Correspondence to Michael Whitaker: michael.whitaker{at}ncl.ac.uk
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Introduction |
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The source of calcium for signals during mitosis is the ER (Ross et al., 1989; Ciapa et al., 1994). The ER gathers around the nucleus as mitosis approaches and is closely associated with the mitotic spindle (Harel et al., 1989). The ERspindle complex can be isolated and shown to sequester calcium (Silver et al., 1980). ER membranes pervade the mitotic spindle (Harris, 1975), so is possible that calcium released very locally to calcium-binding sites over micron length scales may provide signals at the chromosomes and spindle poles. Very local signals of this kind are probably not detectable with current imaging technologies.
During the early syncytial nuclear divisions of Drosophila melanogaster embryos, ER becomes highly concentrated around the nucleus at prophase and is very closely associated with the spindle poles; however, the ER does not invade the spindle itself (Bobinnec et al., 2003). This circumstance offers the opportunity to image calcium concentrations within the nucleus and mitotic spindle without the complication of colocalized ER. It also offers the opportunity to test whether the interaction between ER and mitotic spindle creates a calcium-signaling environment that is distinct from bulk cytoplasm.
The amenable genetics of Drosophila has allowed the identification of a plethora of gene products that are directly involved in regulating the cell division cycle (Gonzalez et al., 1994; Sullivan and Theurkauf, 1995). Many are homologues of regulators that are important in controlling mammalian cell cycles. A number of cell cycle regulatory genes were first identified through their effects on the cell cycles of various early embryos (Evans et al., 1983; Gautier et al., 1988; Sunkel and Glover, 1988; Glover et al., 1991, 1995; Edgar and Lehner, 1996). Calcium gradients may help determine the dorsoventral axis in Drosophila (Creton et al., 2000), but nothing is known about calcium signaling in the fly's early embryonic cell cycles. In this study, we demonstrate that calcium regulates nuclear division during early embryonic cell cycles and go on to show that the ER surrounding the nuclear compartment encloses a calcium-signaling microenvironment that controls mitosis.
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Results |
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Calcium changes occur in syncytial Drosophila embryos in fixed phase relation with the nuclear division cycle
Fig. 1 A shows fluorescence signals in a Drosophila embryo as it passes through cell cycles 811. 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 0.51 µM, green colors represent 0.10.5 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.
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Calcium changes take the form of slow calcium waves that travel from pole to equator during each nuclear division cycle and precede the cortical contraction
When displayed at higher temporal resolution, the [Cai] changes have a spatial substructure. Fig. 2 A shows two examples of the spatial pattern of [Cai] increase. In the top panel, two [Cai] waves are arriving from poles at the equator of the embryo and are annihilating there. In Fig. 2 B, the initiation of a calcium wave at the anterior pole is followed by progression of the wave toward the equator and out of the frame. Thus, the calcium signal shows the same behavior as mitotic waves, originating at both embryonic poles and moving toward the equator. Table II gives the mean wave velocity during syncytial division cycles from cycles 912. The wave speed slows with each cycle, decreasing from 0.45 to 0.29 µm/s. As the nuclear division cycle time slows, the wave becomes progressively slower; the product of wave speed and cycle time remains constant, which is a further indication of the entrainment of calcium wave and nuclear cycle. Fig. 2 C shows a single [Cai] wave in a cycle 8 embryo before the nuclei have reached the cortex. As the wave progresses toward the equator, it is followed by a cortical constriction that represents a cortical contraction travelling at the same velocity. The constriction can be seen in the confocal section, as the movement of the plasma membrane away from the vitelline membrane creates a dye-free perivitelline space that appears black beneath the autofluorescent vitelline membrane. The time that elapsed between the leading edge of the calcium wave and the leading edge of the constriction is 90 s. We observed the association between wave and constriction in three of three embryos, suggesting that the [Cai] increase causes the cortical contraction.
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Because nuclei in the syncytial embryo are not separated by plasma membranes, the syncytial embryo offers the possibility of generating a gradient of inhibitor within the cytoplasm so that individual nuclei will experience different concentrations of inhibitor. GFP::p130 is a PLC orthologue that is catalytically inactive, binds InsP3 with high affinity by virtue of its pleckstrin homology domain, and inhibits calcium signaling when overexpressed in cells or when added to permeabilized cells (Takeuchi et al., 2000). The GFP::p130 chimera was localized to plasma membrane and apparently to ER in interphase, entered the nucleus at prophase, and associated with the mitotic spindle (Fig. 3 C i). By microinjecting GFP::p130 at one pole of the embryo during early anaphase of cycle 11, we were able to generate a gradient of GFP::p130 of 210 µM, which was confirmed by the distribution of fluorescence (Fig. 3 C ii) that persisted for the course of the experiment. The outcome was striking. Nuclei that were exposed to 10 µM GFP::p130 arrested before nuclear envelope breakdown (NEB) in cycle 12. Nuclei that were exposed to intermediate concentrations continued through NEB of cycle 12 but arrested after mitosis entry and were unable to complete mitosis, whereas nuclei that were exposed to 2 µM progressed through mitosis of cycle 12 and arrested before mitosis in cycle 13. Simultaneous imaging of histone and GFP signals demonstrated that nuclei entered metaphase but failed to enter anaphase rapidly, just as we had found with the InsP3 sponge (Fig. 3 C iii). Chromatin decondensation occurred in the arrested nuclei after a delay (Fig. 3 C iii) and elongated, and dumbell-shaped nuclei were also seen (not depicted), although this occurred a few minutes later than we had observed after the microinjection of InsP3 sponge constructs. These observations demonstrate that InsP3 signaling plays a role in mitosis entry at NEB as well as in mitosis exit in Drosophila embryos. In GFP::p130-injected embryos, the mitotic wave (the wave of NEB and anaphase onset) travelled in the opposite direction to that observed in controls (that is, from the farther embryonic pole), indicating that InsP3 is involved in the initiation and propagation of the wave.
Heparin and Xestospongin C are agents that inhibit the interaction of InsP3 with the InsP3 receptor (Ghosh et al., 1988; Gafni et al., 1997). Embryos that were microinjected with either the inhibitor of InsP3-induced calcium release, heparin (80 µg/ml gave half maximum inhibition; n = 4; Groigno and Whitaker, 1998), or Xestospongin C (10 mM of pipette concentration; n = 6; Hu et al., 1999) also showed a block in mitosis that was similar to what we observed with both the InsP3 sponge and GFP::p130 (unpublished data). As far as is known, the InsP3 receptor is the sole signaling target of InsP3 in cells (for review see Fukuda and Mikoshiba, 1997; Mikoshiba, 1997).
These experiments demonstrate that InsP3-triggered calcium release is a signal that is necessary for both entry into mitosis and for anaphase onset in syncytial Drosophila embryos, as it is in sea urchin embryos (Twigg et al., 1988a; Ciapa et al., 1994; Groigno and Whitaker, 1998).
Microdomains of elevated calcium that are separated by ER-rich low calcium domains are observed in cortical buds
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.
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The interphase [Cai] increase occurs at the very periphery of cortical buds that surround the interphase nuclei (Fig. 4 E i). Cortical mitotic spindles are anchored by actin caps that surround each nucleus in interphase (Fig. 4 E ii, cartoon; Sullivan and Theurkauf, 1995; Foe et al., 2000). The actin caps are pushed further apart as the spindles extend at anaphase. In late telophase, the actin wraps around the reforming interphase nucleus to give twice as many actin caps as were present before nuclear division (Fig. 4 E ii). Nuclei, therefore, occupy a greater area of the cortex in mitosis compared with interphase, which gives rise to substantial oscillatory translation of nuclei in the plane of the cortex as the mitotic wave progresses along the embryo (Zalokar and Erk, 1976). When localization of the interphase [Cai] increase, actin, and ER distribution are compared in confocal sections normal to the surface (Fig. 4 E, i and ii), it is evident that the [Cai] increase occurs throughout the cortex in each cap but is markedly higher in the regions of highest actin concentration.
As predicted, the halo of ER that surrounds the nucleus and mitotic spindle appears to separate two distinct calcium microdomains: a region of high calcium in the subcortex, which is associated with actin and contraction in interphase, and a region of lower nuclear calcium. Calcium concentrations are lowest where the ER is most dense.
[Cai] increases occur in the nucleus and mitotic spindle microdomains at both prophase and anaphase
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.
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These data demonstrate the existence of nuclear microdomains of calcium concentration that act as triggers for mitosis entry and exit.
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Discussion |
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InsP3 and the InsP3 receptor are essential for nuclear division
We determined that InsP3 receptors were functional in early embryos by eliciting calcium release in response to InsP3 injection. A genetic approach to determine the importance of InsP3 signaling during rapid syncytial nuclear divisions of the Drosophila embryo does not easily present itself. In fact, despite the ubiquity and importance of calcium signaling (Berridge et al., 2000), very few genetic disorders that are caused by defects in calcium-signaling components have been identified; the strong assumption is that an overwhelming majority of genetic calcium signaling defects are embryonic lethals (Rizzuto and Pozzan, 2003). Instead, we microinjected constructs that have been shown to chelate InsP3. We used a GFP-tagged InsP3-binding protein to determine the cytoplasmic concentration of injected proteins. We determined the inhibitory concentration that blocks both NEB and anaphase onset to be 210 µM, which are concentrations comparable with those previously observed to block InsP3-mediated events (Takeuchi et al., 2000) and are similar to those observed with an InsP3 sponge (Uchiyama et al., 2002). Thus, InsP3 signaling leading to calcium transients is essential for NEB and anaphase onset, as it is in early sea urchin embryos (Poenie et al., 1985; Steinhardt and Alderton, 1988; Twigg et al., 1988b; Ciapa et al., 1994; Wilding et al., 1996; Groigno and Whitaker, 1998). As observed in the sea urchin embryo (Groigno and Whitaker, 1998), the block to anaphase onset was characterized by absence of chromatin disjunction, but spindle elongation and chromatin decondensation did occur, often with a delay. The ER isolates the nucleus during mitosis and generates local nuclear calcium signals via InsP3.
Cell cycle calcium signals that govern mitosis are not prominent in syncytial Drosophila embryos. We show that this is because the ER generates calcium-signaling microdomains within the cortical bud: one beneath the plasma membrane of the cortical buds and the other within the nucleus and mitotic spindle. There is a real possibility that the very different molecular environments are, in part, responsible for the different fluorescence signals that we measured in these different microdomains. However, at metaphase, the calcium concentrations that are reported by fluorescence reporters are uncorrelated, implying that calcium rises in only the spindle microdomain.
Although it has been clear for some time that ER associates with the nucleus and spindle (Terasaki and Jaffe, 1991), this has been interpreted as a mechanism to ensure proportionate inheritance of ER when cells divide (Barr, 2002). In this study, we demonstrate an additional, essential, and novel functionthat of maintaining distinct calcium microdomains during cell division. Nuclear calcium has also been shown in some cell types to be regulated differentially to cytoplasmic calcium (Badminton et al., 1998; MacDonald, 1998), but this is thought to be a result of the properties of the nuclear envelope rather than of an accumulation of ER around the nucleus. Although it was originally proposed that a nuclear envelope persisted throughout mitosis as a spindle envelope during syncytial nuclear divisions (Harel et al., 1989), it is now clear that the nucleus becomes permeable to high molecular weight molecules early in prophase (that is, at the same time as in other cells) but that nuclear lamins persist until metaphase, disappearing before anaphase onset (Paddy et al., 1996). Thus, the nuclear envelope does not exist during mitosis to provide a diffusion barrier that would allow the mechanisms regulating calcium in intact nuclei to operate. On the other hand, the persistence of nuclear lamins may explain why the ER remains outside the spindle until late anaphase in syncytial embryos.
We show that it is possible to apply cell physiology methods to early Drosophila embryos to study calcium signaling. Our data clearly demonstrate for the first time in a protostome embryo that maneuvers designed to prevent calcium signals arrest the nuclear division cycle and that calcium signals are responsible for the waves of mitosis observed in syncytial Drosophila embryos. We also show for the first time that the nucleus and spindle exist within a calcium-signaling microdomain and that calcium increases that are necessary for progress through mitosis are small and localized. This has been possible because ER is excluded from the Drosophila spindle during mitosis. In other embryos and in mammalian somatic cells, ER is an intimate spindle component. Signals that are local to the spindle are less readily detected, perhaps explaining why calcium signals are not always observed during mitosis in some cell types.
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Materials and methods |
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Chemicals
5,5'dibromoBAPTA (tetrapotassium salt) and fluorescent dyes were purchased from Invitrogen. Cycloheximide and Xestospongin C were purchased from Calbiochem. The majority of all other chemicals were purchased from Sigma-Aldrich.
Microinjection
Drawn borosilicate glass micropipettes (GC150F-10; Clarke Electromedical) were loaded with injection solution and advanced toward immobilized Drosophila embryos by using an Eppendorf microinjection system. All fluorescent probes for microinjection were dissolved in injection solution (Ashburner et al., 2005) except Xestospongin C (Gafni et al., 1997), which was dissolved in DMSO for microinjection. The embryos were injected using gas pressure (pneumatic picopump; World Precision Instruments, Inc.). Cytoplasmic concentrations were calibrated by first measuring the size of droplets that were injected into the oil before injection into the embryo. Embryos are 470 x 160 µm but can vary in length and diameter considerably. The approximate volume of an embryo is 6.5 nl, which was calculated by considering the volume of an ellipsoid of the above dimensions. The volume of liquids that were injected into the embryo was estimated by measuring the diameter of a droplet injected under mineral oil. This was 28 µm, giving an injected volume of 12 pl (i.e.,
1:500 embryo vol). The concentration gradient of injected fluorescent protein was calibrated by diffusion modeling (http://www.nrcam.uchc.edu/) to calculate the intraembryonic gradient of protein 15 min after microinjection of a 12-pl vol of 200 µM GFP::p130. The fitted diffusion constant was 3 µm/s1. The gradient remained stable from 10 min after microinjection and for the rest of the time course of the experiment.
Fluorescence measurements
An inverted confocal microscope (model DMIRBE; Leica) and either 20x PL Fluotar NA 0.5 or 40x PL Apo NA 1.25 objectives (Leica) were used for all described experiments. The light source was an argonkrypton laser with two excitation beams, which are available at 488 and 568 nm. Calcium measurements were performed using two fluorescent dyes: one was calcium sensitive (10 kD CaGr) and the other was calcium insensitive (10 kD tetramethylrhodamine dextran [TMR]). CaGr was excited at 488 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 longpass. Images were acquired by using Scanware 5.1 software (Leica). Ratio images were performed for each image pair after background subtraction. All image processing was performed on a silicon graphics computer using IDL software (Research Systems International, Ltd.), and background-subtracted pixel values were displayed in pseudocolor using monochrome or rainbow look-up tables. Images were merged by using either Adobe Photoshop or Metamorph software. All experiments were performed at 18°C.
The ER was labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC18) to determine its distribution (molecular mass of 933.8 D; Invitrogen). This dye was also used to visualize the location of the ER throughout early development of the Drosophila embryo. DiI was made up to a saturated concentration in soybean oil and was microinjected into cells in an oil droplet. The dye diffused along the continuous ER. DiI was excited by the 568-nm line of the argonkrypton laser. The maximum emission wavelength of DiI is 575 nm. Excitation and emission wavelengths were separated by a DD488/568-nm beamsplitter. The emission light was passed back through the beamsplitter and through a barrier filter before entering the photomultiplier tube.
To monitor microtubule dynamics, 10 mg/ml rhodamine-labeled tubulin (Cytoskeleton, Inc.) was microinjected into the embryos (dye/protein heterodimer stochiometry of 1.0). Identical settings that were used to record TMR fluorescence were used to measure rhodamine tubulin fluorescence.
5 mg/ml rhodamine-labeled histone H1 was prepared (Harlow and Lane, 1999) and injected into embryos to monitor the chromatin configuration to permit precise scoring of the stages of mitosis (dye protein stochiometry of 1.0). Identical settings that were used to record TMR fluorescence was used to measure histone H1 fluorescence.
To measure actin dynamics, we used rhodamine or fluorescein-conjugated rabbit nonmuscle actin (10 mg/ml dye/actin; labeling stochiometry of 1:1; Cytoskeleton, Inc.).
Calibration of ratiometric calcium signals
The fluorescence intensity of CaGr1 (made up in injection buffer) was determined in the absence of Ca2+ and in the presence of saturating Ca2+. The fluorescence enhancement (fluorescence in saturating Ca2+/fluorescence in the absence of Ca2+) was found to be 2.55. Single wavelength calcium dyes cannot readily be calibrated absolutely in Drosophila embryos, so calcium concentrations were estimated by using the following approach (Isenberg et al., 1996):
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Protein expression
The GFP::p130 domain construct (Takeuchi et al., 2000) was obtained from M. Katan (Imperial College, London, UK) and was cloned into the expression vector pGEX-6-p1 (GE Healthcare) as follows: GFP::p130 was cut with HindIII, and the 1.3-kb fragment was cloned into pBC SK (+) digested with HindIII. The 1.3-kb EcoRI-SalI fragment was then cloned in frame into pGEX-6-p1 that was cut with the same enzymes. Protein expression and purification were performed in accordance with the supplied manual (GE Healthcare). InsP3 sponge constructs (wild-type and control sponge) were subcloned from the supplied pGEM-T vector (Howard Baylis, University of Cambridge, Cambridge, UK; Walker et al., 2002) into the expression vector pCal-n (Stratagene). The NcoI-SalI fragment was subcloned in frame into pCal-n that was digested with the same enzymes. Expression and purification was performed in accordance with the supplied manual.
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Acknowledgments |
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We also thank the Biotechnology and Biological Sciences Research Council and Wellcome Trust for financial support.
Submitted: 24 March 2005
Accepted: 1 September 2005
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References |
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