Visualization of the cytoplasmic Ca2+ gradient in Fucus serratus rhizoids:

correlation with cell ultrastructure and polarity

COLIN BROWNLEE and ANN L. PULSFORD

Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth PLl 2PB, UK

Summary

Fura-2 has been used with digital image analysis tovisualize and quantify the cytoplasmic Ca2+ gradi-ent in rhizoid cells of Fucus serratus. Ca2+ washigher at the growing rhizoid tip in about 50-60 %of cells studied to date. Considerable variation inthe pattern of the Ca2+ gradient has been found.Verapamil reduced but did not abolish the Ca2+

gradient. Nifedipine had no effect on Ca2+ distri-bution during the experimental period. RemovingCa2+ from the growth medium permeabilized theplasmalemma and allowed fura-2 to efflux from thecytoplasmic compartment, permitting an assess-

ment of the degree of sequestration of the dye intovacuoles and vesicles. Comparisons of ratio imageswith electron micrographs suggested that lowerCa2+ levels were associated with the nuclear region,but there was no direct correlation between Ca2+

levels and vesicle distribution in healthy cells. Therole of localized differences in Ca2+ distribution inthe control of polarity is discussed.

Key words: cytoplasmic Ca2+, fura-2, digital image analysis,polarity.

Introduction

The acquisition and expression of polarity is a fundamen-tal process involved in the growth and development ofvirtually all multicellular organisms. The Fucus zygote isan ideal system for studying regulation of cell functions,especially the control of polarity and development. Therecently fertilized zygote dramatically displays severalfeatures, most notably the fixation of the polar axis andexpression of polarity. A few hours after fertilization,zygotes growing in unidirectional light differentiate into arhizoid and thallus end. This polarity is manifest even atthe single-cell stage. An understanding of these processesis essential to the elucidation of the mechanisms involvedin the development and growth of multicellular plants.The role of cytoplasmic free Ca2+ (Ca2+cyt) in thecontrol of plant cell functions has been widely postulated(e.g. see Gilroy et al. 1987; Hepler & Wayne, 1985;Trewavas, 1986). There is strong evidence for a role ofCa2+ in the control of polarized growth in Fucus rhizoids(e.g. see Jaffe, 1986; Brownlee & Wood, 1986) and pollentubes (Nobiling & Reiss, 1987; Reiss & Herth, 1985).With few exceptions, however, the much-neededmeasurements of [Ca2+cyt] have not been made. Thereare several reasons for this (Brownlee, 1987). The smallsize, presence of cell wall, vacuole and turgor pressure inmost plant cells have made microinjection of indicatorssuch as aequorin difficult, if not impossible. Indeed, only

Journal of Cell Science 91, 249-256 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

one report of the use of aequorin in intact plant cellsexists (Williamson & Ashley, 1982). The same factorshave limited the use of Ca -selective microelectrodes(Brownlee, 1986; Brownlee & Wood, 1986; Miller &Sanders, 1987). The use of acetoxymethyl esters offluorescent indicators such as quin-2 and fura-2 has metwith limited success (Cork, 1985; Brownlee & Wood,1986), except in diatom cells (Brownlee et al. 1987),pollen tubes (Nobiling & Reiss, 1987) and Haemanthusendosperm cells (Keith et al. 1985). Gilroy et al. (1986)have successfully incorporated quin-2 into protoplastsusing an electroporation technique and have measuredphysiologically low Ca2+ levels.

Work with Ca +-selective microelectrodes has indi-cated the presence of a gradient of [Ca2+cyt] in theFucusrhizoid with higher concentrations at the growing tip(Brownlee & Wood, 1986). Kropf & Quatrano (1987)demonstrated enhanced chlorotetracycline fluorescencein growing rhizoid tips of Fucus distichus, indicatinghigher levels of membrane-associated Ca2+ in this region.This method, however, does not give quantitative infor-mation and the physiological significance of such gradi-ents remains speculative. Fura-2 is well suited for Ca2+

measurements in single cells. It can be used in a dualwavelength mode, so eliminating errors due to unequalcell thickness and dye distribution (Tsien et al. 1985).The fluorescence properties of fura-2 are such that theratio of fluorescence at 350 and 385 nm excitation is

249

Ca2+-dependent (Grynkiewicz et al. 1985). In the pres-ent paper, fura-2 is used to map Ca2+ in the Fucusrhizoid and monitor induced changes in the distributionof Ca2+, which may be correlated with disruption ofpolarity.

Materials and methods

Zygote cultureGametes were liberated from mature Fucus senatus fronds asdescribed by Quatrano (1980). Zygotes were grown on glasscoverslips in continuous light at 16°C in filtered sea water.

Fura-2 microinjectionRhizoid cells were impaled with filamented microelectrodes(=0-25 [im tip diameter, 15-20 MQ if filled with 3 M - K C 1 )containing l-OniM-fura-2 in their tips. After recording stableresting potentials (-80 to — 90 mV), fura-2 was injected ionto-phoretically (1-0 nA negative pulses, 2 Hz, 200 ms duration for3-5 min), giving a final estimated fura-2 concentration in thecell of around 100,UM (Purves, 1981). This injection protocol,when applied to cells that show vigorous cytoplasmic streaming(e.g. tomato root hairs), did not affect the streaming rate,indicating little or no modification of [Ca2+cyt] (Clarkson et al.1988). Rhizoid cells were impaled =20 fim behind the apex inorder to avoid any possible confusion between naturally occur-ring Ca2+ gradients and artificially elevated Caz+ levels in theregion of impalement. Following microinjection, the electrodewas removed and the cell was allowed to recover for at least5 min.

Fluorescence microscopyCa2+-dependent fura-2 fluorescence was monitored with dualwavelength fluorescence microscopy (e.g. see Williams et al.1985; Poenieef al. 1986; Brownlee et al. 1987) using a modifiedLeitz fluorescence microscope with a 200 W mercury vapourlamp and a Zeiss Neofluar X40 water-immersion objective.Excitation wavelengths were 350 and 385 nm (8 nm band width,u.v. line filters, Schott). Emission was recorded at 500nm(45 nm bandwidth) using an image-intensified NewviconCCTV camera (Panasonic WV-1900) modified to give variablemanual control of gain. Images were analysed either directlyfrom the camera or stored on video tape. Autofluorescence ateach excitation wavelength and background video signal wererecorded prior to injection. Images of dye-loaded cells at 350and 385 nm were recorded for at least 15 s. Filters were changedmanually. Dye bleaching was negligible during recording.

Image analysisImages were digitized (512X512pixels, 256 grey levels) using aKontron (W. Germany) image analyser with IBAS software. Atotal of 30-60 frames from each image were averaged to improvesignal-to-noise ratios and reduce errors due to temporal vari-ations in lamp output. Spatial variations in camera response andexcitation intensity were monitored as described by Brownlee etal. (1987). After subtraction of mean dark video signal and anyautofluorescence, 350/385 nm ratio images were obtained.Quantitative ratio profiles across chosen transects were com-pared with calibration curves (see below) to calculate pCavalues.

CalibrationIn vitro calibration was carried out using 100-jUl drops ofcalibration buffers (Tsien & Rink, 1980) containing 50 ,UM-

fura-2. For in situ calibration, cells were incubated inionomycin for 10 min and equilibrated with pCa buffers (Wil-liams et al. 1985; Poenie et al. 1986).

Electron micmscopyZygotes growing on cellophane substrates were fixed in situwith 2-5 % glutaraldehyde in sea water with 1 % paraformal-dehyde and 1% sucrose (Evans & Holligan, 1972). Afterwashing in 0-1 M-cacodylate buffer they were postfixed with 1 %osmium tetroxide, dehydrated and embedded in Spurr resin.Sections were mounted on Formvar-coated 1 mm copper slotgrids, stained with uranyl acetate and lead citrate and viewedwith a Philips EM300 transmission electron microscope.

Fig. 1. Bright-field (A) and fluorescence (B,C) images of3-day-old F. senatus rhizoid cells 10 min after microinjectionwith fura-2. B. At 350nm excitation; C, at 385 nm excitation.Arrow indicates site of microinjection. Bar, 10 f.im.

250 C. Brownlee and A. L. Pulsford

Results

Dye distribution following injectionMicroinjection of fura-2 into rhizoid cells resulted inincreased fluorescence at 350 and 385 nm excitation(Fig. 1). Fluorescence was more intense at both wave-lengths in the nuclear and perinuclear regions (cf.Fig. 7), indicating an accumulation of dye in this region.Two kinds of response following injection have beenfound. In the majority of cells, as in Fig. 1, dye distri-bution remained diffuse and fluorescence could be moni-tored for at least 1 h following injection. However,occasional cells showed punctate fluorescence after15-20 min accompanied by a reduced overall fluor-escence (Fig. 2). For the experiments described here,only cells that maintained diffuse fluorescence were used.Dye was generally restricted to the apical cell in 3-day-oldzygotes, but in 2-day-old zygotes microinjection into theapical cell resulted in distribution throughout the wholezygote (Fig. 3). Treatment of fura-2-loaded zygotes withCa^+-free sea water resulted in a loss of fluorescence fromthe cells monitored at 365 nm excitation (isosbesticpoint). Dye was initially rapidly lost from the apical cell(Fig. 3) and much more slowly from the more highlyvacuolate first rhizoid cell. After 15 min in Ca2+-freemedium, the remaining fluorescence in the mature rhi-zoid cell began to assume a paniculate appearance. Thiswas more pronounced in cells that had remained loadedfor longer times (>30min).

Ca2+ distribution from ratio imagesIn situ and in vitro calibration curves differed by no morethan 5 % (Fig. 4) and are comparable in shape to thosepresented by Williams et al. (1985). Fig. 5 shows350/385 nm ratio images of the cell shown in Fig. 1,together with quantitative longitudinal profiles taken10 min following microinjection to allow equilibration ofCa2+cyt following any possible disruption during micro-

injection. Little or no change in Ca2+cyt has beenobserved during this period. [Ca2+cyt] was higher at thegrowing cell apex (Fig. 4A). [Ca2+cyt] ranged from 105(±15, n= 10) nM in the region of the nucleus to 450(±30) nM at the extreme apex. Fig. 5B shows the effect of5xlO~5 M-verapamil on [Ca2+cyt] in the same cell. Areduction in [Ca2+cyt] occurred throughout most of thecell but the effect was more marked at the apex, reducingthe magnitude of the gradient. Verapamil caused themean [Ca2+] in the tip region to fall from 460 to 195(±45) nM (n = 5). No significant change was found in theregion of the nucleus. The effect could be observed 1 minafter application of verapamil. We were unable to detect asignificant change in Ca2+ in response to the sameconcentrations of nifedipine for up to 10 min after ad-dition of nifedipine.

Considerable variation in the magnitude and extent ofthe Ca2+ gradient has been observed. Fig. 6 showsexamples of the types of Ca2+ distribution observed.Rhizoid cells with high Ca2+ localized only at the extremetip occur (Fig. 6A) or with higher Ca2+ only at specificsites at the cell apex (Fig. 6B). Occasionally, cells areobserved without any apparent apically elevated Ca2+

(Fig. 6C). Ca2+ distribution similar to that shown inFig. 4 has been observed in 50-60 % of rhizoids investi-gated to date.

Rhizoid ultrastructureThe rhizoid of the 3-day-old zygote consists of two cellsseparated by a cell wall. The length of the apical cell atthis stage varies between different zygotes (cf.Fig. 7A,B). The growing apical cell shows a typicalorganelle distribution (Fig. 7A; see also Brawley et al.1977). Several distinct types of vesicle can be dis-tinguished, including osmiophilic vesicles, granular andclear vesicles of different sizes and large vesicles or smallvacuoles. There are no large vacuoles in the apical rhizoidcell. There is a general accumulation of vesicles and small

Fig. 2. Rhizoid cell loaded with fura-2 showing diffuse fluorescence (A) at t = 0 but with an indication of dye already being lostfrom the cell apex, followed by an overall loss of fluorescence and particulate nature after 20min (B). Increased backgroundnoise in B is due to increased camera gain to record this image. Arrow indicates site of microinjection. Bar,

Cytoplasmic Ca2+ in Fucus serratus rhizoids 251

Fig. 3. A 2-day-old zygote loaded with fura-2. A. Bright-field image showing apical and sub-apical rhizoid cells.B. Fluorescence at 365 nm excitation lOmin after loading, showing dye distributed throughout the zygote. C. 1 min; and D,15min after treatment with Ca2+-free artificial sea water (SOOmM-NaCl, 0mM-CaCl2, 10niM-MgCl2, 2-5 mM-NaHC03, 10mM-KC1, 1-OmM-EGTA, pH8-0). Arrow indicates site of microinjection. Bar, 20/Um.

1-25

o 1-0

0-75

0-5

0-25

8 7 6 5pCa

Fig. 4. In vitro (•) and in situ (O) calibration curves forfura-2 (see Materials and methods).

vacuoles in the apical and sub-apical regions and in theregion around the nucleus. Endoplasmic reticulum ispresent in the perinuclear region but does not appear tobe present in the apical region. The nucleus usually lies=20-40 (im behind the apex.

Discussion

The results show that a gradient of free [Ca2+] can bevisualized in growing rhizoid cells. The magnitude of thisgradient is such that it is likely to have a role in directingpolarized growth. There are at least two possible conse-quences of the Ca2+ gradient. Jaffe & Nuccitelli (1977)have postulated that a voltage gradient associated withpositive ion entry at the growing tip may induce electro-phoretic movement of vesicles to that region, withsubsequent exocytosis, growth and wall formation. How-

252 C. Brownlee and A. L. Puhford

10Ca (nM)

50 100 1000

pCa

6

Fig. 5. False colour 350/385 nm ratio images of cell shown in Fig. 1, together with corresponding quantitative longitudinalprofiles (straight line a-b) of pCa. 350/385 nm ratios for each pixel were assigned grey values and colour coded as indicated.A. At 10 min after fura-2 microinjection; B, 3 min after addition of 5X 10~5 M-verapamil.

Fig. 6. Bright-field (A,C,E) and corresponding 350/385 nm ratio images of 3-day-old rhizoid cells loaded with fura-2 showingregions of elevated Ca2+ highly localized at the hyphal apex (B,D), or absence of any apical gradient (C). Arrow indicates site ofmicroinjection. Bar, 20 /xm.

ever, it seems likely that interaction with Ca2+-sensitiveprocesses such as calmodulin-regulated events, vesiclefusion and possible regulation of ion fluxes is occurring.

Though mean [Ca2+cyt] values at the rhizoid tip areprobably not high enough to promote responses such asexocytosis and calmodulin-regulated events, other factors

Cytoplasmic Ca2+ in Fucus serratus rhizoids 253

Fig. 7. EM photomicrographs of apical and sub-apical regions of typical 3-day-old rhizoids. A. Single cell; B, dividing cell;C, cell wall; v, vesicles; vac, vacuoles; ov, osmiophilic vesicles; «, nucleus; ch, chloroplast; er, endoplasmic reticulum.Bar, 5 jUm.

may interact (e.g. guanine nucleotides) to reduce the[Ca2+] required for the activation of certain processes(Haslam & Davidson, 1984). Furthermore, the mean[Ca2+] values may not represent the maximum Ca2+

levels in rapidly growing rhizoid cells.The [Ca2+cyt] values reported here are somewhat

lower than those measured with Ca2+-selective micro-electrodes (Brovvnlee & Wood, 1986), especially in the tipregion, but are of similar magnitude to those found byNobiling & Reiss (1987), who used quin-2 to detect agradient of [Ca2+cyt] in pollen tubes. The reason for

higher electrode-derived Ca2+ values is not clear. Severalreports using Ca2+ electrodes in plant and animal cellshave given [Ca2+cyt] up to 1 /UM or more (see Brownlee &Wood, 1986). Leakage of Ca at the site of the Ca2+

electrode impalement may cause artificially high [Ca2+]precisely in the region being monitored. This is difficultto test since such artificially elevated Ca levels are likelyto be highly localized and not resolvable by currentmonitoring techniques. In the Fucus rhizoid leakage andmembrane damage may be more severe at the growing tip(Brownlee & Wood, 1986).

254 C. Brownlee and A. L. Pulsford

[Ca2+] is lower in the region containing the nucleusthan elsewhere. This contrasts with the finding of Wil-liams et al. (1986), who showed elevated [Ca2+] in thenuclear region of smooth muscle cells. It is not clear whysome cells do not show a Ca2+ gradient or show only avery localized gradient. Further work is required todecide whether such cells show low or zero growth. It isalso possible that the gradient may not be static in any onecell. Woods et al. (1987) have shown that stimulation ofrat hepatocytes results in oscillations of Ca2+cyt. It ispossible that Ca2+ oscillations also occur in plant cells,including the Fucus rhizoid. These may involve temporalchanges in the magnitude of the observed Ca2+ gradient.Connor (1986) has visualized Ca2+ gradients in growthprocesses of mammalian nerve cells using fura-2. Gradi-ents were observed only in actively extending cells.

This paper describes a relatively simple and non-disruptive method for direct incorporation of fura-2 intoplant cells. The viability of the cells can be monitoredduring microinjection by measuring membrane potential.Advantages of microinjection of Ca2+ indicators havebeen described (Cobbold & Rink, 1987). Certain pre-cautions to be taken with this technique need to beemphasized, however. In some cells dye is graduallysequestered, presumably into small vacuoles and vesicles(F. serratus rhizoids do not contain large vacuoles).Punctate fluorescence was more frequent in cells thatshowed depolarization during loading and is probably aresponse to cell damage, possibly indicating stimulationof anion transport at vacuole and vesicle membranes.Fura-2 is likely to be transported by anion transporters.The overall loss of fluorescence from the cytoplasm ofthese cells suggests loss of dye by passive leakage or viaactive anion transport at the plasmalemma. Only cellsthat maintained healthy membrane potentials and did notdevelop punctate fluorescence were used in this study.Use of fura-2 and other fluorescent indicators with otherplant cells must be accompanied by visual monitoring ofcompartmentation of the dye within the cell. Significantaccumulation of fura-2 into vacuoles of root hair cells hasbeen observed (Clarkson et al. 1988). Such accumulationmay result in overestimation of [Ca2+cyt] if used in cellsuspensions. If used with single cells and digital imageanalysis, however, cytoplasmic regions of the cell can beanalysed separately, regardless of the degree of vacuola-tion or sequestration of the dye.

It can be argued that spatial inhomogeneities visualizedwith fura-2 may reflect sequestration of the dye intovesicles that may contain a higher Ca and may bepreferentially accumulated in the tip region. However,available evidence strongly suggests that this is not so.First, only cells that showed a diffuse, non-particulatedye distribution were selected. Cells with punctate fluor-escence did not show a Ca2+ gradient. Incubation ofloaded zygotes in Ca2+-free medium caused dye to be lostselectively from different regions of the zygote. Dye wasfirst rapidly (within 1 min) lost from the apical regionsbut was more strongly held in the more vacuolate maturerhizoid cell. We interpret this in terms of increasedplasmalemma permeability in the absence of Ca2+, allow-ing dye to efflux from cytoplasmic regions. Intracellular

membranes (vacuolar and vesicle) are unlikely to beaffected by low Caz+ in the same manner and so dye heldin these compartments will not be lost during thistreatment. It follows that the greater proportion offluorescence signal in the apical cell comes from dye thatis bound only by the plasmalemma, i.e. cytoplasmic.Similar interpretations have been applied to digitonin-permeabilized cells (see Cobbold & Rink, 1987). Second,electron micrographs show that although there is ageneral accumulation of vesicles and small vacuoles in thetip and sub-tip regions, there does not appear to be amarked accumulation of vesicles in the tip. In addition,the nuclear region also shows an accumulation of vesicles(Fig. 6; and Brawley et al. 1977) but this region displayslow [Ca2+]. Third, the reduction of [Ca2+] by verapamilin the tip region can reasonably be explained only interms of a reduction of cytoplasmic [Ca ]. The resultsare also qualitatively in agreement with those obtainedwith Ca2+-selective microelectrodes (Brownlee & Wood,1986).

Cells in the F. serratus zygote are dye-coupled up tothe four-cell stage (see Fig. 3). Thereafter, dye injectedinto the apical cell is restricted to that cell or moves onlyas far as the adjacent cell. Though no defined couplingstructures (plasmodesmata) have been observed in Fucuszygotes, cytoplasmic connections do occur for some timeafter cell division until they are obliterated by the cell wall(Brawley et al. 1977). The dye movements observed hereprobably reflect these structural changes.

Verapamil reduced [Ca2+] in the tip region andreduced the magnitude of the Ca2+ gradient, though thiswas not completely abolished. Verapamil probably actsby blocking voltage-regulated Ca2+ channels (Triggle,1981). In the Fucus rhizoid, the partial dissipation of theCa2+ gradient by verapamil strongly suggests that Ca2+

influx is at least partly responsible for maintaining ahigher [Ca2+] at the growing tip (Gilkey & Jaffe, 1976;Brownlee & Wood, 1986). Verapamil has recently beenshown to block Ca2+ entry and to bind to membranes ofcarrot protoplasts (Graziana et al. 1988). Nifedipine didnot affect the [Ca2+cyt] in short-term experiments in thisstudy. Interestingly, Graziana et al. (1988) showed thatnifedipine did not affect Ca2+ influx in carrot protoplasts.However, prolonged incubation of Fucus zygotes innifedipine did disrupt zygote polarity (data not shown)and effects of nifedipine have been observed on lily pollentube tip growth (Reiss & Herth, 1985) and turgorpressure regulation in Lamprothamnium cells (Okazaki &Tazawa, 1986). No effect on chlorotetracycline-visualizedCa2+ distribution could be observed until 15 min afterapplication of nifedipine to lily pollen tubes (Reiss &Herth, 1985). It is possible that longer-term changes inCa + in response to nifedipine do occur in Fucusrhizoids, which were not detected in the present study.

Kropf & Quatrano (1987) showed that elongation of F.distichus rhizoids was sensitive to channel blockers,whereas polar axis fixation was not, and it seems likelythat axis fixation involves incorporation or migration ofionic channels in the plasma membrane, while polarityexpression (i.e. rhizoid growth) requires these channelsto be functional (Brawley & Robinson, 1985). Work is

Cytoplasmic Ca in Fucus serratus rhizoids 255

now in progress to distinguish further between these twoprocesses.

This work was supported by the NERC.

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{Received 13 June 1988 - Accepted 15 July 1988)

256 C. Brownlee and A. L. Pulsford