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- Pubmed Central
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- Copyright © 2005, The Rockefeller University Press
- ISSN
- 0021-9525
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- 1540-8140
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- 10.1083/jcb.200503139
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Abstract
JCB: ARTICLE Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos Huw Parry, Alex McDougall, and Michael Whitaker Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne Medical School, Newcastle upon Tyne NE2 4HH, England, UK ell cycle calcium signals are generated by the tions calcium signaling during mitosis. We establish that inositol trisphosphate (InsP )–mediated release of the nuclear divisions in syncytial Drosophila embryos are calcium from internal stores (Ciapa, B., D. Pe- accompanied by both cortical and nuclear localized cal- sando, M. Wilding, and M. Whitaker. 1994. Nature. cium transients. Constructs that chelate InsP also prevent 368:875–878; Groigno, L., and M. Whitaker. 1998. nuclear division. An analysis of nuclear calcium concen- Cell. 92:193–204). The major internal calcium store is trations demonstrates that they are differentially regu- the endoplasmic reticulum (ER); thus, the spatial organi- lated. These observations demonstrate that mitotic calcium zation of the ER during mitosis may be important in shap- signals in Drosophila embryos are confined to mitotic ing and defining calcium signals. In early Drosophila microdomains and offer an explanation for the apparent melanogaster embryos, ER surrounds the nucleus and absence of detectable global calcium signals during mitosis mitotic spindle during mitosis, offering an opportunity to in some cell types. determine whether perinuclear localization of ER condi- Introduction Calcium signals have been shown to play an important regula- The source of calcium for signals during mitosis is the tory role in controlling the cell division cycle of early sea urchin ER (Ross et al., 1989; Ciapa et al., 1994). The ER gathers (Ciapa et al., 1994; Wilding et al., 1996; Groigno and Whitaker, around the nucleus as mitosis approaches and is closely associ- 1998), frog (Miller et al., 1993; Snow and Nuccitelli, 1993; Muto ated with the mitotic spindle (Harel et al., 1989). The ER–spindle et al., 1996), and mammalian embryos (Tombes et al., 1992; complex can be isolated and shown to sequester calcium (Sil- Nixon et al., 2002). Cell cycle calcium signals activate calmodu- ver et al., 1980). ER membranes pervade the mitotic spindle lin (Lu and Means, 1993; Takuwa et al., 1995; Török et al., (Harris, 1975), so is possible that calcium released very lo- 1998), and calmodulin kinase II is required for mitosis in both cally to calcium-binding sites over micron length scales may sea urchin embryos (Baitinger et al., 1990) and somatic cells provide signals at the chromosomes and spindle poles. Very (Patel et al., 1999). Nonetheless, despite clear evidence that local signals of this kind are probably not detectable with current blocking calcium signals prevents mitosis, in many cases, puta- imaging technologies. tive mitotic calcium signals are small or undetectable (Tombes During the early syncytial nuclear divisions of Drosoph- and Borisy, 1989; Kao et al., 1990; Tombes et al., 1992; Wilding ila melanogaster embryos, ER becomes highly concentrated et al., 1996; Whitaker and Larman, 2001). The absence of cal- around the nucleus at prophase and is very closely associated cium signals during mitosis in some higher eukaryotic cell types with the spindle poles; however, the ER does not invade the under some conditions implies that calcium regulation of mitosis spindle itself (Bobinnec et al., 2003). This circumstance offers is not a universal signaling mechanism in higher eukaryotes. the opportunity to image calcium concentrations within the nucleus and mitotic spindle without the complication of colo- calized ER. It also offers the opportunity to test whether the Correspondence to Michael Whitaker: [email protected] interaction between ER and mitotic spindle creates a calcium- Abbreviations used in this paper: CaGr, calcium green dextran; [Ca ], intra- i signaling environment that is distinct from bulk cytoplasm. cellular free calcium concentration; DiIC , 1,1-dioctadecyl-3,3,3,3-tetra- The amenable genetics of Drosophila has allowed the methylindocarbocyanine perchlorate; InsP , inositol trisphosphate; NEB, nuclear envelope breakdown; TMR, tetramethylrhodamine. identification of a plethora of gene products that are directly © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 171, No. 1, October 10, 2005 47–59 http://www.jcb.org/cgi/doi/10.1083/jcb.200503139 JCB 47 THE JOURNAL OF CELL BIOLOGY involved in regulating the cell division cycle (Gonzalez et al., Results 1994; Sullivan and Theurkauf, 1995). Many are homologues of regulators that are important in controlling mammalian cell Early Drosophila development is marked by 13 rapid nuclear cycles. A number of cell cycle regulatory genes were first divisions that occur in the same cytoplasm without cytokinesis identified through their effects on the cell cycles of various (Foe and Alberts, 1983; Foe et al., 1993). Dividing nuclei are early embryos (Evans et al., 1983; Gautier et al., 1988; Sunkel first located deep within the embryo; they migrate to the embryo and Glover, 1988; Glover et al., 1991, 1995; Edgar and Lehner, cortex during cycles 8 and 9, and nuclei divide just beneath the 1996). Calcium gradients may help determine the dorso–ventral surface of the embryo during cycles 10–13. During cycles 10–13, axis in Drosophila (Creton et al., 2000), but nothing is known superficial nuclei undergo mitosis asynchronously, giving ap- about calcium signaling in the fly’s early embryonic cell cycles. pearance to mitotic waves that originate simultaneously at In this study, we demonstrate that calcium regulates nuclear both anterior and posterior embryonic poles. At 25C, the division during early embryonic cell cycles and go on to show waves move from pole to equator in 30 s, as determined in that the ER surrounding the nuclear compartment encloses a fast-frozen embryos (Foe and Alberts, 1983). At 18C, we calcium-signaling microenvironment that controls mitosis. find that the mitotic waves are substantially slower, whereas Figure 1. Calcium increases in phase with the interphase cortical contractions in syncytial Drosophila embryos. (A i) [Ca] increases measured by confocal ratio imaging. The three rows of images display CaGr, TMR, and ratio images that represent the spatial distribu- tion of the calcium signals as snapshots in mid- interphase and midmitosis. Interphase and mi- tosis 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 ratio- metric 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 im- age correspond to [Ca ] in the midmicromolar range, as can be seen by comparison with the calibration shown in (ii). (ii) The temporal pat- tern of [Ca] increase in the embryo shown in (i). [Ca ] values are means for the whole em- bryo. Calibrated calcium concentrations are shown at right (see Calibration...signals). Note that as the nuclear division cycle time lengthens, the time between [Ca ] signal peaks also lengthens so that the [Ca ] signal remains in phase with the nuclear cycle. (B i) The nu- clear division cycle length can be increased experimentally by 20% through treatment with cycloheximide. Numbers on x axis repre- sent minutes. Error bars represent SEM. (ii) The [Ca] peaks remain associated with inter- phase after cycloheximide treatment. Temper- ature is 18C. (C) A schematic view of the em- bryo 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. 48 JCB • VOLUME 171 • NUMBER 1 • 2005 Table I. Mean peak and trough [Ca ] in cortical confocal sections in cycles 8–13 Cycle 8 Cycle 9 Cycle 10 Cycle 11 Cycle 12 Cycle 13 Trough Ratio 1.03 0.019 1.02 0.014 1.02 0.025 1.00 0.013 1.00 0.015 1.00 0.009 [Ca ] (M) 0.13 0.018 0.12 0.013 0.12 0.023 0.10 0.012 0.10 0.013 0.10 0.008 Peak Ratio 1.56 0.100 1.57 0.112 1.34 0.025 1.38 0.042 1.36 0.058 1.23 0.033 [Ca ] (M) 1.01 0.305 1.04 0.354 0.52 0.042 0.59 0.078 0.55 0.103 0.35 0.044 n 4 6 10 10 8 3 Assumes K of 1 M for CaGr. cycle times are only slightly lengthened, allowing the waves tions at different points within the confocal section. Red colors to be imaged much more readily with confocal microscopy. represent 1 M calcium, yellow colors represent 0.5–1 M, green colors represent 0.1–0.5 M, and blue colors represent Calcium changes occur in syncytial concentrations 0.1 M (calibration shown in Fig. 1 A ii). [Ca ] Drosophila embryos in fixed phase falls during mitosis and is elevated in the cortex of the embryo. relation with the nuclear division cycle Nuclei migrate to the cortex of the embryo during cycle 10. Once Fig. 1 A shows fluorescence signals in a Drosophila embryo as it the nuclei enter the confocal section, [Ca ] is seen in the ratiomet- passes through cell cycles 8–11. Increased intracellular free cal- ric images to be highest around the nuclei in interphase, but it cium concentration ([Ca ]) is detected by quantitative ratiometric does not increase to the same degree within the nuclei, which ap- imaging in each cell cycle as nuclei enter interphase. The ratio of pear as circular voids. In these and other images, the plane of calcium green dextran (CaGr) and rhodamine dextran fluores- confocal section passes through the embryo cortex at the edges cence quantitatively reflects the intracellular calcium concentra- of the image, whereas the center of the image represents areas Figure 2. The [Ca ] increases are slow, periodic calcium waves that originate at both poles of the embryo and annihilate upon collision. (A) The images display the CaGr/TMR confocal ratio of a section through an em- bryo in cycle 9. Slow calcium waves are seen moving toward the equator of the embryo from the poles and annihilating upon collision. The calcium wave speed in this cycle is 0.4 m/s (Table II). (B) Topographical representation of another slow calcium wave during cycle 10. The polar origin of the calcium wave is evident. The waves propagate cortically toward the equator of the embryo. (C) The slow calcium waves precede the wave of cortical contraction, which is seen as retraction of the plasma membrane away from the vitelline membrane. The images display CaGr/TMR confocal ratio images in a cycle 8 embryo. The slow cortical calcium wave moves vertically downwards away from the anterior pole, as indicated by the movement of arrowheads. The displacement of both the calcium wave and the cortical contraction are progressive. The arrowheads mark the region of highest calcium increase in each successive image, and the white arrow marks the starting position of the calcium increase at time 0. The leading edge of the cortical contraction is marked by a yellow dot. Note that in this image, the embryo equator is beyond the bottom of the images and that the widest part of the image at the top is a property of the confocal section; it is not the embryo equator, which lies beyond the bottom of the images. Temperature is 18C. CALCIUM WAVES IN SYNCYTIAL DROSOPHILA EMBRYOS • PARRY ET AL. 49 Table II. Wave speeds during cycles 9–12 of the wave toward the equator and out of the frame. Thus, the calcium signal shows the same behavior as mitotic waves, Cycle 9 Cycle 10 Cycle 11 Cycle 12 originating at both embryonic poles and moving toward the Embryo 1 0.47 0.37 0.36 0.29 equator. Table II gives the mean wave velocity during syncy- Embryo 2 0.42 0.29 0.32 0.29 tial division cycles from cycles 9–12. The wave speed slows Embryo 3 0.46 0.46 0.42 0.25 with each cycle, decreasing from 0.45 to 0.29 m/s. As the nu- Embryo 4 0.47 0.36 0.36 0.31 clear division cycle time slows, the wave becomes progressively Embryo 5 0.43 0.43 0.40 0.31 Mean speed slower; the product of wave speed and cycle time remains con- (v) 0.45 0.02 0.38 0.09 0.37 0.05 0.29 0.06 stant, which is a further indication of the entrainment of cal- Cycle time cium wave and nuclear cycle. Fig. 2 C shows a single [Ca ] (t; min) 9.3 11.0 11.3 14.4 wave in a cycle 8 embryo before the nuclei have reached the Product (v t) 4.15 4.18 4.18 4.18 cortex. As the wave progresses toward the equator, it is fol- lowed by a cortical constriction that represents a cortical con- Wave speeds in micrometers/second were calculated by measuring the time taken for the center of the wave to cover a fixed distance of 170 m at either the traction travelling at the same velocity. The constriction can be anterior or posterior end (embryo 4 only) of an embryo. All measurements were seen in the confocal section, as the movement of the plasma made on embryos maintained at 18C. membrane away from the vitelline membrane creates a dye- free perivitelline space that appears black beneath the autofluo- several microns deeper in the embryo (Fig. 1 C). The confocal rescent vitelline membrane. The time that elapsed between the images are a window into small areas of the embryo cortex. leading edge of the calcium wave and the leading edge of the This pattern of calcium oscillation, with maximum ratio constriction is 90 s. We observed the association between increases in interphase, was seen in all 29 examined embryos. wave and constriction in three of three embryos, suggesting The oscillation remains in phase during the four nuclear cycles that the [Ca ] increase causes the cortical contraction. as cycle time lengthens with each nuclear division (Fig. 1 A). InsP -induced calcium release is required The increases were analyzed quantitatively in 17 embryos at 3 for mitotic progression various cell cycle stages. The data are shown in Table I. Mean [Ca ] in the trough during mitosis ranged from 0.10 0.008 to Cell cycle calcium signals in other early embryos are triggered 0.13 0.018 m across five nuclear cycles (Table I), and by inositol trisphosphate (InsP ; Ciapa et al., 1994; Muto et al., mean peak [Ca ] was 1.01 0.305 m in cycle 8, falling grad- 1996; Groigno and Whitaker, 1998). Drosophila possesses a ually to 0.35 0.044 m in cycle 13. Also note from the im- single insect-specific InsP receptor isoform (Hasan and Ros- ages of Fig. 1 that local [Ca ] continues to reach micromolar bash, 1992; Yoshikawa et al., 1992). Deletion of the InsP re- i 3 levels during each interphase in areas of the cortex surrounding ceptor arrests larval development at second instar, and embryos the nuclei, even in later nuclear cycles. These observations in- show defects in cell division and endoreplication (Acharya et dicate that calcium oscillations occur in fixed phase relation al., 1997). Embryonic development to second instar also re- with the nuclear cycle in syncytial embryos and that [Ca ] is quires the InsP receptor because no viable eggs or embryos i 3 highest in interphase at the time of maximum cortical contrac- were generated from germ line clones lacking the receptor tion (Foe et al., 1993). (Acharya et al., 1997). Fig. 3 A shows that InsP receptors are Nuclear division cycles in syncytial embryos are sensi- functional in early embryos; the microinjection of InsP leads tive to protein synthesis inhibitors (Boring et al., 1989); at lim- to calcium release. [Ca ] was measured using CaGr, and local- iting concentrations of cycloheximide, the division cycles are ization of the injected InsP was determined by coinjection of slowed but not blocked (Fig. 1 B). We injected cycloheximide rhodamine dextran with InsP . Although InsP was injected 3 3 (0.5 g/ml at final concentration) to increase the duration of into the body of the embryo, [Ca ] rose at the cortex predomi- cycles 8–12 from 65.9 1.2 to 79.1 1.4 min (mean and nantly, and there is also a cortical contraction response. This SEM). Fig. 1 B shows that calcium oscillations continue in experiment shows that InsP -induced calcium release causes phase with the nuclear division cycle in cycloheximide-treated cortical contraction. embryos, demonstrating a close mutual entrainment of calcium It was important to establish that InsP -triggered calcium oscillations and the nuclear division cycle. signals are required for nuclear division in syncytial embryos. One way of specifically interfering with InsP signaling is to Calcium changes take the form of slow use a dominant-negative approach and introduce InsP -binding calcium waves that travel from pole to proteins or binding domains into the cytoplasm (Takeuchi et equator during each nuclear division cycle al., 2000; Walker et al., 2002). We microinjected an InsP and precede the cortical contraction sponge polypeptide consisting of the InsP -binding domain of When displayed at higher temporal resolution, the [Ca ] type 1 InsP receptor (Walker et al., 2002) into embryos at the i 3 changes have a spatial substructure. Fig. 2 A shows two exam- start of cycle 11 and compared its effects with a control sponge ples of the spatial pattern of [Ca ] increase. In the top panel, in which two point substitutions had been made in the InsP - i 3 two [Ca ] waves are arriving from poles at the equator of the binding region to produce a polypeptide with no detectable embryo and are annihilating there. In Fig. 2 B, the initiation of InsP -binding affinity (Walker et al., 2002). Fig. 3 B (i) shows a calcium wave at the anterior pole is followed by progression that 80% of embryos injected with the wild-type sponge arrested 50 JCB • VOLUME 171 • NUMBER 1 • 2005 Figure 3. Functional InsP receptors in Dro- sophila embryos. (A) To determine whether Drosophila embryos had functional InsP cal- cium release channels, InsP was injected into cycle 8 embryos. The site of injection is indi- cated by an asterisk. (i) Microinjection of InsP (pipette concentration of 100 M; final con- centration of 200 nM) at 67 s increases [Ca] 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) InsP was coin- jected 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). InsP microinjection causes a large calcium increase, indicating that functional InsP receptors are present. The cortical contraction is visible in these images by 8–9 s after the injection of InsP (iii) The time course of CaGr fluores- cence increase in the embryo, corresponding to an increase in [Ca ]. The embryo was in cycle 8. (B) Effects of wild-type and control (inactive) InsP sponge. (i) Nuclear cycle pro- gression fails after microinjection of the InsP sponge during interphase of cycle 10 (20 mg/ml in pipette; 40 g/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 fluo- rescein histones during mitosis of cycle 11 us- ing the same microinjection protocol. With wild-type sponge, NEB, chromatin condensa- tion, and metaphase plate formation occur normally, but anaphase onset is delayed, and chromosomes fail to separate. In contrast, em- bryos that were injected with the control sponge in parallel experiments have reformed normal nuclei in cycle 11; chromatin decon- densation after mitosis occurs but is abnormal. (C) Effects of p130 InsP -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 inter- phase of the following cycle, cycle 12. At in- termediate concentrations, nuclei arrest with condensed chromosomes in metaphase of cy- cle 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 M 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 18C. In these experiments, the time of pole bud formation was not recorded. Bars, 30 m. their nuclear division within one cycle, whereas the remainder nuclei rapidly sank from the field of view and could not be fol- arrested in the next division cycle. A comparison of histone- lowed further but remained in a state of arrest for as long as they tagged chromatin indicates that the embryos microinjected with were visible in deep confocal sections (not depicted). wild-type sponge form normal metaphase figures but that ana- Because nuclei in the syncytial embryo are not separated phase failed to take place. The spindle and chromatin also elon- by plasma membranes, the syncytial embryo offers the possibil- gated (some with the dumbell shape that is characteristic of ana- ity of generating a gradient of inhibitor within the cytoplasm so phase bridges). Chromosomes failed to separate, decondensing to that individual nuclei will experience different concentrations of form the same number of nuclei as before (Fig. 3 B ii). Then, the inhibitor. GFP::p130 is a PLC orthologue that is catalytically CALCIUM WAVES IN SYNCYTIAL DROSOPHILA EMBRYOS • PARRY ET AL. 51 Figure 4. 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 [Ca ] 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 DiIC 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 52 JCB • VOLUME 171 • NUMBER 1 • 2005 inactive, binds InsP with high affinity by virtue of its pleck- These experiments demonstrate that InsP -triggered cal- 3 3 strin homology domain, and inhibits calcium signaling when cium release is a signal that is necessary for both entry into mi- overexpressed in cells or when added to permeabilized cells tosis and for anaphase onset in syncytial Drosophila embryos, (Takeuchi et al., 2000). The GFP::p130 chimera was localized as it is in sea urchin embryos (Twigg et al., 1988a; Ciapa et al., to plasma membrane and apparently to ER in interphase, en- 1994; Groigno and Whitaker, 1998). tered the nucleus at prophase, and associated with the mitotic Microdomains of elevated calcium that spindle (Fig. 3 C i). By microinjecting GFP::p130 at one pole are separated by ER-rich low calcium of the embryo during early anaphase of cycle 11, we were able domains are observed in cortical buds to generate a gradient of GFP::p130 of 2–10 M, which was confirmed by the distribution of fluorescence (Fig. 3 C ii) that Once nuclei reach the surface, it is possible to stage the nuclear persisted for the course of the experiment. The outcome was cycle precisely. Fig. 4 shows the spatial distribution of the inter- striking. Nuclei that were exposed to 10 M GFP::p130 ar- phase [Ca ] increase from metaphase through interphase to rested before nuclear envelope breakdown (NEB) in cycle 12. metaphase of the next cycle, as seen in glancing tangential con- Nuclei that were exposed to intermediate concentrations con- focal sections (Fig. 1 C); this is compared with the disposition of tinued through NEB of cycle 12 but arrested after mitosis en- mitotic spindles, ER, and actin. CaGr fluorescence (Fig. 4 A, i try and were unable to complete mitosis, whereas nuclei that and ii) indicates that the major [Ca ] increase occurs in inter- were exposed to 2 M progressed through mitosis of cycle 12 phase in a cortical region surrounding the nuclei but is separated and arrested before mitosis in cycle 13. Simultaneous imaging from interphase nuclei by a region of low calcium concentra- of histone and GFP signals demonstrated that nuclei entered tion. As nuclei enter mitosis, the cortical [Ca ] levels fall overall, metaphase but failed to enter anaphase rapidly, just as we had and the signal becomes confined to narrower regions surround- found with the InsP sponge (Fig. 3 C iii). Chromatin decon- ing the mitotic spindle. [Ca ] in the nucleus and mitotic spindle 3 i densation occurred in the arrested nuclei after a delay (Fig. 3 appears higher than in the circumnuclear region but much lower C iii) and elongated, and dumbell-shaped nuclei were also than in the cortical region. These images cannot be compared di- seen (not depicted), although this occurred a few minutes later rectly to those of Fig. 1, as ratiometric methods cannot be used than we had observed after the microinjection of InsP sponge when simultaneously measuring rhodamine-tagged cytoskeletal constructs. These observations demonstrate that InsP signal- components. Moreover, the increased detection sensitivity that ing plays a role in mitosis entry at NEB as well as in mitosis is required to visualize CaGr fluorescence in the nucleus and exit in Drosophila embryos. In GFP::p130-injected embryos, spindle leads to saturation of the cortical CaGr signal because of the mitotic wave (the wave of NEB and anaphase onset) trav- the limited dynamic range of the confocal microscope. elled in the opposite direction to that observed in controls (that Fig. 4 B shows, in a separate experiment, changes in ER is, from the farther embryonic pole), indicating that InsP is distribution during the nuclear division cycle, which was visu- involved in the initiation and propagation of the wave. alized using DiI, a lipophilic carbocyanine dye that labels ER Heparin and Xestospongin C are agents that inhibit the and other elements of the ER/Golgi/endosome system (Terasaki interaction of InsP with the InsP receptor (Ghosh et al., 1988; and Jaffe, 1991). The pattern of DiI fluorescence is identical to 3 3 Gafni et al., 1997). Embryos that were microinjected with ei- that reported for an ER-localized GFP-tagged protein in early ther the inhibitor of InsP -induced calcium release, heparin (80 Drosophila embryos (Bobinnec et al., 2003). The ER extends g/ml gave half maximum inhibition; n 4; Groigno and Whit- diffusely into the space between nuclei during interphase and is aker, 1998), or Xestospongin C (10 mM of pipette concentra- markedly concentrated immediately around the mitotic spindle tion; n 6; Hu et al., 1999) also showed a block in mitosis that during mitosis (Fig. 4 D). It is excluded from the spindle until was similar to what we observed with both the InsP sponge late telophase, when ER invades the spindle in the region of the and GFP::p130 (unpublished data). As far as is known, the midbody. The pattern of distribution of cortical ER (Fig. 4 B) is InsP receptor is the sole signaling target of InsP in cells (for linked to the pattern of [Ca ] increase, with [Ca ] being highest 3 3 i i review see Fukuda and Mikoshiba, 1997; Mikoshiba, 1997). in the interstices between ER accumulation around the nuclei rhodamine-labeled actin and CaGr to determine the spatial relation between actin and [Ca] . (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] increase follows the pattern of actin distribution closely throughout the nuclear cycle. Embryo is in cycle 11. Bars, 50 m. (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 [Ca ], embryos were coinjected with fluorescein-labeled actin and DiIC 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 [Ca ] levels during interphase, when the actin caps are present in cortical buds, with those at ana- phase. The [Ca] 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 [Ca ] 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 DiIC 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 [Ca ] 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 18C. CALCIUM WAVES IN SYNCYTIAL DROSOPHILA EMBRYOS • PARRY ET AL. 53 Figure 5. 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 [Ca ] in the nucleus and mitotic spindle microdomain (circles) with [Ca ] in the embryo section as a whole (bars) i i 54 JCB • VOLUME 171 • NUMBER 1 • 2005 and spindles (Fig. 4, A i and C i). Fig. 4 C (ii) shows the pattern at the very periphery of the deep section through the embryo of distribution of cortical actin during the nuclear division cycle, (Fig. 5 A). Fig. 5 demonstrates that an increase in nuclear [Ca ] which was visualized using rhodamine-actin simultaneously occurs at a time that coincides with the aforementioned larger with CaGr (Fig. 3 C i). There is a close correspondence between global cortical interphase [Ca ] increase, and it falls as nuclei the distribution of actin and regions of highest [Ca ] increase. enter prometaphase. Peak [Ca ] was less than that observed in i i During mitosis, actin is localized to the interstices between ER the whole embryo (Fig. 5 B). In addition, we detected a second that were noted above. The ER appears to isolate the nucleus [Ca ] increase in the mitotic spindle at around the time of and mitotic spindle from these regions of highest [Ca ] as nuclei anaphase onset (Fig. 5 B). When we tracked nuclei using ratio- enter and progress through mitosis (Fig. 4 D). metric imaging with the 70-kD form of CaGr, which is ex- The interphase [Ca ] increase occurs at the very periphery cluded from the nucleus during interphase, we observed a lo- of cortical buds that surround the interphase nuclei (Fig. 4 E i). cal [Ca ] increase in the spindle at anaphase. However, the Cortical mitotic spindles are anchored by actin caps that sur- NEB-associated signal was absent (unpublished data), con- round each nucleus in interphase (Fig. 4 E ii, cartoon; Sullivan firming that the local [Ca ] increase at prophase occurred and Theurkauf, 1995; Foe et al., 2000). The actin caps are within the nucleus. pushed further apart as the spindles extend at anaphase. In late To make a direct comparison between cortical and nuclear telophase, the actin wraps around the reforming interphase nu- calcium concentrations in individual cortical buds, we used ratio- cleus to give twice as many actin caps as were present before metric imaging with confocal sections at the shallower level used nuclear division (Fig. 4 E ii). Nuclei, therefore, occupy a in Fig. 4 to enable us to visualize both cortical and nuclear cal- greater area of the cortex in mitosis compared with interphase, cium simultaneously (level 1; Fig. 5 D). We found the calcium which gives rise to substantial oscillatory translation of nuclei concentration in the nuclear microdomain to be significantly in the plane of the cortex as the mitotic wave progresses along lower than cortical calcium at prophase and significantly higher the embryo (Zalokar and Erk, 1976). When localization of the than cortical calcium levels at anaphase onset (Fig. 5 C). These interphase [Ca ] increase, actin, and ER distribution are com- experiments also confirmed that the peak of nuclear calcium at pared in confocal sections normal to the surface (Fig. 4 E, i and prophase coincided with the peak of cortical calcium concentra- ii), it is evident that the [Ca ] increase occurs throughout the tion. Simultaneous imaging of [Ca ] and ER at metaphase just i i cortex in each cap but is markedly higher in the regions of before anaphase onset showed that [Ca ] in the spindle was highest actin concentration. confined to the space enclosed by ER (Fig. 5 E). As predicted, the halo of ER that surrounds the nucleus These data demonstrate the existence of nuclear micro- and mitotic spindle appears to separate two distinct calcium domains of calcium concentration that act as triggers for mitosis microdomains: a region of high calcium in the subcortex, entry and exit. which is associated with actin and contraction in interphase, and a region of lower nuclear calcium. Calcium concentrations Discussion are lowest where the ER is most dense. The interphase [Ca ] signal is linked to [Ca ] increases occur in the nucleus and contraction, the actin cytoskeleton, and mitotic spindle microdomains at both the cortical ER prophase and anaphase The interphase [Ca ] increase occurs very close to the surface To confirm that calcium increases occurred at prophase and of the embryo in the space between the nucleus and its cap that anaphase, as would be predicted from observations in sea ur- contains both actin and ER. High [Ca ] correlates with the chin embryos (Ciapa et al., 1994; Wilding et al., 1996; Groigno phase of cortical contraction that is associated with interphase and Whitaker, 1998), we used ratiometric calcium imaging of nuclei. Alternating bands of contraction/relaxation pass along single nuclei. We screened for [Ca ] increases by tracking the the embryo as the calcium signal progresses, giving rise to [Ca ] changes in and around individual nuclei during a nuclear large oscillations in nuclear position that were observed in the cycle in cycle 10 in six different embryos (Fig. 5, A and B). cortex, which are referred to as yolk contractions (Foe et al., Note that individual nuclei travel quite large distances along 1993) and are inhibited by cytochalasins (Hatanaka and Odada, the cortex of the embryo as nuclear divisions progress (Zalo- 1991). Progressive, slow calcium waves have been observed in kar and Erk, 1976). We chose a level of confocal section that the cleavage furrows of early fish embryos (Webb and Nucci- was deeper in the buds than that shown in Fig. 4 (level 2; Fig. telli, 1985; Fluck et al., 1991; Chang and Meng, 1995; Webb et 5 D) in order to image nuclear calcium; at this level of con- al., 1997; Lee et al., 2003; Webb and Miller, 2003), and [Ca ] focal section, the cortical increase in [Ca ] can be seen only increases have also been recorded in frog embryos (Steinhardt during cycle 11 in five embryos at level 1, which is shown in D. The interphase peak of nuclear [Ca ] coincides with the cortical interphase [Ca ] peak but i i is of lower magnitude. Note the shallow confocal section that is illustrated schematically and is similar to that in Figs. 1–4. Temperature is 18C. Error bars represent SEM. (D) Simultaneous imaging of [Ca ] using CaGr and of ER using DiIC . Two pairs of images from an image series are shown, illustrating i 18 prophase and metaphase just before anaphase onset in cycle 10. Green, CaGr; red, DiIC . 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 [Ca ], as they are nonratiometric. Temperature is 18C. CALCIUM WAVES IN SYNCYTIAL DROSOPHILA EMBRYOS • PARRY ET AL. 55 et al., 1974; Miller et al., 1993; Snow and Nuccitelli, 1993; that of maintaining distinct calcium microdomains during cell Muto and Mikoshiba, 1998). A calcium signal at cytokinesis division. Nuclear calcium has also been shown in some cell has also been shown to be essential for the insertion of new types to be regulated differentially to cytoplasmic calcium membrane into the cleavage furrow in the sea urchin embryo (Badminton et al., 1998; MacDonald, 1998), but this is thought (Shuster and Burgess, 2002). During pseudocleavage in syncy- to be a result of the properties of the nuclear envelope rather tial Drosophila embryos, membrane addition from endosomes than of an accumulation of ER around the nucleus. Although it is essential for actin recruitment and furrow elongation (Riggs was originally proposed that a nuclear envelope persisted et al., 2003). Calcium signals have been found to be associated throughout mitosis as a spindle envelope during syncytial nu- with cortical contraction in ascidian and fish embryos (Roegiers clear divisions (Harel et al., 1989), it is now clear that the nu- et al., 1995; Leung et al., 1998). cleus becomes permeable to high molecular weight molecules early in prophase (that is, at the same time as in other cells) but InsP and the InsP receptor are 3 3 that nuclear lamins persist until metaphase, disappearing before essential for nuclear division anaphase onset (Paddy et al., 1996). Thus, the nuclear envelope We determined that InsP receptors were functional in early em- does not exist during mitosis to provide a diffusion barrier that bryos by eliciting calcium release in response to InsP injection. would allow the mechanisms regulating calcium in intact nu- A genetic approach to determine the importance of InsP signal- clei to operate. On the other hand, the persistence of nuclear ing during rapid syncytial nuclear divisions of the Drosophila lamins may explain why the ER remains outside the spindle embryo does not easily present itself. In fact, despite the ubiq- until late anaphase in syncytial embryos. uity and importance of calcium signaling (Berridge et al., 2000), We show that it is possible to apply cell physiology meth- very few genetic disorders that are caused by defects in calcium- ods to early Drosophila embryos to study calcium signaling. Our signaling components have been identified; the strong as- data clearly demonstrate for the first time in a protostome em- sumption is that an overwhelming majority of genetic calcium bryo that maneuvers designed to prevent calcium signals arrest signaling defects are embryonic lethals (Rizzuto and Pozzan, the nuclear division cycle and that calcium signals are responsi- 2003). Instead, we microinjected constructs that have been ble for the waves of mitosis observed in syncytial Drosophila shown to chelate InsP . We used a GFP-tagged InsP -binding embryos. We also show for the first time that the nucleus and 3 3 protein to determine the cytoplasmic concentration of injected spindle exist within a calcium-signaling microdomain and that proteins. We determined the inhibitory concentration that blocks calcium increases that are necessary for progress through mitosis both NEB and anaphase onset to be 2–10 M, which are con- are small and localized. This has been possible because ER is ex- centrations comparable with those previously observed to block cluded from the Drosophila spindle during mitosis. In other em- InsP -mediated events (Takeuchi et al., 2000) and are similar to bryos and in mammalian somatic cells, ER is an intimate spindle those observed with an InsP sponge (Uchiyama et al., 2002). component. Signals that are local to the spindle are less readily Thus, InsP signaling leading to calcium transients is essential detected, perhaps explaining why calcium signals are not always for NEB and anaphase onset, as it is in early sea urchin embryos observed during mitosis in some cell types. (Poenie et al., 1985; Steinhardt and Alderton, 1988; Twigg et al., 1988b; Ciapa et al., 1994; Wilding et al., 1996; Groigno Materials and methods and Whitaker, 1998). As observed in the sea urchin embryo (Groigno and Whitaker, 1998), the block to anaphase onset was Preparation of embryos for microinjection characterized by absence of chromatin disjunction, but spindle Drosophila embryos (strained with Oregon R) were used for all presented experiments. Flies were kept at RT in plastic bottles containing a solid food elongation and chromatin decondensation did occur, often with base (Elgin and Miller, 1980) with breathable stoppers. Optimum egg lay- a delay. The ER isolates the nucleus during mitosis and gener- ing occurred 21 d after egg deposition. The adult flies were transferred to a ates local nuclear calcium signals via InsP . 3 fresh glass bottle containing a 5 10-cm strip of chromatography paper (3 MM; Whatman) to provide a place for the flies to rest and to decrease hu- Cell cycle calcium signals that govern mitosis are not midity. The bottle was capped with a 2.5% agar plate (small petri dishes fit prominent in syncytial Drosophila embryos. We show that this the bottle necks) that was inverted and left for 30 min. The first collection is because the ER generates calcium-signaling microdomains was discarded, and subsequent collections were used experimentally. Ad- hesive tape (magic 3M; Scotch) was affixed to one side of a 22 64-mm within the cortical bud: one beneath the plasma membrane of coverslip by double-sided tape. Glue was prepared by dissolving the adhe- the cortical buds and the other within the nucleus and mitotic sive of Scotch tape in heptane, and the glue–heptane solution was pipetted spindle. There is a real possibility that the very different molec- onto the center of the coverslip and allowed to dry. Embryos were placed on the adhesive-coated coverslip. The coverslip supporting the embryos was ular environments are, in part, responsible for the different transferred to a large petri dish containing silica gel crystals. The embryos fluorescence signals that we measured in these different micro- were desiccated for 10 min (causing the loss of 5% cell volume) and cov- domains. However, at metaphase, the calcium concentrations ered with mineral oil during injection and imaging (halocarbon oil; 50% ha- locarbon 27 and 50% halocarbon 700; Sigma-Aldrich) to prevent further that are reported by fluorescence reporters are uncorrelated, desiccation. Any embryos that developed wrinkles during desiccation were implying that calcium rises in only the spindle microdomain. discarded. The embryos were injected immediately after desiccation. Although it has been clear for some time that ER associ- ates with the nucleus and spindle (Terasaki and Jaffe, 1991), Chemicals 5,5dibromoBAPTA (tetrapotassium salt) and fluorescent dyes were pur- this has been interpreted as a mechanism to ensure proportionate chased from Invitrogen. Cycloheximide and Xestospongin C were purchased inheritance of ER when cells divide (Barr, 2002). In this study, from Calbiochem. The majority of all other chemicals were purchased from we demonstrate an additional, essential, and novel function— Sigma-Aldrich. 56 JCB • VOLUME 171 • NUMBER 1 • 2005 2 Microinjection where F is the fluorescence at “resting” calcium ([Ca ] ), which we have r r Drawn borosilicate glass micropipettes (GC150F-10; Clarke Electromedi- taken to be 100 nM during mitosis of cycle 10. Gillot and Whitaker for CaGr1 (when coupled to a 10-kD dextran) to cal) were loaded with injection solution and advanced toward immobi- (1994) calculated the K for CaGr1 in the Drosophila em- lized Drosophila embryos by using an Eppendorf microinjection system. be 2 M in the sea urchin egg. The K All fluorescent probes for microinjection were dissolved in injection solu- bryo is likely to be lower as a result of the ionic strength of the Drosophila tion (Ashburner et al., 2005) except Xestospongin C (Gafni et al., 1997), embryo’s cytoplasm, which is intermediate between that of marine and which was dissolved in DMSO for microinjection. The embryos were in- vertebrate embryos (Van der Meer and Jaffe, 1983). Accordingly, the cal- jected using gas pressure (pneumatic picopump; World Precision Instru- cium concentration in Fig. 1 has been calibrated by using two values for of 1 and 2 M, respectively. ments, Inc.). Cytoplasmic concentrations were calibrated by first measur- the K ing the size of droplets that were injected into the oil before injection into Protein expression the embryo. Embryos are 470 160 m but can vary in length and di- The GFP::p130 domain construct (Takeuchi et al., 2000) was obtained ameter considerably. The approximate volume of an embryo is 6.5 nl, from M. Katan (Imperial College, London, UK) and was cloned into the which was calculated by considering the volume of an ellipsoid of the expression vector pGEX-6-p1 (GE Healthcare) as follows: GFP::p130 was above dimensions. The volume of liquids that were injected into the em- cut with HindIII, and the 1.3-kb fragment was cloned into pBC SK ( ) di- bryo was estimated by measuring the diameter of a droplet injected under gested with HindIII. The 1.3-kb EcoRI-SalI fragment was then cloned in mineral oil. This was 28 m, giving an injected volume of 12 pl (i.e., frame into pGEX-6-p1 that was cut with the same enzymes. Protein ex- 1:500 embryo vol). The concentration gradient of injected fluorescent pression and purification were performed in accordance with the sup- protein was calibrated by diffusion modeling (http://www.nrcam.uchc.edu/) plied manual (GE Healthcare). InsP sponge constructs (wild-type and to calculate the intraembryonic gradient of protein 15 min after microin- control sponge) were subcloned from the supplied pGEM-T vector jection of a 12-pl vol of 200 M GFP::p130. The fitted diffusion constant (Howard Baylis, University of Cambridge, Cambridge, UK; Walker et al., . The gradient remained stable from 10 min after microin- was 3 m/s 2002) into the expression vector pCal-n (Stratagene). The NcoI-SalI frag- jection and for the rest of the time course of the experiment. ment was subcloned in frame into pCal-n that was digested with the same Fluorescence measurements enzymes. Expression and purification was performed in accordance with An inverted confocal microscope (model DMIRBE; Leica) and either 20 PL the supplied manual. Fluotar NA 0.5 or 40 PL Apo NA 1.25 objectives (Leica) were used for We thank Pierre Leopold for his early interest in this project, Maureen Sinclair all described experiments. The light source was an argon–krypton laser and Trevor Jowett for help with Drosophila, Howard Baylis and Matilda Katan with two excitation beams, which are available at 488 and 568 nm. Cal- for InsP -binding protein constructs, and Michael Aitchison for help with prep- cium measurements were performed using two fluorescent dyes: one was aration of the figures. calcium sensitive (10 kD CaGr) and the other was calcium insensitive We also thank the Biotechnology and Biological Sciences Research (10 kD tetramethylrhodamine dextran [TMR]). CaGr was excited at 488 Council and Wellcome Trust for financial support. nm, and TMR was excited at 568 nm with a dichroic mirror at 580 nm. Emission filters were a 530 30 nm FITC bandpass and a 590 nm long- Submitted: 24 March 2005 pass. Images were acquired by using Scanware 5.1 software (Leica). Ratio Accepted: 1 September 2005 images were performed for each image pair after background subtraction. 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The Journal of Cell Biology – Pubmed Central
Published: Oct 10, 2005