Intranuclear Ca2+ signals within individual nuclear lobes ofneutrophils

Robert Hodges, Iraj Laffafian, Maurice B. Hallett*

Molecular Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, CF14 4XN, Cardiff, UK

Received 15 April 2002; revised 4 November 2002; accepted 23 December 2002

Abstract

Cytosolic free Ca2+ and intranuclear Ca2+ behave similarly in human neutrophils. However, conventional laser scanning at350 ms/frame resolution at lower than physiological temperatures demonstrates that (i) the nuclear fluo3–Ca2+ signal persists longerthan the cytosolic signal in some (but not all) nuclear lobes, (ii) the neutrophil nuclear membrane and fine inter-lobe filamentspresent barriers to diffusion of fluo3–Ca2+ and lucifer yellow, and (iii) the diffusion barrier correlates with condensed chromosomalmaterial on the nuclear envelope and blockage of the movement of fluo3–Ca2+ into individual nuclear lobes.� 2003 Elsevier Science Ltd. All rights reserved.

1. Introduction

The interaction between cytosolic free Ca2+ andintracellular organelles is clearly important to gainunderstanding of Ca2+ signalling within cells. Inneutrophils, which have little endoplasmic reticulum andfew mitochondria, the major organelle is the nucleus(Schmid-Schonbein et al., 1998). The neutrophil nucleusis unusual in that it is segmented. There are usuallybetween three and six major nuclear lobes, each 2–3 µmin diameter, which are connected by filaments (inter-lobefilaments) as fine as 0.1 µm in diameter (Bessis, 1993). Itis this characteristic of the nucleus that gives this celltype its fuller classification as a poly-morpho-nuclearneutrophilic leucocyte. The nuclear lobes contain partlycondensed DNA, with chromosomal material randomlydistributed among the lobes (Campbell et al., 1995;Sanchez et al., 1997).

The neutrophil is unable to divide and is programmedfor death. In some ways, the morphology of the neutro-phil nucleus is similar to that of other cells under-going apoptosis. Being a small cell (10 µm in diameter),the multi-lobed nucleus dominates the geometry ofthe neutrophil, occupying nearly a quarter of the

cytoplasmic volume (Schmid-Schonbein et al., 1998) (inliver cells, the nucleus is just 9% of the cell). The mannerin which Ca2+ is transported through or around thenucleus of the neutrophil is therefore clearly importantfor understanding the distribution of Ca2+ signals in thiscell. The distinctive nuclear shape of the neutrophil alsoprovides an opportunity to unequivocally image Ca2+

within the nucleoplasm, and visualise the movement ofintranuclear Ca2+ between nuclear lobes.

In this study, we investigated intranuclear Ca2+

signals and found that, at a hypothermic temperature(25 (C), the nuclear fluo3–Ca2+ signal in some nuclearlobes persisted longer than the cytosolic signal. Also, thenuclear envelope and filaments between nuclear lobespresented barriers to the diffusion of both fluo3–Ca2+

and lucifer yellow. This diffusion barrier correlated withthe presence of condensed and packed chromosomalmaterial.

2. Materials and Methods

2.1. Neutrophil isolation

Neutrophils were isolated from heparinised bloodof healthy volunteers, as described previously (Hallettet al., 1990). Following dextran sedimentation, centri-fugation through Ficoll–Paque (Pharmacia) and

* Corresponding author. Tel.: +44-1222-742-748;fax: +44-1222-761-623

E-mail address: HallettMB@Cardiff.ac.uk (M. Hallett).

Cell Biology International 27 (2003) 395–402

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hypotonic lysis of red cells, neutrophils were washed andresuspended in Krebs buffer (120 mM NaCl, 4.8 mMKCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2,25 mM HEPES and 0.1% bovine serum albumin,adjusted to pH 7.4 with NaOH).

2.2. Measurement of nuclear free Ca2+ concentration

Neutrophils were loaded with fluo3 from theacetoxymethyl ester as previously described (Hallettet al., 1996), to give an intracellular concentration ofabout 100 µM. With a kd in neutrophils of 864 nM, anda cytosolic Ca2+ buffering capacity of 3000–1000:1, fluo3increased the buffering capacity of the cells by less than10% (Al-Mohanna and Hallett, 1988). Cytosolic andnuclear fluo3 were visualised by confocal laser scanningmicroscopy (Leica CLSM) through the midpoint of theneutrophil (optical section thickness approx. 0.5 µm).Ca2+ concentrations were calculated from regions of theimage generated by excitation by the 488 nm laser line,using the conventional equation (Hallett et al., 1996).The speed of image acquisition was maximised on theinstrument by reducing the field of laser scanning and bycollecting data on both the forward and return paths ofthe laser scan. The return scan data was correctlyaligned with the forward scan data using a linear testobject (an electron microscope sample grid, imaged byreflectance).

When the two sets of data (forward and reverse) werecorrectly aligned, sharp images of fluo3 loaded neutro-phils were acquired at average rates of 1 frame/350 ms(for a 128�128 pixel area) or 1 frame/600 ms (256�256area). Using a �100 objective, the complete image ofa single round neutrophil (10 µm diameter) occupied a128�128 pixel area, and thus Ca2+ imaging wasachieved at a time resolution of 350 ms/frame and aspatial resolution of 78 nm/pixel. As previously reportedby PerezTerzic et al. (1997a), the spectral properties offluo3 within the nucleus are altered, so excitation spectrafor fluo3 within the neutrophil cytosol and nucleus wereacquired using a rapid access monochromator system(Delta RAM, PTI, Surbiton, UK) coupled to a conven-tional fluorescence microscope (Nikon Eclipse). Imagesover the excitation range 400–500 nm were acquired at1 nm intervals. Data from the cytosolic and nuclearregions of the image were acquired and analysed usingImageMaster software (PTI).

2.3. Nuclear staining using acridine orange

After recording Ca2+ changes in the selected neutro-phil, the laser power and voltage to the detection PMTwere reduced to levels at which the fluo3 signal wasundetectable. The position of the nuclear lobes withinthe cell was then determined by the addition of acridineorange (5 µg/ml, 5 min) to the microscope incubation

chamber. The acridine orange/DNA fluorescence wasfar greater than the fluo3, and it was often necessary toreduce the voltage PMT still further. As the microscopestage was not moved, and the nuclear image wasacquired using the same laser line and filters (488 nmexcitation) as the Ca2+ images, the two images coincidedand were used to map the movement of Ca2+ into thenuclear lobes.

2.4. Micro-injection of lucifer yellow

Lipid-assisted micro-injection (SLAM) of adherentneutrophils was achieved using lipid-coated micro-pipettes with an internal pressure of 5–20 mbar,as previously described (Laffafian and Hallett, 1998).Gentle contact between this micro-pipette and theneutrophil plasma membrane resulted in fusion of thelipid at the pipette tip with the cell, and transfer ofthe aqueous contents of the pipette to the cytosol. Thepressure was held constant during the injection and theprocedure had no effect on cell viability (Laffafian andHallett, 1998; Hallett et al., 1999). In order to gainkinetic data of lucifer yellow entry into the nucleus, theSLAM-pipette was advanced towards a neutrophilwhile recording fluorescent images using a video capturesystem (25 frames/s). The movement of lucifer yellowacross the cell and into the nucleus was thereforeobserved with a time resolution of 40 ms.

3. Results

3.1. Ca2+ entry into the nucleus from localised Ca2+

release

Changes in cytosolic free Ca2+ concentrationoccurred uniformly throughout the neutrophil cytosoland nuclear space at 37 (C, and it was not possible todetermine the nuclear-cytosolic boundary from imagesof changes in free Ca2+ concentration (see e.g. Hallettet al., 1996). However, at lower than physiologicaltemperatures (20–26 (C), stimulation with f-met-leu-pheoften produced prominent nuclear shapes in the con-focal image of fluo3-loaded cells, which lasted for severalhundreds of milliseconds longer than the cytosolic Ca2+

signal (Fig. 1). This differential effect of nuclear andcytosolic fluo3 signal was probably due to a difference inthe affinity of the indicator in the two locations (Perez-Terzic et al., 1997). However, the persistent nuclear fluo3signal was often restricted to two or three out of thefour or five nuclear lobes that the neutrophil possesses(Fig. 1).

In order to investigate the nature of this sub-nuclearrestriction, the relationship of this signal to cytosolic freeCa2+ release was established using rapid bi-directionallaser scanning. This approach demonstrated that therelease of Ca2+ was from a site in the cytosol close to

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the nucleus, and preceded the elevation of Ca2+ withinthe nuclear space (Fig. 2a). The release site was outsidethe nuclear lobes and localised to a single site nearthe nucleus. This perinuclear Ca2+ release event haspreviously been observed using a variety of imagingtechniques (Davies et al., 1991; Pettit and Hallett, 1998).However, in this study, as with the other approaches, itwas not always possible to “catch” the Ca2+ releaseevent, presumably because it occurred between thelaser scanning intervals or at a confocal plane otherthan that being imaged. However, in every case in whichthe juxtanuclear Ca2+ release event induced by f-met-leu-phe was observed (5 out of 35), an elevation ofnuclear Ca2+ followed (Fig. 2a).

It has previously been shown that, when neutrophilscontact �2 integrin-engaging surfaces, small Ca2+ releaseevents also occur towards the cell periphery (Pettit andHallett, 1996, 1998). Under these conditions, localisedCa2+ release events were detected approximately 3–4 µmfrom the cytosol-nuclear boundary, but these never (9out of 9) resulted in an elevation of nuclear Ca2+. Inorder to exclude the possibility that �2 integrin stimu-lation prevented entry of Ca2+ into the nucleus, a neu-trophil in which �2 integrin-mediated Ca2+ puffs weregenerated by local contact with a platelet (Sheikh and

Nash, 1996) was chosen for subsequent stimulation withf-met-leu-phe (Fig. 2b). Although the �2 integrin-mediated Ca2+ puffs did not enter the nuclear space, thesubsequent f-met-leu-phe-generated Ca2+ signal near thenucleus did elevate the nuclear Ca2+ signal (Fig. 2b). Itwas therefore concluded that �2 integrin stimulation didnot inhibit Ca2+ entry into the nucleus, but that contactbetween the puff of elevated Ca2+ and the nuclear bound-ary was required for an elevation of nuclear Ca2+.

3.2. Persistence of the nuclear Ca2+ signal

Although the rate of increase in fluorescence intensitywithin the nucleus and cytosol were similar, fluo3 fluor-escence in the cytosol and some of the nuclear lobesreturned to resting levels more quickly than in othernuclear lobes (Fig. 1). It was this persistence (at 25 (C)of nuclear fluo3 fluorescence that enabled the nuclearshape to be clearly distinguished in some fluo3 images. Itwas noted that nuclear lobes connected by large inter-nuclear filaments often had similar signals, while thoseseparated by a thin filament did not (see e.g. Fig. 1). Onepossible explanation for the localised “Ca2+ persistence”phenomenon was that the thin connections renderednuclear lobes permeable to fluo3–Ca2+, while other

Fig. 1. Cytosolic and nuclear fluo3–Ca2+ changes during stimulation with the chemotactic peptide, f-met-leu-phe. Changes in cytosolic and nuclearfluo3–Ca2+ in a single confocal slice during stimulation with f-met-leu-phe (1 µM) are shown in the images in the upper panel at the times indicated(in seconds). The images have been pseudo-coloured to emphasise the distributional differences in fluo3 fluorescence, with blue as the resting leveland yellow above the threshold of the stimulated cell. The lower panel shows AO fluorescence, indicating the position of the four lobed-nucleus atthe same confocal plane within the cell, and a labelled map of the lobes, together with the time course of fluo3–Ca2+ changes (plotted as the ratioof fluorescence to initial fluorescence, F/Fo) in nuclear lobes labelled N1 and N2 (N1+N2), N3 and N4 (N3+N4) and the region excluding N1, N2,N3 and N4 (cytosol). The arrow shows the point at which f-met-leu-phe was added (labelled CP). The position of the nuclear lobes on the fluo3images is also shown.

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connections could insulate lobes from this diffusion. Assome lobes tracked the cytosolic free Ca2+ concentrationaccurately, this suggested that there could also be differ-ences in diffusion across the nuclear-cytosol boundary.These possibilities also suggested that Ca2+ in somenuclear lobes may be isolated from that in other lobes,perhaps as a result of the anatomical restriction at theinterlobe filament.

3.3. Sub-nuclear fluo3 Ca2+ and condensed DNAdistribution

In order to investigate the possibility that somenuclear lobes were diffusionally isolated as a result oftheir nuclear microanatomy, the nuclear shape wasexamined in more detail using acridine orange (AO).Inter-lobe filaments often tapered to very narrowdiameters (Fig. 3), which were below accurate quantifi-cation (<0.1 µm). While this would significantly reducediffusion from one nuclear lobe to another, it may nottotally prevent it. However, as these structures could bevisualised by their DNA content (AO fluorescence), it ispossible that the chromosomal material they containedalso constituted the barrier.

An estimate of the density of chromosomal materialin the filament was made by assuming the structure hada circular cross-section. With a diameter of less than0.1 µm, it occupies only 1/5 of the 0.5 µm opticalsection thickness, and hence the AO intensity should be

increased by a factor of at least five to accurately reflectDNA density. This analysis suggests that the inter-lobefilaments are heavily packed with chromosomal material(Fig. 3).

In order to determine whether the condensedchromosomal material excluded the elevated intra-nuclear fluo3–Ca2+, the distribution of DNA within thenuclear lobes was determined. This was achieved bylaser bleaching the nuclear AO to degrade the image inorder to reveal the non-uniform nuclear distribution ofcondensed DNA. Chromosomal material was con-densed on to the nuclear envelope and often condensedinto a single spot within the lobe (Figs. 1, 2 and 4). Thepersistently elevated nuclear fluo3–Ca2+ signal corre-sponded not to the outer boundary of the nuclearenvelope, but to the intranuclear space free of condensedDNA (see Figs. 2 and 4). Nuclear fluo3–Ca2+wasexcluded from the areas that subsequently stained mostheavily with AO (Fig. 4), giving a “smoke ring” appear-ance to the nuclear fluo3–Ca2+ signals in some cells (seeFig. 2b and 4). The blockage of fluo3–Ca2+ diffusionfrom one nuclear lobe to another corresponded to thepresence of this condensed chromosomal material at thenarrow nuclear filaments (Fig. 1). In the example shownin Fig. 1, the two lobes which reflected cytosolic freeCa2+ concentration were separated from the other twolobes which retained high nuclear fluo3–Ca2+ by a thinnuclear filament, whereas each pair of lobes were linkedby larger inter-lobe connections.

Fig. 2. Cytosolic Ca2+ puffs and nuclear fluo3–Ca2+. Series of Ca2+ images at 350 ms intervals from (a) a round neutrophil (i.e. before polarisationand cell spreading) and (b) a polarised neutrophil contacting a platelet (marked “p”). The series shows the effect of stimulation with f-met-leu-phe(1 µM) added at the arrow (CP). The asterisk (*) in both sets of images marks the position of the initial Ca2+ puff induced by CP, which occurs beforenuclear Ca2+ signalling. In the lower set of images, the cross (+) marks the position of the Ca2+ puff triggered by contact with the platelet (p). Theimages have been pseudo-coloured to emphasise the distributional differences in fluo3 fluorescence, with blue as the resting level and yellow abovethe threshold of the stimulated cell. In the final image of each sequence, AO fluorescence is shown, indicating the position of the lobed nucleus atthe same confocal plane within the cell, and the positions of the Ca2+ puffs and the platelet are indicated.

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3.4. Nuclear permeability to lucifer yellow

In order to establish whether the nuclear membraneand condensed chromosomal material provided ageneral barrier to diffusion, or was selective for fluo3and Ca2+ ions, the movement of other small moleculeswas investigated. Micro-injection of fura2 (Hallett et al.,1990) or lucifer yellow (Laffafian and Hallett, 1998), orester-loading with fura2 (fura2-AM), fluo3 (fluo3-AM)or fluorescein (fluorescein diacetate) demonstrated thatmolecules of these sizes were able to permeate the

neutrophil nuclear membrane and condensed chromo-somal material. As lucifer yellow (450 kDa) wasinsensitive to both free Ca2+ concentration and thenuclear environment (lucifer yellow-injected cells showno sign of the nuclear shape at equilibrium), this probewas used to visualise entry into the nucleus. At videorate (40 ms resolution), the entry of lucifer yellow intothe nucleus was followed during SLAM-injection(Fig. 5). In thinly spread neutrophils in which thenuclear lobes could be visualised, conventional (non-confocal) imaging showed that the entry of lucifer

Fig. 3. Localisation of condensed chromosomal material at the nuclear boundary and intra-lobe filament. An image of a five-lobed neutrophilnucleus (AO) is shown to demonstrate condensation of chromosomal material at the nuclear boundary and the thinness of some intra-lobe filaments.In the graph on the right, the relative intensities of AO staining on a line straddling the interlobe filament and condensed material at the lobeperiphery are shown. The intensity is corrected for the filament diameter, to demonstrate the high density of chromosomal material at the“bottle-neck” nuclear filament.

Fig. 4. Persistent nuclear fluo3–Ca2+ is restricted to the intranuclear space and excluded from the chromosomal condensate. Fluo3 images are shown(a) before and (b) 700 ms after stimulation with f-met-leu-phe (1 µM). The images have been pseudo-coloured to emphasise the distributionaldifferences in fluo3 fluorescence, with blue as the resting level and yellow above the threshold of the stimulated cell. In the final image, AOfluorescence indicating the distribution of condensed chromosomal material within the three-lobed nucleus at the same confocal plane is shown. Inthe panel below, the intensities of AO staining (thin line) and fluo3–Ca2+ images (thick line) before (Ca2+

R) and after stimulation (Ca2+S) are

compared across a nuclear lobe, as indicated in the insert. The “smoke-ring” effect seen in the fluo3 image corresponds with the inner, DNA-freeintranuclear space.

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yellow into the cytosol from the micro-pipette occurredbefore its entry into the nuclear lobes (Fig. 5).

Once within the nuclear space, lucifer yellow diffusedrapidly within an individual nuclear lobe, but was some-times excluded from particular lobes. In the exampleshown (Fig. 5), lucifer yellow entered two nuclear lobeswith an interconnecting nuclear filament, but failed toenter the third lobe. This third lobe was temporarilyisolated from both the cytosolic and intranuclear dye. Itis unlikely that this effect was artefactually caused byclosure of nuclear pores as a result of an injection-mediated elevation of cytosolic free Ca2+, as this washardly affected by SLAM injection, only transiently(4–6 s) rising to no more than 150 nM (from a restinglevel of 100 nM). This suggested that, under physiologi-cal conditions, dye entry into the nuclear space occurredmore readily into some lobes than others. Dye wasprevented from entering where there were barriers togeneral diffusion at both the cytosol–nuclear boundaryand between lobes at “inter-lobe bottlenecks”.

4. Discussion

In this paper, we show Ca2+ changes in the neutro-phil nucleus that originate from cytosolic Ca2+ puffsgenerated by the release of Ca2+ from a juxtanuclearCa2+ store. We found no evidence for Ca2+ signalsoriginating within the nucleus, but releases of Ca2+ nearthe nuclear boundary were able to penetrate into thenucleus. Release of Ca2+ from this site fed into thenucleus when the Ca2+ puff touched a permeable part ofthe nuclear envelope. More distant Ca2+ release eventsthat did not touch the nucleus did not provoke nuclearCa2+ signals. This was consistent with the source of therise in nuclear Ca2+ being the cytosol. In some cells, such

as mouse and starfish oocytes (Pesty et al., 1998; Santellaet al., 1998) and HeLa (Badminton et al., 1996, 1998),there is evidence that Ca2+ signals in the nucleus aregenerated independently of the cytosol. However,the evidence here suggests that nuclear Ca2+ in theneutrophil reflects cytosolic Ca2+, particularly in theregion of the Ca2+ release site. Lipp et al. (1997), usingrapid laser scanning in HeLa cells, showed that Ca2+

puffs near the nucleus enter the nucleoplasm, and thenucleus reflects the cytosolic Ca2+ concentration. Differ-ent cell types may thus handle nuclear Ca2+ differently,and it may thus be unwise to extrapolate data on nuclearCa2+ from one cell type to another.

The speed of entry of Ca2+ from the localised cyto-solic release site near the nucleus into the nucleus itselfwas rapid, and within the time resolution of imaging (i.e.within 350 ms). This suggested that diffusion of Ca2+

within the nucleus was rapid. Since it was possible, atthis time resolution, to sometimes “catch” localised Ca2+

events in the cytosol but never within nuclear lobes, itwas concluded that Ca2+ was less well buffered in thenucleoplasm than in the cytoplasm. In a study onhepatocyte nuclei, it was shown that Ca2+ in the nucleusmoved as a wave, at a velocity of about 26 µm/s (Foxet al., 1997). This speed would fill an individual nuclearlobe (2 µm) of a neutrophil in 100 ms (and four lobes in200 ms, if the Ca2+ source was central), which is withinthe time resolution of our fastest imaging (350 ms). Wesuspect that this time may be shorter in neutrophils, as“xt” laser scanning has shown that the Ca2+ rise time(from rest to maximum Ca2+ signal throughout the cell)in individual neutrophils is about 500 ms (Pettit andHallett, 1995).

The unusual feature of the nuclear fluo3–Ca2+ signalsseen at hypothermic temperatures (25 (C) was theirpersistence in some nuclear lobes after cytosolic free

Fig. 5. Slam-injection of lucifer yellow reveals nuclear lobe diffusional blockage. The upper panel shows a neutrophil (approx. 10 µm diameter) inwhich three nuclear lobes can be clearly distinguished (labelled in first image), and the tip of a micro-pipette containing lucifer yellow making contactwith the cell. The lower panel shows the fluorescence image at contact, where initially only the brightly glowing micro-pipette tip was visible,200 ms later when the lucifer yellow entered the cytosol, 1 s later when a clear nuclear shape showed that two of the three nuclear lobes were freelyconnected, and 10 s later when the fluorescence was uniform throughout the cell. After 1 s, the lucifer yellow was able to cross one thin intranuclearfilament connecting two of the lobes, but not the filament connecting the third.

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Ca2+ declined towards base-line. This may be explicableif the nuclear envelope was relatively impermeable tofluo3–Ca2+ ions over the majority of its surface, or thenuclear pores were closed by Ca2+ (Lee et al., 1998;PerezTerzic et al., 1997b). From previous studies, it hasbeen shown that the nuclear pore is large enough to letmolecules as large as 40 kDa pass though (Paine et al.,1975). In this study, we also showed that lucifer yellowand fluo3 are clearly able to permeate the nuclearmembrane (Fig. 5). However, it appears that, either as aresult of condensed DNA on the nuclear envelope or thenon-uniform distribution of pores, the nucleus may bemore permeable at some locations, such as near thenuclear cleft (Fig. 5). This may be “strategically” placedfor the Ca2+ storage/release organelle that is located atthis site (Davies et al., 1991; Pettit and Hallett, 1998).

The Ca2+ permeability of the nuclear membrane maybe reduced by deposited chromosomal material more inneutrophils than in other cells. This material excludedfluo3–Ca2+ (but not fluo3). Although it acts as a physi-cal barrier to diffusion (also excluding lucifer yellowtransfer), it may also be a high capacity immobile Ca2+

chelator. There is probably a high density of negativelycharged Ca2+ binding molecules, such as histones andpossibly other molecules (Gilchrist et al., 1994), in thecondensed material. As this material prevents transfer offluo3–Ca2+ from one lobe to another, it may act as aCa2+ blocker. It was interesting to note that thosenuclear lobes which were insulated from changes influo3–Ca2+ in neighbouring nuclear lobes via an intra-lobe nuclear filament, were also less permeable to fluo3–Ca2+ in the surrounding cytosol. This again sug-gests that, away from the special region, the nuclearmembrane of some lobes may present a boundary tofluo3–Ca2+ movement.

This low permeability is in contrast to some of theother nuclear lobes, which were more permeable to fluo3and Ca2+ and accurately reflected changes in free Ca2+

in the cytosol. These lobes are thus similar to nerve cells(O’Malley, 1994), which are permeable to Ca2+, whereasthe impermeant nuclear lobes are more similar topancreatic nuclei (Gerasimenko et al., 1994) and neuro-blastoma cells (Al-Mohanna et al., 1994), which areinsulated from changes in cytosolic free Ca2+ andexclude short pulses of Ca2+. This spatial difference inCa2+ permeability may explain the speed with whichCa2+ penetrates the nucleus when released from thejuxta-nuclear Ca2+ store, but the slow subsidence time insome lobes, as Ca2+ slowly diffuses across other parts ofthe nuclear membrane.

Since the major Ca2+ release site for chemotacticstimuli is located close to the nucleus within the folds ofnuclear lobes (Davies et al., 1991; Pettit and Hallett,1998), the opportunity for Ca2+ tunnelling to occurthrough the nucleus exists. In HeLa cells, Ca2+ whichenters the nucleus at one site re-emerges at a distant site,

thus “by-passing” the cytosolic buffering (Lipp et al.,1997). Although the persistence of nuclear fluo3–Ca2+

signals reported here were observable only at 25 (C, it ispossible that Ca2+ tunnelling also occurs at physio-logical temperatures. Such tunnelling may provide anexplanation for the signalling of polarisation of neutro-phil morphology that occurs when these cells becomechemotactic. The nucleus lies towards the back of theneutrophil when undergoing chemotaxis, with the singlejuxta-nuclear Ca2+ release site close to it, but towardsthe front of the cell (Brundage et al., 1991; Hallett 1997).The organisation of these two organelles may give aCa2+ signal polarity throughout the cell as a result of anasymmetry of Ca2+ diffusion by trans-nuclear tunnelling(Hallett 1997). Thus, nuclear Ca2+ tunnelling may play aphysiological role. However, unlike HeLa cells, theunusual impermeability of some lobes of the neutro-phil nucleus and the filament-lobe boundaries, whichcorrelate with the presence of condensed chromo-somal material, may produce morecomplex tunnellingpatterns.

Acknowledgements

We thank the ARC (UK) and BBSRC (Max HanssFund) for supporting this work.

References

Al-Mohanna FA, Hallett MB. The use of fura 2 to determine therelationship between intracellular free Ca2+ and oxidase activationin rat neutrophils. Cell Calcium 1988;8:17–26.

Al-Mohanna FA, Caddy KWT, Bolsover SR. The nucleus is insulatedfrom large cytosolic calcium ion changes. Nature 1994;367:745–50.

Badminton MN, Kendall JM, Rembold CM, Campbell AK. Currentevidence suggests independent regulation of nuclear calcium. CellCalcium 1998;23:79–86.

Badminton MN, Campbell AK, Rembold CM. Differential regulationof nuclear and cytosolic Ca2+ in HeLa cells. J Biol Chem 1996;271:31210–4.

Bessis M. Living blood cells and their ultrastructure. Berlin:Springer-Verlag, 1993.

Brundage RA, Fogarty KE, Tuft RA, Fay FS. Ca2+ gradientsunderlying polarization and chemotaxis of eosinophils. Science1991;254:703–5.

Campbell MS, Lovell MA, Gorbsky GJ. Stability of nuclear seg-mentation in human neutrophils and evidence against a rolefor microfilaments or microtubules in their genesis duringdifferentiation of HL60 myelocytes. J Leuk Biol 1995;58:659–66.

Davies EV, Hallett MB, Campbell AK. Localised superoxide releaseby neutrophils can be provoked by a cytosolic calcium “cloud”.Immunology 1991;73:228–34.

Fox JL, Burgstahler AD, Nathanson MH. Mechanism of long rangeCa2+ signalling in the nucleus of isolated rat hepatocytes. BiochemJ 1997;326:491–4.

Gerasimenko OV, Gerasimenko JV, Petersen OH, Tepikin AV. Shortpulses of acteylcholine stimulation induce cytosolic Ca2+ signalsthat are excluded from the nuclear region in pancreatic acinar cells.Pflugers Arch 1994;432:1055–61.

R. Hodges et al. / Cell Biology International 27 (2003) 395–402 401

Gilchrist JSC, Czubryt MP, Pierce GN. Calcium and Ca2+ bindingproteins in the nucleus. Mol Cell Biochem 1994;135:79–88.

Hallett MB. Controlling the molecular motor of neutrophilchemotaxis. Bioessays 1997;19:615–21.

Hallett MB, Davies EV, Campbell AK. Oxidase activation in indi-vidual neutrophils is dependent on the onset and magnitude of theCa2+ signal. Cell Calcium 1990;11:655–63.

Hallett MB, Davies EV, Pettit EJ. Fluorescent methods for measuringand imaging the cytosolic free Ca2+ in neutrophils. Methods: ACompanion to Methods in Enzymology 1996;9:591–606.

Hallett MB, Hodges R, Cadman M et al. Techniques for measuringand manipulating free Ca2+ in the cytosol and organelles ofneutrophils. J Immunol Methods 1999;232:77–88.

Laffafian I, Hallett MB. Lipid-assisted microinjection: introducingmaterial into the cytosol and membranes of small cells. Biophys J1998;75:2558–63.

Lee MA, Dunn RC, Clapham DE, StehnoBittel L. Calcium regulationof nuclear pore permeability. Cell Calcium 1998;23:91–101.

Lipp P, Thomas D, Berridge MJ, Bootman MD. Nuclear calciumsignalling by individual cytoplasmic calcium puffs. EMBO J 1997;16:7166–73.

O’Malley DM. Calcium permeability of the nuclear envelope:evaluation using confocal volumes and intracellular perfusion.J Neurosci 1994;14:5741–8.

Paine PL, Moore LC, Horowitz SD. Nuclear envelope permeability.Nature 1975;254:109–14.

PerezTerzic C, StehnoBittel L, Clapham DE. Nucleoplasmic andcytoplasmic differences in the flourescence properties of the cal-cium indicator Fluo-3. Cell Calcium 1997a;21:275–82.

PerezTerzic C, Jaconi M, Clapham DE. Nuclear clacium and theregulation of the nuclear pore complex. Bioessays 1997b;21:787–92.

Pesty A, Avazeri N, Lefevre B. Nuclear calcium release by InsP3receptor channels plays a role in meiosis reinitiation in the mouseoocyte. Cell Calcium 1998;24:239–51.

Pettit EJ, Hallett MB. Early Ca2+ signalling events in neutrophilsdetected by rapid confocal laser scanning. Biochem J 1995;310:445–8.

Pettit EJ, Hallett MB. Localised and global cytosolic Ca2+ changes inneutrophils during engagement of CD11b/CD18 integrin visualisedusing confocal laser scanning reconstruction. J Cell Sci 1996;109:1689–94.

Pettit EJ, Hallett MB. Two distinct Ca2+ storage and release sites inhuman neutrophils. J Leuk Biol 1998;63:225–32.

Sanchez JA, Karni RJ, Wangh LJ. Fluorescent in situ hybridization(FISH) analysis of the relationship between chromosome locationand nuclear morphology in human neutrophils. Chromosoma1997;106:168–77.

Santella L, DeRiso L, Gragnaniello G, Kyozuka E. Separate acti-vation of the cytoplasmic and nuclear calcium pools in matur-ing starfish oocytes. Biochem Biophys Res Commun 1998;252:1–4.

Schmid-Schonbein GW, Shih YY, Chen S. Morphometry of humanneutrophils. Blood 1998;56:866–78.

Sheikh S, Nash GB. Continuous activation and deactivation ofCD11b/CD18 during de novo expression enables rolling neutro-phils to immobilise on platelets. Blood 1996;87:5040–50.

R. Hodges et al. / Cell Biology International 27 (2003) 395–402402