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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 the nucleus and spindle microdomains during mitosis. (A) Confocal ratio images of embryos that were injected with CaGr/TMR during cycle 10. Calcium dynamics in and around an individual nucleus were measured in the region of interest that is displayed on the images (white boxes). Note that individual nuclei move tens of microns as the nuclear division cycle progresses. In this region of interest, calcium increases during interphase before NEB (NEB occurs at the prophase/prometaphase boundary) and again during metaphase/anaphase. The time between images varies (see inset schematic for the timing of this cell cycle relative to pole cell formation). Also note that in order to visualize the nuclei in quasi-equatorial section, these confocal sections are deeper than those of Figs. 1–4 (also shown schematically) so that the nucleus-associated calcium changes are more evident than in previous figures. (B) Quantitative analysis of six similar experiments. Data from the regions of interest are expressed as the ratio versus time. The light blue column indicates the peak interphase calcium signal. This column is significantly different from all columns marked with a light blue star (P < 0.05). The red column represents the peak anaphase calcium signal. This calcium increase is significantly different from all columns marked with a red star (P < 0.05). S, S phase. (C) Comparison of [Cai] in the nucleus and mitotic spindle microdomain (circles) with [Cai] in the embryo section as a whole (bars) during cycle 11 in five embryos at level 1, which is shown in D. The interphase peak of nuclear [Cai] coincides with the cortical interphase [Cai] peak but is of lower magnitude. Note the shallow confocal section that is illustrated schematically and is similar to that in Figs. 1–4. Temperature is 18°C. Error bars represent SEM. (D) Simultaneous imaging of [Cai] using CaGr and of ER using DiIC18. Two pairs of images from an image series are shown, illustrating prophase and metaphase just before anaphase onset in cycle 10. Green, CaGr; red, DiIC18. White encircled areas are of equal size and position. These images show the spatial relationship between CaGr fluorescence and ER but are only indicative of [Cai], as they are nonratiometric. Temperature is 18°C.
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fig5: Calcium increases in the nucleus and spindle microdomains during mitosis. (A) Confocal ratio images of embryos that were injected with CaGr/TMR during cycle 10. Calcium dynamics in and around an individual nucleus were measured in the region of interest that is displayed on the images (white boxes). Note that individual nuclei move tens of microns as the nuclear division cycle progresses. In this region of interest, calcium increases during interphase before NEB (NEB occurs at the prophase/prometaphase boundary) and again during metaphase/anaphase. The time between images varies (see inset schematic for the timing of this cell cycle relative to pole cell formation). Also note that in order to visualize the nuclei in quasi-equatorial section, these confocal sections are deeper than those of Figs. 1–4 (also shown schematically) so that the nucleus-associated calcium changes are more evident than in previous figures. (B) Quantitative analysis of six similar experiments. Data from the regions of interest are expressed as the ratio versus time. The light blue column indicates the peak interphase calcium signal. This column is significantly different from all columns marked with a light blue star (P < 0.05). The red column represents the peak anaphase calcium signal. This calcium increase is significantly different from all columns marked with a red star (P < 0.05). S, S phase. (C) Comparison of [Cai] in the nucleus and mitotic spindle microdomain (circles) with [Cai] in the embryo section as a whole (bars) during cycle 11 in five embryos at level 1, which is shown in D. The interphase peak of nuclear [Cai] coincides with the cortical interphase [Cai] peak but is of lower magnitude. Note the shallow confocal section that is illustrated schematically and is similar to that in Figs. 1–4. Temperature is 18°C. Error bars represent SEM. (D) Simultaneous imaging of [Cai] using CaGr and of ER using DiIC18. Two pairs of images from an image series are shown, illustrating prophase and metaphase just before anaphase onset in cycle 10. Green, CaGr; red, DiIC18. White encircled areas are of equal size and position. These images show the spatial relationship between CaGr fluorescence and ER but are only indicative of [Cai], as they are nonratiometric. Temperature is 18°C.

Mentions: To confirm that calcium increases occurred at prophase and anaphase, as would be predicted from observations in sea urchin embryos (Ciapa et al., 1994; Wilding et al., 1996; Groigno and Whitaker, 1998), we used ratiometric calcium imaging of single nuclei. We screened for [Cai] increases by tracking the [Cai] changes in and around individual nuclei during a nuclear cycle in cycle 10 in six different embryos (Fig. 5, A and B). Note that individual nuclei travel quite large distances along the cortex of the embryo as nuclear divisions progress (Zalokar and Erk, 1976). We chose a level of confocal section that was deeper in the buds than that shown in Fig. 4 (level 2; Fig. 5 D) in order to image nuclear calcium; at this level of confocal section, the cortical increase in [Cai] can be seen only at the very periphery of the deep section through the embryo (Fig. 5 A). Fig. 5 demonstrates that an increase in nuclear [Cai] occurs at a time that coincides with the aforementioned larger global cortical interphase [Cai] increase, and it falls as nuclei enter prometaphase. Peak [Cai] was less than that observed in the whole embryo (Fig. 5 B). In addition, we detected a second [Cai] increase in the mitotic spindle at around the time of anaphase onset (Fig. 5 B). When we tracked nuclei using ratiometric imaging with the 70-kD form of CaGr, which is excluded from the nucleus during interphase, we observed a local [Cai] increase in the spindle at anaphase. However, the NEB-associated signal was absent (unpublished data), confirming that the local [Cai] increase at prophase occurred within the nucleus.


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 the nucleus and spindle microdomains during mitosis. (A) Confocal ratio images of embryos that were injected with CaGr/TMR during cycle 10. Calcium dynamics in and around an individual nucleus were measured in the region of interest that is displayed on the images (white boxes). Note that individual nuclei move tens of microns as the nuclear division cycle progresses. In this region of interest, calcium increases during interphase before NEB (NEB occurs at the prophase/prometaphase boundary) and again during metaphase/anaphase. The time between images varies (see inset schematic for the timing of this cell cycle relative to pole cell formation). Also note that in order to visualize the nuclei in quasi-equatorial section, these confocal sections are deeper than those of Figs. 1–4 (also shown schematically) so that the nucleus-associated calcium changes are more evident than in previous figures. (B) Quantitative analysis of six similar experiments. Data from the regions of interest are expressed as the ratio versus time. The light blue column indicates the peak interphase calcium signal. This column is significantly different from all columns marked with a light blue star (P < 0.05). The red column represents the peak anaphase calcium signal. This calcium increase is significantly different from all columns marked with a red star (P < 0.05). S, S phase. (C) Comparison of [Cai] in the nucleus and mitotic spindle microdomain (circles) with [Cai] in the embryo section as a whole (bars) during cycle 11 in five embryos at level 1, which is shown in D. The interphase peak of nuclear [Cai] coincides with the cortical interphase [Cai] peak but is of lower magnitude. Note the shallow confocal section that is illustrated schematically and is similar to that in Figs. 1–4. Temperature is 18°C. Error bars represent SEM. (D) Simultaneous imaging of [Cai] using CaGr and of ER using DiIC18. Two pairs of images from an image series are shown, illustrating prophase and metaphase just before anaphase onset in cycle 10. Green, CaGr; red, DiIC18. White encircled areas are of equal size and position. These images show the spatial relationship between CaGr fluorescence and ER but are only indicative of [Cai], as they are nonratiometric. Temperature is 18°C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171230&req=5

fig5: Calcium increases in the nucleus and spindle microdomains during mitosis. (A) Confocal ratio images of embryos that were injected with CaGr/TMR during cycle 10. Calcium dynamics in and around an individual nucleus were measured in the region of interest that is displayed on the images (white boxes). Note that individual nuclei move tens of microns as the nuclear division cycle progresses. In this region of interest, calcium increases during interphase before NEB (NEB occurs at the prophase/prometaphase boundary) and again during metaphase/anaphase. The time between images varies (see inset schematic for the timing of this cell cycle relative to pole cell formation). Also note that in order to visualize the nuclei in quasi-equatorial section, these confocal sections are deeper than those of Figs. 1–4 (also shown schematically) so that the nucleus-associated calcium changes are more evident than in previous figures. (B) Quantitative analysis of six similar experiments. Data from the regions of interest are expressed as the ratio versus time. The light blue column indicates the peak interphase calcium signal. This column is significantly different from all columns marked with a light blue star (P < 0.05). The red column represents the peak anaphase calcium signal. This calcium increase is significantly different from all columns marked with a red star (P < 0.05). S, S phase. (C) Comparison of [Cai] in the nucleus and mitotic spindle microdomain (circles) with [Cai] in the embryo section as a whole (bars) during cycle 11 in five embryos at level 1, which is shown in D. The interphase peak of nuclear [Cai] coincides with the cortical interphase [Cai] peak but is of lower magnitude. Note the shallow confocal section that is illustrated schematically and is similar to that in Figs. 1–4. Temperature is 18°C. Error bars represent SEM. (D) Simultaneous imaging of [Cai] using CaGr and of ER using DiIC18. Two pairs of images from an image series are shown, illustrating prophase and metaphase just before anaphase onset in cycle 10. Green, CaGr; red, DiIC18. White encircled areas are of equal size and position. These images show the spatial relationship between CaGr fluorescence and ER but are only indicative of [Cai], as they are nonratiometric. Temperature is 18°C.
Mentions: To confirm that calcium increases occurred at prophase and anaphase, as would be predicted from observations in sea urchin embryos (Ciapa et al., 1994; Wilding et al., 1996; Groigno and Whitaker, 1998), we used ratiometric calcium imaging of single nuclei. We screened for [Cai] increases by tracking the [Cai] changes in and around individual nuclei during a nuclear cycle in cycle 10 in six different embryos (Fig. 5, A and B). Note that individual nuclei travel quite large distances along the cortex of the embryo as nuclear divisions progress (Zalokar and Erk, 1976). We chose a level of confocal section that was deeper in the buds than that shown in Fig. 4 (level 2; Fig. 5 D) in order to image nuclear calcium; at this level of confocal section, the cortical increase in [Cai] can be seen only at the very periphery of the deep section through the embryo (Fig. 5 A). Fig. 5 demonstrates that an increase in nuclear [Cai] occurs at a time that coincides with the aforementioned larger global cortical interphase [Cai] increase, and it falls as nuclei enter prometaphase. Peak [Cai] was less than that observed in the whole embryo (Fig. 5 B). In addition, we detected a second [Cai] increase in the mitotic spindle at around the time of anaphase onset (Fig. 5 B). When we tracked nuclei using ratiometric imaging with the 70-kD form of CaGr, which is excluded from the nucleus during interphase, we observed a local [Cai] increase in the spindle at anaphase. However, the NEB-associated signal was absent (unpublished data), confirming that the local [Cai] increase at prophase occurred within the nucleus.

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