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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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Calcium increases in phase with the interphase cortical contractions in syncytial Drosophila embryos. (A i) [Cai] increases measured by confocal ratio imaging. The three rows of images display CaGr, TMR, and ratio images that represent the spatial distribution of the calcium signals as snapshots in midinterphase and midmitosis. Interphase and mitosis in cycles 8 and 9 are inferred from the timing of the cortical contractions (I and M). In the later two cycles, when the nuclei have come to the egg cortex, their presence is marked by areas of low calcium concentration. They are sometimes faintly visible in the ratiometric image (arrows), and the correlation between nuclei in interphase (I), the cortical contractions, and the calcium increase was noted. The pixel values in the ratiometric image are represented by a conventional rainbow scale, with higher calcium concentrations shown as warmer tones. Red colors in the image correspond to [Cai] in the midmicromolar range, as can be seen by comparison with the calibration shown in (ii). (ii) The temporal pattern of [Ca]i increase in the embryo shown in (i). [Cai] values are means for the whole embryo. Calibrated calcium concentrations are shown at right (see Calibration…signals). Note that as the nuclear division cycle time lengthens, the time between [Cai] signal peaks also lengthens so that the [Cai] signal remains in phase with the nuclear cycle. (B i) The nuclear division cycle length can be increased experimentally by ∼20% through treatment with cycloheximide. Numbers on x axis represent minutes. Error bars represent SEM. (ii) The [Ca]i peaks remain associated with interphase after cycloheximide treatment. Temperature is 18°C. (C) A schematic view of the embryo showing the plane of the confocal section in this and other figures. Note that the periphery of the section through the embryo provides images of the cortex and that the center of the section looks deeper into the embryo. Images are oriented with the anterior pole being uppermost.
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fig1: Calcium increases in phase with the interphase cortical contractions in syncytial Drosophila embryos. (A i) [Cai] increases measured by confocal ratio imaging. The three rows of images display CaGr, TMR, and ratio images that represent the spatial distribution of the calcium signals as snapshots in midinterphase and midmitosis. Interphase and mitosis in cycles 8 and 9 are inferred from the timing of the cortical contractions (I and M). In the later two cycles, when the nuclei have come to the egg cortex, their presence is marked by areas of low calcium concentration. They are sometimes faintly visible in the ratiometric image (arrows), and the correlation between nuclei in interphase (I), the cortical contractions, and the calcium increase was noted. The pixel values in the ratiometric image are represented by a conventional rainbow scale, with higher calcium concentrations shown as warmer tones. Red colors in the image correspond to [Cai] in the midmicromolar range, as can be seen by comparison with the calibration shown in (ii). (ii) The temporal pattern of [Ca]i increase in the embryo shown in (i). [Cai] values are means for the whole embryo. Calibrated calcium concentrations are shown at right (see Calibration…signals). Note that as the nuclear division cycle time lengthens, the time between [Cai] signal peaks also lengthens so that the [Cai] signal remains in phase with the nuclear cycle. (B i) The nuclear division cycle length can be increased experimentally by ∼20% through treatment with cycloheximide. Numbers on x axis represent minutes. Error bars represent SEM. (ii) The [Ca]i peaks remain associated with interphase after cycloheximide treatment. Temperature is 18°C. (C) A schematic view of the embryo showing the plane of the confocal section in this and other figures. Note that the periphery of the section through the embryo provides images of the cortex and that the center of the section looks deeper into the embryo. Images are oriented with the anterior pole being uppermost.

Mentions: Fig. 1 A shows fluorescence signals in a Drosophila embryo as it passes through cell cycles 8–11. Increased intracellular free calcium concentration ([Cai]) is detected by quantitative ratiometric imaging in each cell cycle as nuclei enter interphase. The ratio of calcium green dextran (CaGr) and rhodamine dextran fluorescence quantitatively reflects the intracellular calcium concentrations at different points within the confocal section. Red colors represent ≥1 μM calcium, yellow colors represent 0.5–1 μM, green colors represent 0.1–0.5 μM, and blue colors represent concentrations <0.1 μM (calibration shown in Fig. 1 A ii). [Cai] falls during mitosis and is elevated in the cortex of the embryo. Nuclei migrate to the cortex of the embryo during cycle 10. Once the nuclei enter the confocal section, [Cai] is seen in the ratiometric images to be highest around the nuclei in interphase, but it does not increase to the same degree within the nuclei, which appear as circular voids. In these and other images, the plane of confocal section passes through the embryo cortex at the edges of the image, whereas the center of the image represents areas several microns deeper in the embryo (Fig. 1 C). The confocal images are a window into small areas of the embryo cortex.


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

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

Calcium increases in phase with the interphase cortical contractions in syncytial Drosophila embryos. (A i) [Cai] increases measured by confocal ratio imaging. The three rows of images display CaGr, TMR, and ratio images that represent the spatial distribution of the calcium signals as snapshots in midinterphase and midmitosis. Interphase and mitosis in cycles 8 and 9 are inferred from the timing of the cortical contractions (I and M). In the later two cycles, when the nuclei have come to the egg cortex, their presence is marked by areas of low calcium concentration. They are sometimes faintly visible in the ratiometric image (arrows), and the correlation between nuclei in interphase (I), the cortical contractions, and the calcium increase was noted. The pixel values in the ratiometric image are represented by a conventional rainbow scale, with higher calcium concentrations shown as warmer tones. Red colors in the image correspond to [Cai] in the midmicromolar range, as can be seen by comparison with the calibration shown in (ii). (ii) The temporal pattern of [Ca]i increase in the embryo shown in (i). [Cai] values are means for the whole embryo. Calibrated calcium concentrations are shown at right (see Calibration…signals). Note that as the nuclear division cycle time lengthens, the time between [Cai] signal peaks also lengthens so that the [Cai] signal remains in phase with the nuclear cycle. (B i) The nuclear division cycle length can be increased experimentally by ∼20% through treatment with cycloheximide. Numbers on x axis represent minutes. Error bars represent SEM. (ii) The [Ca]i peaks remain associated with interphase after cycloheximide treatment. Temperature is 18°C. (C) A schematic view of the embryo showing the plane of the confocal section in this and other figures. Note that the periphery of the section through the embryo provides images of the cortex and that the center of the section looks deeper into the embryo. Images are oriented with the anterior pole being uppermost.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Calcium increases in phase with the interphase cortical contractions in syncytial Drosophila embryos. (A i) [Cai] increases measured by confocal ratio imaging. The three rows of images display CaGr, TMR, and ratio images that represent the spatial distribution of the calcium signals as snapshots in midinterphase and midmitosis. Interphase and mitosis in cycles 8 and 9 are inferred from the timing of the cortical contractions (I and M). In the later two cycles, when the nuclei have come to the egg cortex, their presence is marked by areas of low calcium concentration. They are sometimes faintly visible in the ratiometric image (arrows), and the correlation between nuclei in interphase (I), the cortical contractions, and the calcium increase was noted. The pixel values in the ratiometric image are represented by a conventional rainbow scale, with higher calcium concentrations shown as warmer tones. Red colors in the image correspond to [Cai] in the midmicromolar range, as can be seen by comparison with the calibration shown in (ii). (ii) The temporal pattern of [Ca]i increase in the embryo shown in (i). [Cai] values are means for the whole embryo. Calibrated calcium concentrations are shown at right (see Calibration…signals). Note that as the nuclear division cycle time lengthens, the time between [Cai] signal peaks also lengthens so that the [Cai] signal remains in phase with the nuclear cycle. (B i) The nuclear division cycle length can be increased experimentally by ∼20% through treatment with cycloheximide. Numbers on x axis represent minutes. Error bars represent SEM. (ii) The [Ca]i peaks remain associated with interphase after cycloheximide treatment. Temperature is 18°C. (C) A schematic view of the embryo showing the plane of the confocal section in this and other figures. Note that the periphery of the section through the embryo provides images of the cortex and that the center of the section looks deeper into the embryo. Images are oriented with the anterior pole being uppermost.
Mentions: Fig. 1 A shows fluorescence signals in a Drosophila embryo as it passes through cell cycles 8–11. Increased intracellular free calcium concentration ([Cai]) is detected by quantitative ratiometric imaging in each cell cycle as nuclei enter interphase. The ratio of calcium green dextran (CaGr) and rhodamine dextran fluorescence quantitatively reflects the intracellular calcium concentrations at different points within the confocal section. Red colors represent ≥1 μM calcium, yellow colors represent 0.5–1 μM, green colors represent 0.1–0.5 μM, and blue colors represent concentrations <0.1 μM (calibration shown in Fig. 1 A ii). [Cai] falls during mitosis and is elevated in the cortex of the embryo. Nuclei migrate to the cortex of the embryo during cycle 10. Once the nuclei enter the confocal section, [Cai] is seen in the ratiometric images to be highest around the nuclei in interphase, but it does not increase to the same degree within the nuclei, which appear as circular voids. In these and other images, the plane of confocal section passes through the embryo cortex at the edges of the image, whereas the center of the image represents areas several microns deeper in the embryo (Fig. 1 C). The confocal images are a window into small areas of the embryo cortex.

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