices between spindles. During interphase, ER is absent from the nuclei, as might be expected, and is dispersed relative to mitosis. Embryo is in cycle 10. (C) Further embryos were coinjected with rhodamine-labeled actin and CaGr to determine the spatial relation between actin and [Ca]i. (i) Confocal images that display the pattern of calcium increase from mitosis to mitosis. (ii) The distribution of actin in the same confocal sections. The pattern of [Ca]i increase follows the pattern of actin distribution closely throughout the nuclear cycle. Embryo is in cycle 11. Bars, 50 μm. (D) Simultaneous imaging of ER (DiI fluorescence) and the mitotic spindle (fluorescein tubulin fluorescence). The images show that the ER surrounds the spindle as the nucleus enters mitosis. NEB, nuclear envelope breakdown; Pm, prometaphase; M, metaphase. (E) To determine the spatial relationships between the ER, actin, and [Cai], embryos were coinjected with fluorescein-labeled actin and DiIC18 and were compared with other embryos that were injected with CaGr/TMR ratios in confocal z-sections. (i) Confocal ratiometric images normal to the surface of the embryo compare the cortical [Cai] levels during interphase, when the actin caps are present in cortical buds, with those at anaphase. The [Ca]i increase occurs through this thickness of cortex in interphase but is very prominent just beneath the plasmalemma within the cortical bud. During anaphase, cortical buds are absent and [Cai] levels are both lower and more uniform beneath the cortex. (ii) Cartoons displaying the distribution of actin, microtubules, and chromosomes during metaphase, anaphase, telophase, and interphase accompanied by images of actin and ER in cortical buds in sections normal to the cortex (z sections) as mitosis progresses. Embryos were coinjected with fluorescein-actin and DiIC16 to visualize the cortical actin and ER during mitosis. Confocal merged images reveal that the actin (green) and ER (red) are in close apposition but do not overlap significantly. ER was found below the actin cap. A comparison with (i) indicates that [Cai] is highest in the region of the actin cap. ER wraps around the nucleus and mitotic spindle. Results shown are representative of data from at least three embryos in separate experiments. Temperature is 18°C.
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fig4: Spatial correlation of cortical calcium signals, the cortical cytoskeleton, and ER after the arrival of nuclei at the cortex. (A) Embryo coinjected with CaGr and rhodamine tubulin. (i) CaGr confocal images from metaphase of cycle 10 to metaphase of cycle 11. Increasing detector sensitivity in order to better detect the nuclear CaGr signal, which leads to saturation of the signal in the cortex. The [Cai] increase during interphase is highest in the region surrounding the nuclei. (ii) Simultaneous rhodamine tubulin confocal images from the same sections that display the microtubule configuration and permit determination of the phase of the cell cycle during mitosis. Embryo is in cycle 9. (B) Confocal images revealing the distribution of the ER during mitosis in a different embryo. Embryos were injected with the lipophilic dye DiIC18 to label the ER. During mitosis, the ER is concentrated around the mitotic spindle, at the poles during metaphase and anaphase, and at the midbody in telophase. Also note the ER-free interstices between spindles. During interphase, ER is absent from the nuclei, as might be expected, and is dispersed relative to mitosis. Embryo is in cycle 10. (C) Further embryos were coinjected with rhodamine-labeled actin and CaGr to determine the spatial relation between actin and [Ca]i. (i) Confocal images that display the pattern of calcium increase from mitosis to mitosis. (ii) The distribution of actin in the same confocal sections. The pattern of [Ca]i increase follows the pattern of actin distribution closely throughout the nuclear cycle. Embryo is in cycle 11. Bars, 50 μm. (D) Simultaneous imaging of ER (DiI fluorescence) and the mitotic spindle (fluorescein tubulin fluorescence). The images show that the ER surrounds the spindle as the nucleus enters mitosis. NEB, nuclear envelope breakdown; Pm, prometaphase; M, metaphase. (E) To determine the spatial relationships between the ER, actin, and [Cai], embryos were coinjected with fluorescein-labeled actin and DiIC18 and were compared with other embryos that were injected with CaGr/TMR ratios in confocal z-sections. (i) Confocal ratiometric images normal to the surface of the embryo compare the cortical [Cai] levels during interphase, when the actin caps are present in cortical buds, with those at anaphase. The [Ca]i increase occurs through this thickness of cortex in interphase but is very prominent just beneath the plasmalemma within the cortical bud. During anaphase, cortical buds are absent and [Cai] levels are both lower and more uniform beneath the cortex. (ii) Cartoons displaying the distribution of actin, microtubules, and chromosomes during metaphase, anaphase, telophase, and interphase accompanied by images of actin and ER in cortical buds in sections normal to the cortex (z sections) as mitosis progresses. Embryos were coinjected with fluorescein-actin and DiIC16 to visualize the cortical actin and ER during mitosis. Confocal merged images reveal that the actin (green) and ER (red) are in close apposition but do not overlap significantly. ER was found below the actin cap. A comparison with (i) indicates that [Cai] is highest in the region of the actin cap. ER wraps around the nucleus and mitotic spindle. Results shown are representative of data from at least three embryos in separate experiments. Temperature is 18°C.

Mentions: Once nuclei reach the surface, it is possible to stage the nuclear cycle precisely. Fig. 4 shows the spatial distribution of the interphase [Cai] increase from metaphase through interphase to metaphase of the next cycle, as seen in glancing tangential confocal sections (Fig. 1 C); this is compared with the disposition of mitotic spindles, ER, and actin. CaGr fluorescence (Fig. 4 A, i and ii) indicates that the major [Cai] increase occurs in interphase in a cortical region surrounding the nuclei but is separated from interphase nuclei by a region of low calcium concentration. As nuclei enter mitosis, the cortical [Cai] levels fall overall, and the signal becomes confined to narrower regions surrounding the mitotic spindle. [Cai] in the nucleus and mitotic spindle appears higher than in the circumnuclear region but much lower than in the cortical region. These images cannot be compared directly to those of Fig. 1, as ratiometric methods cannot be used when simultaneously measuring rhodamine-tagged cytoskeletal components. Moreover, the increased detection sensitivity that is required to visualize CaGr fluorescence in the nucleus and spindle leads to saturation of the cortical CaGr signal because of the limited dynamic range of the confocal microscope.


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

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

Spatial correlation of cortical calcium signals, the cortical cytoskeleton, and ER after the arrival of nuclei at the cortex. (A) Embryo coinjected with CaGr and rhodamine tubulin. (i) CaGr confocal images from metaphase of cycle 10 to metaphase of cycle 11. Increasing detector sensitivity in order to better detect the nuclear CaGr signal, which leads to saturation of the signal in the cortex. The [Cai] increase during interphase is highest in the region surrounding the nuclei. (ii) Simultaneous rhodamine tubulin confocal images from the same sections that display the microtubule configuration and permit determination of the phase of the cell cycle during mitosis. Embryo is in cycle 9. (B) Confocal images revealing the distribution of the ER during mitosis in a different embryo. Embryos were injected with the lipophilic dye DiIC18 to label the ER. During mitosis, the ER is concentrated around the mitotic spindle, at the poles during metaphase and anaphase, and at the midbody in telophase. Also note the ER-free interstices between spindles. During interphase, ER is absent from the nuclei, as might be expected, and is dispersed relative to mitosis. Embryo is in cycle 10. (C) Further embryos were coinjected with rhodamine-labeled actin and CaGr to determine the spatial relation between actin and [Ca]i. (i) Confocal images that display the pattern of calcium increase from mitosis to mitosis. (ii) The distribution of actin in the same confocal sections. The pattern of [Ca]i increase follows the pattern of actin distribution closely throughout the nuclear cycle. Embryo is in cycle 11. Bars, 50 μm. (D) Simultaneous imaging of ER (DiI fluorescence) and the mitotic spindle (fluorescein tubulin fluorescence). The images show that the ER surrounds the spindle as the nucleus enters mitosis. NEB, nuclear envelope breakdown; Pm, prometaphase; M, metaphase. (E) To determine the spatial relationships between the ER, actin, and [Cai], embryos were coinjected with fluorescein-labeled actin and DiIC18 and were compared with other embryos that were injected with CaGr/TMR ratios in confocal z-sections. (i) Confocal ratiometric images normal to the surface of the embryo compare the cortical [Cai] levels during interphase, when the actin caps are present in cortical buds, with those at anaphase. The [Ca]i increase occurs through this thickness of cortex in interphase but is very prominent just beneath the plasmalemma within the cortical bud. During anaphase, cortical buds are absent and [Cai] levels are both lower and more uniform beneath the cortex. (ii) Cartoons displaying the distribution of actin, microtubules, and chromosomes during metaphase, anaphase, telophase, and interphase accompanied by images of actin and ER in cortical buds in sections normal to the cortex (z sections) as mitosis progresses. Embryos were coinjected with fluorescein-actin and DiIC16 to visualize the cortical actin and ER during mitosis. Confocal merged images reveal that the actin (green) and ER (red) are in close apposition but do not overlap significantly. ER was found below the actin cap. A comparison with (i) indicates that [Cai] is highest in the region of the actin cap. ER wraps around the nucleus and mitotic spindle. Results shown are representative of data from at least three embryos in separate experiments. Temperature is 18°C.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Spatial correlation of cortical calcium signals, the cortical cytoskeleton, and ER after the arrival of nuclei at the cortex. (A) Embryo coinjected with CaGr and rhodamine tubulin. (i) CaGr confocal images from metaphase of cycle 10 to metaphase of cycle 11. Increasing detector sensitivity in order to better detect the nuclear CaGr signal, which leads to saturation of the signal in the cortex. The [Cai] increase during interphase is highest in the region surrounding the nuclei. (ii) Simultaneous rhodamine tubulin confocal images from the same sections that display the microtubule configuration and permit determination of the phase of the cell cycle during mitosis. Embryo is in cycle 9. (B) Confocal images revealing the distribution of the ER during mitosis in a different embryo. Embryos were injected with the lipophilic dye DiIC18 to label the ER. During mitosis, the ER is concentrated around the mitotic spindle, at the poles during metaphase and anaphase, and at the midbody in telophase. Also note the ER-free interstices between spindles. During interphase, ER is absent from the nuclei, as might be expected, and is dispersed relative to mitosis. Embryo is in cycle 10. (C) Further embryos were coinjected with rhodamine-labeled actin and CaGr to determine the spatial relation between actin and [Ca]i. (i) Confocal images that display the pattern of calcium increase from mitosis to mitosis. (ii) The distribution of actin in the same confocal sections. The pattern of [Ca]i increase follows the pattern of actin distribution closely throughout the nuclear cycle. Embryo is in cycle 11. Bars, 50 μm. (D) Simultaneous imaging of ER (DiI fluorescence) and the mitotic spindle (fluorescein tubulin fluorescence). The images show that the ER surrounds the spindle as the nucleus enters mitosis. NEB, nuclear envelope breakdown; Pm, prometaphase; M, metaphase. (E) To determine the spatial relationships between the ER, actin, and [Cai], embryos were coinjected with fluorescein-labeled actin and DiIC18 and were compared with other embryos that were injected with CaGr/TMR ratios in confocal z-sections. (i) Confocal ratiometric images normal to the surface of the embryo compare the cortical [Cai] levels during interphase, when the actin caps are present in cortical buds, with those at anaphase. The [Ca]i increase occurs through this thickness of cortex in interphase but is very prominent just beneath the plasmalemma within the cortical bud. During anaphase, cortical buds are absent and [Cai] levels are both lower and more uniform beneath the cortex. (ii) Cartoons displaying the distribution of actin, microtubules, and chromosomes during metaphase, anaphase, telophase, and interphase accompanied by images of actin and ER in cortical buds in sections normal to the cortex (z sections) as mitosis progresses. Embryos were coinjected with fluorescein-actin and DiIC16 to visualize the cortical actin and ER during mitosis. Confocal merged images reveal that the actin (green) and ER (red) are in close apposition but do not overlap significantly. ER was found below the actin cap. A comparison with (i) indicates that [Cai] is highest in the region of the actin cap. ER wraps around the nucleus and mitotic spindle. Results shown are representative of data from at least three embryos in separate experiments. Temperature is 18°C.
Mentions: Once nuclei reach the surface, it is possible to stage the nuclear cycle precisely. Fig. 4 shows the spatial distribution of the interphase [Cai] increase from metaphase through interphase to metaphase of the next cycle, as seen in glancing tangential confocal sections (Fig. 1 C); this is compared with the disposition of mitotic spindles, ER, and actin. CaGr fluorescence (Fig. 4 A, i and ii) indicates that the major [Cai] increase occurs in interphase in a cortical region surrounding the nuclei but is separated from interphase nuclei by a region of low calcium concentration. As nuclei enter mitosis, the cortical [Cai] levels fall overall, and the signal becomes confined to narrower regions surrounding the mitotic spindle. [Cai] in the nucleus and mitotic spindle appears higher than in the circumnuclear region but much lower than in the cortical region. These images cannot be compared directly to those of Fig. 1, as ratiometric methods cannot be used when simultaneously measuring rhodamine-tagged cytoskeletal components. Moreover, the increased detection sensitivity that is required to visualize CaGr fluorescence in the nucleus and spindle leads to saturation of the cortical CaGr signal because of the limited dynamic range of the confocal microscope.

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