J. Cell Sd. 79, 217-235 (1985) 217Printed in Great Britain © The Company of Biologists Limited 1985

SUBPLASMALEMMAL CALCIUM-BINDING

MICROREGIONS IN ACANTHAMOEBA

A. SOBOTADepartment of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str.,02-093 Warsaw, Poland

SUMMARY

Acanthamoeba cells, fixed with glutaraldehyde supplemented with calcium ions, show Ca-dependent, electron-dense deposits (CaDD) at the cytoplasmic surface of the plasma membrane.The formation of CaDD is stimulated by pre-incubation and fixation of the cells in the presence ofATP. Chemical analysis of the trichloroacetic acid extracts of CaDD-containing cells demonstratesthat adenosine monophosphate, pyrophosphate and inorganic phosphate are probably the com-pounds involved in the formation of CaDD. Treatment of CaDD-containing cells with exogenousphosphatase evokes the disappearance of electron-dense material and a fine fibrillar network can beobserved inside the 'empty' CaDD. The ability to restore CaDD in the presence of calcium ionswith either pyrophosphate or orthophosphate confirms the suggestion that calcium/phosphate saltsmay be deposited in special subplasmalemmal calcium-binding microregions.

INTRODUCTION

Cytochemical studies of the cell surface reveal calcium-dependent electron-densedeposits (CaDD) at the cytoplasmic surface of the plasma membrane (Clawson &Good, 1971; Oschman & Wall, 1972). These deposits are found at the plasmamembrane of cells active in endocytosis (Przelecka & Sobota, 1976; Sobota,Hrebenda & Przelecka, 1977; Stockem & Klein, 1979; de Chastellier & Ryter, 1982),exocytosis (Plattner & Fuchs, 1975), secretion (Skaer, Peters & Emmines, 1974), cellmovement (Stockem & Klein, 1979), contraction (Oschman & Wall, 1972; Nicaise,Hernandez-Nicaise & Malaval, 1982) and excitation processes (Hillman & Llinas,1974; Oschman, Hall, Peters & Wall, 1974; Politoff, Rose & Pappas, 1974; DeAraujo Jorge, De Souza & Machado, 1979).

It is suggested that the appearance of Ca-dependent deposits reflects the presenceof membrane-bound calcium stores, which may be a source of trigger calcium for theactivation or inhibition of submembraneous contractile elements (Cohen & de Vries,1973; Stockem & Klein, 1979). Calcium stores at the plasma membrane have beenalso predicted from physiological and biochemical studies (Hallett, Schneider &Carbone, 1972; Josefsson, 1975; St Louis & Sulakhe, 1976; Feldman & Weinhold,1977; Limas, 1977; Kusamran, Mattox & Thompson, 1980; Stolze & Schultz, 1980;Glowacka, Sobota & Przelecka, 1985).

Elemental analysis of CaDD by the X-ray microanalysis technique reveals calciumand phosphorus as the main elements of these structures (Oschman et al. 1974;

Key words: calcium-binding microregions, plasma membrane, filamentous matrix.

218 A. Sobota

Skaer et al. 1974; Sobota, Przelecka & Janossy, 1978), the atomic ratio of theseelements being similar to that of the dicalcium ATP salt (Skaer et al. 1974). Ourprevious X-ray microanalysis studies suggest that besides ATP, other low molecularweight phosphates might also be engaged in CaDD (Sobota et al. 1978).

The role of phosphoproteins and phospholipids as constituents of the electron-dense deposits has also been considered (Oschman et al. 1974; Geyer, Linss &Stibenz, 1978; Belitser, Zaalishvilli & Sytnianskaja, 1982). Nevertheless, the natureand origin of calcium phosphorus-containing, electron-dense deposits remainobscure.

In the present study we tried to characterize the chemical nature of CaDD bycytochemical and biochemical methods, and consider what kind of phosphorus isinvolved in the formation of the deposits. Conditions have been found under whichthe destruction and restoration of Ca deposits may occur. Ultrastructural andcytochemical studies reveal filamentous material inside the 'empty' CaDD, whichmight serve as a nucleation centre for the deposition of an excess of calcium ionsentering the cells.

MATERIALS AND METHODS

Culture

The experiments were performed on a small, free-living soil amoeba, Acanthamoeba castellanii(Neff strain). Acanthamoeba cells were cultured axenically as previously described (Sobota,Burovina, Pogorelov & Solus, 1984). For experiments 6- to 7-day-old cultures (~106 cells per ml)were used.

Formation of CaDDCalcium-dependent, electron-dense deposits were examined with an electron microscope as

described earlier (Sobota et al. 1977). Briefly, the cells were fixed in 2-5 % glutaraldehyde (Serva,•Az35/280= 1, or freshly distilled) containing 5mM-CaCl2, 50mM-NaCl, and buffered with 50 mM-collidine buffer, pH 7-4 (glutaraldehyde/Ca fixative) for 1 h at 0°C or at room temperature. Thenthe samples were rinsed with solution containing 5mM-CaCl2, lOOmM-NaCl and buffered withSO mM-collidine, pH7-4 (Ca-washing solution) and postfixed with osmium/Ca fixative ( 1 %osmium tetroxide, 5 mM-CaCl2, lOOmM-NaCl, 50mM-collidine buffer, pH7-4). Subsequently, thecells were dehydrated in an ethanol series, passed though propylene oxide and finally embedded inEpon 812. Thin sections, with or without subsequent counterstaining, were examined in a JEM100 B electron microscope at 80 kV. In some experiments the cells were fixed with glutaraldehydefixative in which CaCl2 was replaced by 0-1 or 3-0 mM-Pb(NO3)2 or by 0-1 or 1 -0 mM-Ce2(SO4)2. Inthese cases, the washing solutions and osmium fixative did not contain Pbz + and Ce3+, as well asCa2+. Ce3+ and Pb2 + are useful trapping cations for inorganic phosphate (Wachstein & Meisel,1957; Robinson & Karnovsky, 1983).

Destruction of CaDDThe glutaraldehyde/Ca-fixed cells containing CaDD were rinsed either with 5 % trichloroacetic

acid (TCA) for 5 min at 0°C or with a solution containing lOmM-EDTA, 2-5 % glutaraldehydeand 50 mM-phosphate buffer, pH 7-0 (EDTA/phosphate solution), 3X2 min at 0°C or 20°C as indi-cated in the Results. Then the samples were rinsed with Ca-washing solution, postfixed withosmium/Ca fixative and prepared for electron-microscopic examination as described above.

The effect of alkaline phosphatase on the destruction of CaDD was checked in the glutar-aldehyde/Ca-fixed cells. For this purpose the fixed cells were rinsed (2x5 min) with Ca-washing

Calcium-binding micmregions 219

solution supplemented with lOmM-lysine. The pelleted cells were treated with dry acetone at—20 °C for 5 min. Under these conditions the plasma membrane is partly destroyed, which allowsmacromolecules to enter the cells (Gadasi & Korn, 1980). After washing, the samples wereresuspended in a solution containing 5mM-CaCl2, lOOmM-NaCl, and 50 mM-collidine buffer,pH7-4, and supplemented with alkaline phosphatase ( lmgml"1 , Sigma). The samples weretreated with alkaline phosphatase for 3 h at 25 °C. The control samples were incubated with heat-denatured (100°C, 3 min) enzyme. The pelleted samples were rinsed with Ca-washing solution,postfixed with osmium/Ca fixative and processed for electron-microscopic examination.

Extraction oflipidsAfter glutaraldehyde/Ca fixation the cells were treated with chloroform/methanol mixture

(2:l ,v/v) for 30min at room temperature. The samples were then rinsed with Ca-washingsolution, postfixed with osmium/Ca fixative for 45 min at room temperature and prepared forelectron-microscopic examination.

Restoration of electron-dense depositsCells fixed with glutaraldehyde/Ca fixative (0°C, 1 h) were rinsed with either 5 % TCA or

EDTA/phosphate solution (3x2min, at 0°C). Such treatments resulted in the destruction ofCaDD. Without any other washes the samples were resuspended in a solution containing lOmM-CaCl2, 2-5% glutaraldehyde and buffered with collidine, pH7-4 (second glutaraldehyde/Cafixative). One hour later the samples were washed once in a solution containing 10mM-CaCl2,lOOmM-NaCl, 50mM-collidine buffer, pH7-4, and subsequently postfixed with osmium/Cafixative for 45 min. All the above procedures were carried out at 0°C. The control experiments aredescribed in the Results.

Localization of Ca-ATPase activityAcanthamoeba cells were fixed with 2-5% glutaraldehyde containing lOOmM-sucrose, 50 mM-

Hepes buffer, pH 7-4, during 1 h at room temperature. The samples were rinsed in solutioncontaining lOOmM-sucrose, 50mM-NaCl, 50mM-Hepes buffer, pH7-4 (3x5min at room tem-perature). The enzyme activity was detected in a medium containing 2mM-ATP, 3mM-CaCl2,1 mM-Pb(NO3)2, lOOmM-sucrose, 50 mM-Hepes buffer, pH7-4. The incubation was carried out for15 min at 25°C. After that the samples were rinsed three times in a solution containing 100 mM-sucrose, 50mM-NaCl, 50 mM-Hepes buffer, pH7-4, and postfixed with 1% osmium tetroxidesolution containing lOOmM-NaCl, 50mM-Hepes buffer, pH7-4. The samples were then preparedas usual for electron-microscopic examination.

Analytical methodsAdenine-related compounds labelled with 3H in TCA extracts of Acanthamoeba cells fixed with

glutaraldehyde were separated by descending paper chromatography on Whatman no. 1 paper(3cmX37cm) using isobutyric acid/29% NH^OH/lOOmM-EDTA/^O (66:1:1:32, by vol.)system (Gruda, Pollender & Therien, 1983). The spots were detected under ultraviolet light. Thesheets were cut into 2-cm strips and extracted with 0-8 ml of 0-2M-HC1 for 2h with shaking. TheHC1 extracts were put into scintillation vials and dried. The dry material was dissolved in 10 ml ofscintillation solution (650 ml toluene, 350 ml Triton X-100, 7g 2,5-diphenyloxazole (PPO), 0-5 gl,4-bis-2(5-phenyloxazolyl)benzene (POPOP)) and counted in a liquid scintillation counter.

Phosphates were separated, depending on their chain length, by descending chromatography onWhatman no. 1 paper (3 cmX37 cm). The chromatograms were developed with a diluted mixtureof 2-propanol/trichloracetic acid/29 % NH4OH/H2O (735 ml/50 g/2-5 ml/265 ml); 100 ml of thismixture was diluted with 25 ml H2O before use as a chromatographic solvent (Griffin, Davidian &Penniall, 1965). Rp values of phosphate standards with different chain lengths (mono-, di-, poly-phosphates) were taken from Griffin et al. (1965). The analysis of 3ZP distribution was performedby counting 1-cm strips in a Geiger-Muller counter.

Inorganic phosphate was estimated by the method of Fiske & Subbarow (1925).

220 A. Sobota

RESULTS

Formation of CaDD: effects ofPb2+, ATP and pyrvphosphate

As demonstrated previously (Sobota et al. 1977), fixation of Acanthamoeba cellswith 2-5 % glutaraldehyde solution containing calcium ions revealed numerous elec-tron-dense deposits at the cytoplasmic surface of the plasma membrane (Fig. 1). Thetwo-dimensional distribution of these deposits is shown in Figure 2. When thefixative contained 3 mM-Pb2+ instead of calcium ions, no electron-dense depositswere observed at the cytoplasmic surface of the plasma membrane (Fig. 3). How-ever, under these conditions abundant electron-dense deposits were visible inmitochondria, in the space between inner and outer mitochondrial membranes, andat the external surface of the plasma membrane (Fig. 3). Decreasing the Pb2+

concentration in the fixative to 0 1 mM reduced significantly the number of electron-dense granules and only a few of them could be observed at the external surface of theplasma membrane. When the fixative solution contained 1-0 or (MmM-Ce3+, nodeposits were observed, either inside the mitochondria or at the plasma membrane.

The number of calcium-dependent deposits at the plasma membrane increasedwhen the cells were incubated with 3 mM-ATP before fixation and when theglutaraldehyde/Ca fixation was also carried out in the presence of ATP. The analysisof the frequency of CaDD distribution along the plasma membrane showed that thepresence of ATP in the fixative increased the number of CaDD 1-4 times in com-parison with the control value. The examination of ATP-enhanced CaDD with theelectron microscope revealed that some deposits were linked to a filamentousmaterial, which seemed to be inserted in the CaDD (Fig. 4). The stimulatory effectof ATP on the appearance of CaDD suggested that ATP or products of ATPhydrolysis might be precipitated as calcium salts in the deposits. ATP might alsostimulate the phosphorylation of some subplasmalemmal constituents, whichfacilitated binding of calcium/phosphate salts.

The detection of relatively high amounts of pyrophosphate, inorganic phosphateand AMP, and small amounts of ATP in the extracts from CaDD-containing cells(see below) suggested that these compounds might be constituents of CaDD.Therefore, the effect of these compounds on the formation of CaDD was checked oncells prefixed with Ca2+-free glutaraldehyde fixative (0°C, 15min). After suchprefixation electron-dense deposits were never observed. The prefixed cells werethen loaded with either 3 mM-AMP, 3 mM-ATP, 3 mM-KH2PO4 or 3 mM-potassium

Fig. 1. Electron micrograph of cell fixed with glutaraldehyde/Ca solution. CaDD areobserved at the cytoplasmic surface of the plasma membrane. X30 000.Fig. 2. Periphery of a flattened cell showing two-dimensional distribution of CaDD. Thecell, after fixation for 5 min, was flattened directly on a Formvar/carbon-coated grid. Thesample was washed for 1 s in water and examined under the electron microscope withoutosmium fixation and staining. CaDD differing in contrast, probably belong to twoopposing cell membranes. X47 000.Fig. 3. Section of the cell fixed with glutaraldehyde/Pb2+. No electron-dense depositsare observed at the cytoplasmic surface of the plasma membrane. Abundant deposits arevisible in mitochondria, cytoplasmic vesicle, and at the outer surface of the plasmamembrane. X66 000.

Calcium-binding microregions 221

t•m

A. Sobota

pyrophosphate, and 1 h later, without any washing, the pelleted cells were incubatedwith glutaraldehyde/Ca fixative (1 h, 20°C). The results were that the electron-densedeposits could be seen in the cells incubated with pyrophosphate (Fig. 5). Thedeposits were similar in size and had a similar frequency of distribution along theplasma membrane to that of typical CaDD (compare Fig. S with Fig. 1).

Paper chromatographic analysis of TCA extracts of CaDD

As shown above, the fixation of the cells with glutaraldehyde/Ca solutionsupplemented with ATP enhanced the appearance of CaDD. Therefore, to study thekind of compound involved in the formation of CaDD, the cells were fixed in thepresence of [3H]ATP or [y-3ZP]ATP, and TCA extracts of these cells were analysedby paper chromatography. Cells prefixed for 5min with glutaraldehyde/1 mM-EDTA/ATP solution and subsequently fixed in the presence of calcium ions (glutar-aldehyde/Ca/ATP fixative), were devoid of CaDD and therefore these samples wereused as controls.

Fig. 4. Cell fixed with glutaraldehyde/Ca/ATP shows filamentous material (arrows)associated with CaDD. X310000.

Calcium-binding microregions 223

Fig. 5. Electron-dense deposits at the plasma membrane of the cell loaded withpyrophosphate and subsequently postfixed with glutaraldehyde/Ca. X 27 000.

Addition of tritium-labelled ATP to the fixative allowed us to analyse the role ofATP and adenine-related compounds in the formation of CaDD. Chromatographicanalysis of TCA extracts of CaDD-containing cells showed that the label migratedmainly with an Rp value of 0-5, which corresponded to AMP; small amounts of thelabel were also found at Rp values of 0-75 and 0-18, which corresponded to adenosineand ATP, respectively (Fig. 6). In TCA extracts of the control samples only a smallquantity of ATP was detected (Fig. 6).

Analysis of the supematants of incubation media after glutaraldehyde/Ca/ATPfixation demonstrated a high level of ATP, a lower level of ADP, and small amountsof AMP and adenosine (data not presented).

Fixation of the cells in the presence of [y-32P] ATP allowed us to analyse the role ofphosphate residues of ATP in the formation of CaDD. The label extracted from theCaDD-containing cells migrated mainly at Rp 0-53, which corresponded to pyro-phosphate; small amounts of the label were also found at Rp 0-4, which probablycorresponded to ATP, and at Rp 0 68, which corresponded to inorganic phosphate(Fig. 7). A similar distribution of the label was detected in extracts from controlsamples, but the level of the radioactivity at RF 0-53 and 0-4 corresponded to half ofthe values for the experimental samples (Fig. 7)

TCA extracts of the CaDD-containing cells and of the cells devoid of CaDD wereanalysed chemically for total content of inorganic phosphate. The content of inorg-anic phosphate of the CaDD-containing samples was ~6-0/ig P; per 10 cells,whereas the control samples contained 3-8 ̂ g P; per 106 cells.

Coincidental distribution of CaDD and Ca-ATPase

Cytochemically, using the modified Wachstein-Meisel medium, the activity of Ca-ATPase was detected at the cytoplasmic surface of the plasma membrane (Fig. 8).

224 A. Sobota

The lead phosphate globules, the products of Ca-ATPase activity, seemed to belocalized in the same microregions at the plasma membrane as were observed forCaDD (compare Fig. 8 with Fig. 1). The frequency of distribution was 3-4 leadphosphate globules per (Jxn of plasma membrane profile, and 3-7 CaDD per /im of themembrane (50 profiles of membrane were analysed for each case).

Co-localization of Ca-ATPase activity and CaDD at the plasma membrane mightsuggest that CaDD appeared as a result of Ca-ATPase activity In this case Ca2+

added to the glutaraldehyde fixative might serve to stimulate the enzyme activity, aswell as precipitating inorganic phosphate liberated from endogenous ATP by theenzyme. To solve this question, the cells were briefly prefixed (lOmin, 20°C) with

3000

8

1000

o o o o

|I

•oI

0-2 0-4 0-6 0-8 10

Fig. 6. Chromatography of TCA extracts of the glutaraldehyde-fixed cells in thepresence of 1 mM-ATP. The cells were preincubated (20min) in the growth mediumsupplemented with lmM-[3H]ATP (25/iCimT1 of medium). Half of the cells were thenincubated in glutaraldehyde/Ca fixative supplemented with 1 mM-[3H] ATP for 45 min at20 °C and after washing with Ca-washing solution were extracted with S % TCA (CaDD-containing cells, • — • ) . The other half were prefixed with glutaraldehyde/1 mM-EDTA solution supplemented with lmM-[3H]ATP and, 5 min later, the samples werefixed with glutaraldehyde/Ca/[3H]ATP solution as above (CaDD-lacking cells,O O). Reference compounds: A, ATP; B, ADP; C, AMP; D, adenosine.

Calcium-binding tnicroregions 225

1600 -

Fig. 7. Chromatography of TCA extracts of the cells preincubated with ATP and fixedwith glutaraldehyde solution containing lmM-[y-32P]ATP (S^Ciml"1 of incubationmedium). The experiments were carried out as described for Fig. 6. • • , CaDD-containing cells, O O, CaDD-lacking cells.

8

Fig. 8. Cytochemical localization of Ca-ATPase activity. The lead phosphate productsreflecting the enzyme activity are localized at the plasma membrane. Unstained section.X 35 000.

226 A. Sobota

glutaraldehyde without calcium ions and then fixation was continued in the presenceof 5 mM-Ca2+ and 3 mM-ATP, conditions that permit Ca-ATPase activity. As a resultof this procedure no electron-dense deposits were observed at the plasma membrane(Fig. 9). However, if the above Ca/ATP/glutaraldehyde medium was supplementedwith lmM-Pb2+, lead phosphate granules, reflecting the activity of Ca-ATPase,were visible at the membrane (Fig. 10). These results indicated that CaDD werenot formed as a result of the precipitation of inorganic phosphate liberated in conse-quence of Ca-ATPase activity.

Fine structure of CaDD

Assuming the presence of phosphorylated constituents in CaDD, cells fixed withglutaraldehyde/Ca were.treated with exogenous alkaline phosphatase in the presence

%

•»'

Figs 9, 10. Glutaraldehyde-prefixed cells were incubated either in glutaraldehyde/collidine/Ca/ATP solution or in glutaraldehyde/collidine/Ca/ATP/Pb medium. In thefirst case, no electron-dense deposits are observed in the cell (Fig. 9); in the second,electron-dense granules at the cytoplasmic surface of the plasma membrane are visible(Fig. 10). Unstained sections. Fig. 9, X71000; Fig. 10, X45 000.

Calcium-binding miavregions 227

of 5mM-CaCl2. After such treatment the deposits were significantly clearer; theelectron-dense material disappeared and only the delineated edges of the depositscould be observed, indicating the places where CaDD were localized (Fig. 11). Insome cases the electron-dense material was partly removed and a delicate matrixinside the 'empty' deposits was visible (Figs 11, 12). The matrix was composed of

Pigs 11, 12. Effect of alkaline phosphatase on CaDD. Electron-dense material of thedeposits is decomposed (arrows). In some of the fine organized fibrillous matrix of theempty deposits can be observed (arrowheads). Section stained. Inset: schema showingfine organization of CaDD matrix and its association with plasma membrane. Fig. 11,x240000;Fig. 12, X460000.

A. Sobota

Table 1. Conditions of the CaDD restoration procedure

SeeAB0D

Firstglutaraldehyde

fixation (60min)

Ca2+, 0°CCa2+, 0°CCa2+, 20°CEDTA, 0°C

EDTA/phosphatewashing

3x2min,0°C3x2min, 0°C3x2min, 0°C3x2min,0°C

Secondglutaraldehyde/Cafixation (90 min)

0°COmitted

0°C0°C

Restoration+-

—

three hemispheres of different sizes, inserted into one another and attached to theplasma membrane. The inner, middle, and outer hemispheres were cross-linkedradially by fibrillar material, which was also finally joined to the plasma membrane(Fig. 12). Along the hemispheres regularly distributed, more intensive electron-dense spots could be distinguished. In the control samples, which were treated withheat-inactivated alkaline phosphatase in the presence of 5 mM-CaCl2, no decompo-sition of CaDD was observed.

When the CaDD-containing cells were treated intensively with chloroform metha-nol mixture, distinct round transparent areas inside CaDD appeared. Under theseconditions much damage to the cytoplasm was also apparent (Fig. 13).

Restoration of CaDD

Treatment of the CaDD-containing cells with TCA or EDTA/phosphate solutionresulted in a complete disappearance of electron-dense deposits (compare Fig. 14 and

13

Fig. 13. Chloroform/methanol treatment of CaDD. Transparent areas inside CaDD canbe observed. Also, the damaged cytoplasm is visible. Unstained section. X200000.

Calcium-binding microregions 229

\

15

• V

16

• . * . '

Figs 14-16. Restoration of CaDD. Glutaraldehyde/Ca-fixed cells show CaDD at theplasma membrane (Fig. 14). Treatment of CaDD-containing cells with EDTA/phosphate solution leads to complete disappearance of CaDD (Fig. IS). Reincubation ofEDTA/phosphate-treated cells in glutaraldehyde/Ca solution leads to reappearing of thedeposits (Fig. 16). Unstained sections. X50000.

230 A. Sobota

Fig. 15). When the EDTA/phosphate-treated samples were again incubated inglutaraldehyde/Ca solution the deposits reappeared at similar places on the plasmamembrane (Fig. 16). It should be noted, however, that restoration of the depositswas successful only if all the treatments were carried out at 0°C. The conditions forthe subsequent steps of restoration procedure are presented schematically in Table 1,set A. No restoration of CaDD was observed in the following cases: (a) when thecells fixed with glutaraldehyde/Ca at 0°C, and washed with EDTA/phosphatesolution were not subsequently incubated in the second glutaraldehyde/Ca solution,but were postfixed at once with osmium/Ca fixative (Table 1, set B); (b) when thefirst glutaraldehyde/Ca fixation of the cells was carried out at 20°C, though subse-quent steps of restoration procedure (i.e. washing with EDTA/phosphate solution,the second fixation with glutaraldehyde/Ca) were performed at 0°C (Table 1, set C);(c) when the cells were fixed at 0°C with glutaraldehyde/EDTA solution (calciumions omitted), though the second fixation with glutaraldehyde was performed in thepresence of calcium ions (Table 1, set D). Also, no restored deposits were observedwhen CaDD-containing cells were washed with TCA before the restoration process.

DISCUSSION

The appearance of calcium-dependent, electron-dense deposits at the plasmamembrane indicates the existence of special sites at the cytoplasmic surface of theplasma membrane where binding of calcium ions may take place (Oschman & Wall,1972; Hillman & Llinas, 1974; Sobotaefa/. 1977; Stockem & Klein, 1979; Sobota &Przelecka, 1981). On the basis of X-ray microanalysis data, phosphorus, in additionto calcium, is another major element detected in CaDD (Oschman et al. 1974; Skaeret al. 1974; Plattner & Fuchs, 1975; Sobota et al. 1978). Therefore, it is reasonableto assume that the cellular phosphate ions or phosphate-containing compounds arequite likely to capture calcium ions when high amounts of calcium flow into the cells(Shoenberg, Goodford, Wolowyk & Wooton, 1973).

What is the nature of the phosphorus compounds that form CaDD? Our resultshave shown that adenosine monophosphate might be involved in CaDD, althoughthis nucleotide does not form insoluble salts with calcium ions.

On the other hand, significant amounts of inorganic phosphate and pyrophosphatedetected in TCA extracts of CaDD-containing cells and the ability of the cells torestore CaDD in the medium with either inorganic phosphate or pyrophosphatesuggest that these anions may be essential for the formation of electron-densedeposits at the plasma membrane.

The absence of deposits at the cytoplasmic surface of the plasma membranewhen Ca2+ in the fixative solution was replaced by Pb2+ or Ce3+, cations known toform practically insoluble precipitates with inorganic phosphate (Wachstein &Meisel, 1957; Robinson & Karnovsky, 1983), suggests that microareas at the plasmamembrane involved in the formation of CaDD show specific affinity for calcium/phosphate salts but not for lead or cerous salts.

Calcium-binding microregions 231

The coincidental distribution of Ca-ATPase activity and CaDD at the plasmamembrane suggests that this enzyme is responsible for the appearance of CaDD.However, as shown above, the formation of CaDD and the formation of the productsof Ca-ATPase activity occur independently and may be easily distinguished from oneanother, indicating that inorganic phosphate liberated by Ca-ATPase is unlikely tobe responsible for the appearance of CaDD.

The regular distribution of CaDD at the plasma membrane (3-7 CaDD per fim ofmembrane profile) and the uniform size of the deposits suggest that calcium/phosphate salts are bound at special sites. Such special microregions at themembrane were observed by Nicaise et al. (1982), who showed that after Ca-freefixation the plasma membrane was completely free of electron-dense material but atthe same time empty micropapillae (55 pan in diameter) were visible. These emptystructure seemed to contain certain 'pale' material, which could serve as a matrix forthe binding of Ca/P salts. The proteinaceous character of the matrix and its role as anucleation centre for binding Ca/P salts in electron-dense granules in the cytoplasmof unicellular organisms were discussed by Pautard (1970), Simkiss (1976), Nilsson& Coleman (1977). The presence of protein-like material as an essential constituentof CaDD was demonstrated by Belitser et al. (1982), who showed that followingproteolytic digestion of sections of Epon-embedded material the CaDD were de-stroyed, and also observed that CaDD were sensitive to the action of phospholipaseA. The results suggest that proteins and phospholipids are probably involved in theformation of the matrix of CaDD.

The stimulatory effect of ATP on the formation of CaDD indicates a possibleinvolvement of phosphorylation of subplasmalemmal constituents in the formationof CaDD. A partial decomposition of CaDD after treatment with exogenous alkalinephosphatase supports the suggestion of an important role for phosphorylated con-stituents in the binding of Ca/P salts in CaDD. The participation of membrane-associated phosphoproteins in binding of calcium ions was demonstrated by Wolf &Siegel (1972) as well as by Geyer et al. (1978).

de Chastellier & Ryter (1981) studied the distribution of CaDD in Dictyosteliumcells and observed that CaDD were more abundant in the regions where filopodiaand phagocytic cups were formed. These cellular regions were rich in microfila-mentous network. On the other hand, CaDD were not observed at the phagosomemembrane, which is known to contain no actin or spectrin molecules (Korn et al.1974; Hardy & Schrier, 1978; Tokoyasu, Schekman & Singer, 1979). An increasedamount of CaDD was detected in cells during endocytosis and locomotion (Przelecka& Sobota, 1976; Stockem & Klein, 1979; de Chastellier & Ryter, 1982). Thesephysiological phenomena also involve the formation and disintegration of corticalfilamentous network (Stockem, Naib-Majani & Wohlfarth-Bottermann, 1983).

Knowledge of the fine substructure in CaDD, i.e. the presence of filamentous androd-like material that is cross-linked and attached to the plasma membrane (Fig. 12),as well as the association of CaDD with cortical filamentous material, allows us tospeculate how, under certain physiological and experimental conditions, the matrixof CaDD could be laid down.

232 A. Sobota

The formation of CaDD seems to be under the control of anionic sites on theexternal surface of the membrane. Short exposure (10 s) of living cells to cationicferritin/Ca solution (before glutaraldehyde/Ca fixation) prevents the appearance ofCaDD (unpublished results). A similar kind of relationship between the formation ofCaDD at the cytoplasmic surface of the plasma membrane and the changed structureof the external mucous layer in Amoeba proteus has been suggested (Stockem &Klein, 1979). The transmembrane association between negatively charged groups onthe outer surface of erythrocytes and the spectrin and actin molecules covering thecytoplasmic surface of the membrane, is well documented (Nicolson, 1976).

Considering such properties of CaDD as: (a) the localization at the cytoplasmicsurface of the plasma membrane; (b) the enhancement of their appearance by ATP;(c) the decomposition of CaDD as a result of treatment with phosphatase; (d) thepresence of filamentous and rod-like materials as constituents of the matrix ofCaDD; (e) the association of CaDD with cytoplasmic filamentous material; (f) theabundance of CaDD in microfilament-rich regions; and (g) the absence of CaDD atthe membrane of phagosomes, one assumes that contractile proteins (e.g. phos-phorylated spectrin and actin protofilaments) may serve as essential constituents ofthe matrix of CaDD.

Spectrin is a fibrous and flexible molecule that is able to form oligomers ofdifferent size, depending on the ionic conditions (Ungewickell & Gratzer, 1978;Tyler, Anderson & Branton, 1980). Tetramers of spectrin cross-link actin proto-filaments, which form a meshwork in the subplasmalemmal layer (Brenner & Korn,1980; Cohen, Tyler & Branton, 1980). This protein might also aggregate at thecytoplasmic surface of the plasma membrane (Nicolson, 1976), and might bephosphorylated by protein kinase (Wolfe & Lux, 1978; O'Connell & Swislocki, 1983)and dephosphorylated by exogenous alkaline phosphatase (Brenner & Korn, 1979).Spectrin as well as actin is depleted in the phagosome membrane (Korn et al. 1974;Hardy & Schrier, 1978; Tokuyasu et al. 1979). Recent studies have shown thatspectrin is present not only in erythrocytes but also in non-erythroid cells (Burridge,Kelly & Mangeat, 1982; Glenney & Glenney, 1983), including Acanthamoeba(Pollard, 1984).

In the presence of millimolar levels of calcium ions, spectrin and actin mol-ecules aggregated in small patches on the cytoplasmic surface of the erythrocyteghost membrane (Elgsaeter, Shotton & Branton, 1976). The possible role ofphosphorylated spectrin as a calcium binding constituent of the ghost membrane wasconsidered by Geyer et al. (1978), who showed that despectrination of themembranes significantly reduced the number and size of CaDD.

Assuming that phosphorylated spectrin and actin molecules are importantconstituents of the CaDD matrix, it seems probable that during cell fixation thecalcium ions entering might stimulate the formation of spectrin/actin patches, whichserve as a matrix in which the binding of calcium/phosphate salts occurs and wherethe electron-dense, calcium/phosphorus-containing granules are observed at thecytoplasmic surface of the plasma membrane.

Calcium-binding microregions 233

The author thanks Mrs K. Mrozinska for excellent technical assistance and Professor Dr A.Przelecka for discussions.

REFERENCES

BELTTSER, N. V., ZAALISHVUJ, G. V. & SYTNIANSKAJA, N. P. (1982). Ca2+-binding sites and Ca-ATPase activity in barley root tip cells. Protoplasma 111, 63-78.

BRENNER, S. L. & KORN, E. D. (1979). Spectrin-actin interaction. Phosphorylated anddephosphorylated spectrin tetramer cross-link F-actin. J. biol. Chem. 254, 8620—8627.

BRENNER, S. L. & KORN, E. D. (1980). Spectrin-actin complex isolated from sheep erythrocytesaccelerates actin polymerization by simple nucleation. Evidence for oligomeric actin in theerythrocyte cytoskeleton. J. biol. Chem. 255, 1670-1676.

BURRIDGE, K., KELLY, T. & MANGEAT, P. (1982). Nonerythrocyte spectins: Actin-membraneattachment proteins occurring in many cell types. J. Cell Biol. 95, 478—486.

CLAWSON, C. C. & GOOD, R. A. (1971). Micropapillae. A surface specialization of human leuco-cytes. J . Cell Biol. 48, 207-211.

COHEN, C. M., TYLER, J. M. & BRANTON, D. (1980). Spectrin-actin association studied byelectron microscopy of shadowed preparations. Cell 21, 875—883.

COHEN, I. & DE VRIES, A. (1973). Platelet contractile regulation in an isometric system. Nature,Lond. 246, 36-37.

D E ARAUJO JORGE, T. C , D E SOUZA, W. & MACHADO, R. D. (1979). Ultrastructural localizationof calcium-binding sites in the electrocyte oiElectrophorus electricus (L)._J. CellSci. 38, 97—104.DE CHASTELLIER, C. & RYTES, A. (1981). Calcium-dependent deposits at the plasma membraneof Dictyostelium discoideum and their possible relation with contractile proteins. Biol. Cell 40,109-118.

DE CHASTELLIER, C. & RYTER, A. (1982). Calcium deposits at the plasma membrane ofDictyostelium discoideum during phagocytosis and cell motility. Biol. Cell 43, 121-128.

ELGSAETER, A., SHOTTON, D. & BRANTON, D. (1976). Intramembranal particle aggregation inerythrocyte ghosts. II. The influence of spectrin aggregation. Biochim. biophys. Ada 426,101-122.

FELDMAN, D. L. & WEINHOLD, P. A. (1977). Calcium binding to rat heart plasma membranes:Isolation and purification of a lipoprotein component with a high calcium binding capacity.Biochemistry 16, 3470-3475.

FISKE, C. H. & SUBBAROW, Y. (1925). The colometric determination of phosphorus. J. biol. Chem.66, 375-400.

GADASI, H. & KORN, E. D. (1980). Evidence for differential intracellular localization of theAcanthamoeba myosin isoenzymes. Nature, Lond. 286, 452—456.

GEYER, G., LINSS, W. & STIBENZ, D. (1978). Phosphorylated spectrins are likely constituents ofmajor Ca2+ affinity sites. Acta histochem. 61, 135-141.

GLENNEY, J. R. JR.& GLENNEY, P. (1983). Fodrin is the general spectrin-like protein found in mostcells whereas spectrin and TW protein have a restricted distribution. Cell 34, 503-512.

GLOWACKA, S. K., SOBOTA, A. & PRZELECKA, A. (1985). Displacement of cell-surface associatedcalcium inhibits phagocytosis and Ca-ATPase activity in amoeba. Cell Biol. Int. Rep. 9, 183-191.

GRIFFIN, J. B. ,DAVIDIAN, N. M. & PENNIALL, R. (1965). Studies of phosphorus metabolism byisolated nuclei. VII. Identification of polyphosphate as a product. J. biol. Chem. 240,4427-4434.

GRUDA, J., POLLENDER, J. M. & THEJUEN, H. M. (1983). Effect of liver plasma membranes onG-actin. I. Possible implication of membrane NPases in inactivation of G-actin. Can. J.Biochem. Cell Biol. 61, 85-92.

HALLETT, M., SCHNEIDER, A. S. & CARBONE, E. (1972). Tetracycline fluorescence as a calcium-probe for nerve membrane with some model studies using erythrocyte ghosts. J. Membrane Biol.10, 31-44.

HARDY, B. & SCHRIER, S. L. (1978). The role of spectrin in erythrocyte ghost endocytosis.Biochem. biophys. Res. Comrnun. 81, 1153-1161.

HILLMAN, D. E. & LLINAS, R. (1974). Calcium-containing electron-dense structures in the axonsof the squid giant synapse. J. Cell Biol. 61, 146-155.

234 A. Sobota

JOSEFSSON, J. O. (1975). Studies on the mechanism of induction of pinocytosis in Amoeba proteus.Actaphysiol. scand. 432, 1-65.

KORN, E. D., BOWERS, B., BATZRI, S., SIMMONS, S. R. & VICTORIA, E. J. (1974). Endocytosis and

exocytosis: role of microfilaments and involvement of phospholipids in membrane fusion.J. supramolec. Struct. 2, 517-528.

KUSAMRAN, K., MATTOX, S. M. & THOMPSON, G. A. (1980). Studies on the size, location andturnover of calcium pools accessible to growing Tetrakymena cells. Biochim. bipohys. Acta 598,16-26.

LIMAS, C. J. (1977). Calcium-binding sites in rat myocardial sarcolemma. Archs Biochem. Biopkys.179, 302-309.

NICAISE, G., HERNANDEZ-NICAISE, M. L. & MALAVAL, L. (1982). Electron microscopy and X-raymicroanalysis of calcium-binding sites on the plasma membrane of Berve giant smooth musclefibre, jf. CellSci. 55, 353-364.

NICOLSON, G. L. (1976). Transmembrane control of the receptors on normal and tumor cells. I.Cytoplasmic influence over cell surface components. Biochim. biophys. Acta 457, 57-108.

NiLSSON, I. R. & COLEMAN, J. R. (1977). Calcium-rich, retractile granules in Tetrahymenapyriformis and their possible role in the intracellular ion regulation. J. Cell Set. 24, 311-325.

O'CONNELL, M. A. & SwiSLOCKI, N. I. (1983). Spectrin phosphorylation in senescent raterythrocytes. Mech. Ageing Dev. 22, 51-70.

OSCHMAN, J. L., HALL, T . A., PETERS, P. D. & WALL, B. J. (1974). Association of calcium withmembranes of squid axon. Ultrastructure and microprobe analysis..?. CellBiol. 61, 156—165.

OSCHMAN, J. L. & WALL, B. J. (1972). Calcium binding to intestinal membranes. J. Cell Biol. 55,58-73.

PAUTARD, F. G. E. (1970). Calcification in unicellular organisms. lnBiological Calcification (ed.H. Schraer), pp. 105-201. New York: Appleton-Century-Crofts.

PLATTNER, H. & FUCHS, S. (1975). X-ray microanalysis of calcium binding sites in Paramecium.With special reference to exocytosis. Histochemistry 45, 23-47.

POLITOFF, A. L., ROSE, S. & PAPPAS, G. D. (1974). The calcium binding sites of synaptic vesiclesof the frog sartorius neuromuscular junction. J . Cell Biol. 61, 818-823.

POLLARD, T. D. (1984). Actin assembly and actin binding proteins {tomAcanthamoeba. Third Int.Congr. Cell Biol., Tokyo, Japan, Abstracts, SC-2-2.

PRZELECKA, A. & SOBOTA, A. (1976). Calcium dependent deposits at the plasma membrane duringdevelopment of the oocyte of Galleria mellonella. Cytobiologie 13, 182-190.

ROBINSON, J. M. & KARNOVSKY, M. J. (1983). Ultrastructural localization of several phosphataseswith cerium. J. Histochem. Cytochem. 31, 1197-1208.

SHOENBERG, C. F., GOODFORD, P. J., WOLOWYK, M. W. &WOOTON, G. S. (1973). Ionic changesduring smooth muscle fixation for electron microscopy. ,J. mechanochem. Cell Motil. 2, 69—82.

SIMHSS, K. (1976). Intracellular and extracellular routes in biomineralization. Symp. Soc. exp.Biol. 30, 423-444.

SKAER, R. J., PETERS, P. D. & EMMINES, J. P. (1974). The localization of calcium and phosphorusin human platelets. J. CellSci. 15, 679-692.

SOBOTA, A., BUROVTNA, I. V., POGORELOV, A. G. & SOLUS, A. A. (1984). Correlation betweenpotassium and phosphorus content and their nonuniform distribution in Acanthamoebacastellanii. Histochemistry 81, 201-204.

SOBOTA, A., HREBENDA, B. & PRZELECKA, A. (1977). Formation of calcium-dependent deposits atthe plasma membrane of Acanthamoeba castellanii. Cytobiologie 15, 259-268.

SOBOTA, A. &PRZELECKA, A. (1981). Visualization of calcium-binding sites at plasma membrane ofshock-frozen Acanthamoeba cells. Acta histochem. 68, 125-129.

SOBOTA, A., PRZELECKA, A. & JANOSSY, A. G. S. (1978). X-ray microanalysis of calcium-dependent deposits at the plasma membrane of Acanthamoeba castellanii. Cytobiologie 17,464-469.

ST LOUIS, P. J. & SULAKHE, P. V. (1976). Adenosine triphosphate-dependent calcium binding andaccumulation by guinea-pig cardiac sarcolemma. Can. J. Biochem. 54, 946—956.

STOCKEM, W. & KLEIN, H. P. (1979). Pinocytosis and locomotion in amoebae. XV. Demon-stration of Ca++-binding sites during induced pinocytosis in Amoeba proteus. Protoplasma 100,33-43.

Calcium-binding micmregions 235

STOCKEM, W., NAIB-MAJANI, W. & WOHLFARTH-BOTTERMANN, K. E. (1983). Pinpcytosia andlocomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin inAmoeba proteus. Eur.J. Cell Biol. 29, 171-178.

STOLZE, H. & SCHULTZ, I. (1980). Effect of atropine, ouabain, antimycin A and A 23187 on'trigger Ca2+ pool' in exocrine pancreas. Am. J. Pkysiol. 238, G338-G348.

TOKUYASU, K. T. , SCHEKMAN, R. & SINGER, S. J. (1979). Domains of receptor mobility andendocytosis in the membranes of neonatal human erythrocytes are deficient in spectrin. J. CellBiol. 80, 481-486.

TYLER, J. M., ANDERSON, J. M. & BRANTON, D. (1980). Structural comparison of several actin-binding macromolecules. J. Cell Biol. 85, 489-495.

UNGEWICKELL, E. & GRATZER, W. (1978). Self-association of human spectrin. A thermodynamicand kinetic study. Eur.J. Biochem. 88, 379-385.

WACHSTEIN, M. & MEISEL, E. (1957). Histochemistry of hepatic phosphatases at a physiologicalpH. Am.J. din. Path. 11, 13-23.'

WOLF, D. J. & SIEGEL, F. L. (1972). Purification of a calcium-binding phosphoprotein from pigbrain. J . biol. Chem. 247, 4180-4186.

WOLFE, L. C. 8C LUX, S. E. (1978). Membrane protein phosphorylation of intact normal andhereditary spherocytic erythrocytes. J . biol. Chem. 253, 3336-3342.

(Received 12 April 1985 -Accepted 17 June 1985)