J. Cell Sci. 38, 97-104 (1979) 97Printed in Great Britain © Company of Biologists Limited 1979

ULTRASTRUCTURAL LOCALIZATION OFCALCIUM-BINDING SITES IN THEELECTROCYTE OF ELECTROPHORUS

ELECTRICUS (L.)

T. C. DE ARAUJO JORGE, W. DE SOUZA ANDR. D. MACHADO*Laboratdrio de Microscopia EletrOnica, Instituto de Biofisica, Universidade Federal doRio de Janeiro, Cidade Universitdria, 21.941, Rio de Janeiro, Brasil

SUMMARY

Calcium-binding sites were detected in the electrocyte of Electrophones electricus (L.) usingthe Oschman & Wall technique, in which CaCl2 was added to the fixative and washing solutions.Deposits were seen scattered along the plasma membrane of the electrocyte, inside mito-chondria, associated with the post-synaptic membrane and the membrane of synaptic vesicles.

INTRODUCTION

Calcium ions (Ca2+) play an essential role in many biological processes such asdepolarization-secretion coupling (Del Castilho & Katz, 1954; Baker, Hodgkin &Ridgway, 1971), muscular contraction (Katz & Miledi, 1968; Heuser, Katz & Miledi,1971), flagellar or ciliary activity (Plattner, 1975; Fisher, Kaneshiro & Peters, 1976),etc. In general these processes are mediated by interactions between Ca2+ and cellmembranes. The possibility of localizing Ca2+-binding sites by ultrastructuralcytochemistry was suggested by Oschman & Wall (1972) using CaCl2 in the fixativesolutions. This technique has been widely used in recent years with the aim oflocalizing Ca2+-binding sites in several biological structures.

The electrocytes of the electric organ of Electrophorus electricus are highly special-ized syncytial cells able to produce a synchronous electric discharge generatingbioelectric potentials similar to those in nerve and muscle (Esquibel, 1970). Theypresent an innervated posterior face which is electrically and chemically excitableand a non-innervated anterior face which is not excitable (Keynes & Martins-Ferreira,1953) and in both faces there are perpendicularly oriented tubular invaginations of theplasma membrane. Because of the relative scarcity of cytoplasmic organelles, theplasma membrane is the predominant component of the electrocyte. This, therefore,makes it a convenient system for studying membranes and related processes.

It was shown recently (Oliveira, Machado & Chagas, 1978) thai isolated electrocytemembranes have high affinity for Ca2+. In the present paper we describe the resultsobtained by applying Oschman & Wall's technique to localize Ca2+-binding sitesin the cellular structures of intact electrocytes.

• For all correspondence.

98 T. C. De Araujo Jorge, W. De Souza and R. D. Machado

MATERIALS AND METHODS

Fragments of the main electric organ, close to the skin of adult fishes, were removed, dissectedin Ringer solution and fixed by immersion in 2'5 % glutaraldehyde in 80 mM S-collidinebuffer for 2 h at room temperature, washed in the same buffer plus 85 % sucrose and post-fixed in 1 % OsO4 in 80 mM S-collidine buffer plus 7 % sucrose for 1 h at 4 °C. In all solutionsCaCl2 was added at concentrations of 5 or 90 mM. Two controls were used: in one, 1 mM EDTA(ethylenediaminetetra-acetic acid) was added to all solutions; in the second, CaCl2 was addedto the glutaraldehyde solution, but omitted and replaced by 1 mM EDTA in washing andpostfixation solutions. Dehydration was carried out in acetone and embedding in Epon.Tissue fragments were oriented so that sections were obtained transverse to the electrocyte;ultrathin sections were obtained with an Ultratome III LKB ultramicrotome, stained witheither uranyl acetate for 10 min and lead citrate for 3 min, or only with the latter, and observedeither in an AEI EM-6B or in a Philips EM-301 electron microscope. Some sections of purplecolour were picked up on carbon-coated, collodion-covered nylon grids and examined in aJEOL 100-CX microscope equipped with a Kevex energy-dispersive X-ray analysis system.A microprobe was used as generated by the STEM accessory.

RESULTS

As previously described (Machado, De Souza, Cotta-Pereira & Oliveira Castro,1976) the electrocyte has tubular invaginations on the anterior and the posterior faces.At the anterior face they are longer, more numerous and closely packed. At theposterior face many synaptic contacts are observed. They contain a large number of38'5-nm vesicles, sometimes in contact with the presynaptic membrane. In front ofthe synaptic contact the plasma membrane of the electrocyte presents a region ofincreased electron density associated with the inner leaflet of the membrane. Somemitochondria are found mainly surrounding the nuclei. Many microfilaments areseen scattered throughout the cytoplasm.

The Oschman & Wall technique forms electron-dense granules on calcium-bindingsites and the presence of CaCl2 in the fixative and washing solutions was indispensablefor their formation and maintenance. When either calcium was omitted or EDTA wasadded to the solutions no electron-dense deposits were detected (Fig. 1).

In tissue fragments fixed with glutaraldehyde and OsO4 containing calciumnumerous electron-dense deposits were observed at the plasma membrana, includingthe invaginations, in either the anterior or the posterior faces (Figs. 2 and 3, arrows).The aspect and distribution of the granules were the same when either 5 or 90 mMCaCl2 was used. The deposits are globular, 45-50 nm in diameter and are scattered

Fig. 1. Surface of the electrocyte of a control preparation in which EDTA was addedto the washing solutions. No reaction deposit is observed in the membranes, c,cytoplasm; i, invagination. x 52500.Fig. 2. Anterior surface of the electrocyte of a preparation in which CaCl2 was addedto the fixative and washing solutions. Deposits (arrows) are observed associated withthe electrocyte's membrane and cytoplasmic microfilaments ("//). x 45 000.Fig. 3. Posterior surface of the electrocyte in the same preparation as Fig. 2. x 112 500.Fig. 4. Calcium-binding sites (arrows) in the mitochondrion (m) of the electrocyte.x 75 000.

Calcium-binding sites in E. electricus 99

100 T. C. De Araujo Jorge, W. De Souza and R. D. Machado

Figs. 5-8. Posterior face of the electrocyte from the preparation in which CaCl2 wasadded to the fixative and washing solutions. Deposits (arrows) are associated with themembrane of the synaptic vesicles (v) (Figs. $, 7, 8) and post-synaptic membrane (ps).Fig- 5, X75000; Fig. 6, X70000; Fig. 7, X171000; Fig. 8, X171000.

Calcium-binding sites in E. electricus 101

along the membrane. Similar deposits were also seen in the cytoplasm, associated withmicrofilaments (Figs. 2 and 4, arrows) and inside mitochondria (Fig. 4).

At the post-synaptic membranes, however, there was a higher density of granules(Figs. 5, 6), thus suggesting the presence of many Ca2+-binding sites at this region.In the synapse, electron-dense deposits were seen associated with the membrane ofthe synaptic vesicles (Figs. 5, 7, 8). They did not occur in all vesicles and apparentlydid not present preferential orientation with respect to the axis of the nerve terminal.

Electron-probe X-ray analysis of the dense granules (Fig. 2) on the imaginations ofthe membranes showed the presence of Ca2+ and osmium (Fig. 9). A chloride peakappeared in the substrate and is probably due to the plastic support.

Fig. 9. Energy-dispersive X-ray spectrum from a dense granule attached to the plasmamembrane of the electrocyte. Calcium (CA), osmium (OS) and chloride (CL) peaksare indicated.

DISCUSSION

The observation made by Oschman & Wall (1972) that when CaCl2 is added tofixative solutions it is possible to visualize by electron microscopy Ca2+-binding sitesoffers a new possibility for the structural localization of these important sites. Thespecificity of the technique is confirmed by the observations that the deposits areremoved when the tissue is treated with EDTA or EGTA. The use of X-ray micro-analysis also shows the presence of Ca2+ as well as phosphorus in the deposits(Oschman, Hall, Peters & Wall, 1974; Hillman & Lh'nas, 1974; Gambetti, Bulkar,Somlyo & Gonatas, 1975; Fisher et al. 1976). In the electrocyte we could also detectby X-ray analysis the presence of Ca2+ in the dense granules attached to the plasmamembrane, both at the surface and on invaginations. Since the first application of thistechnique to intestinal cells of the cockroach (Oschman & Wall, 1972) it has beenused to detect Ca2+-binding sites in different cell types such as the frog sartoriusneuromuscular junction (Politoff, Rose & Pappas, 1974; Pappas & Rose, 1976), thesquid giant axon (Hillman & Llinas, 1974; Oschman et al. 1974), plasma membrane

102 T. C. De Araujo Jorge, W. De Souza and R. D. Machado

of Acanthamoeba castellanii (Sobota, Hrebenda & Przelecka, 1977), insect oocyte(Przelecka & Sobota, 1976), Paramecium engaged in secretion (Fisher et al. 1976)and in ciliary movement (Plattner, 1975; Fisher et al. 1976), gap junction membranes(Larsen, 1974) and glial cells (Gambetti et al. 1975). Moreover, the deposits have beenfound in areas in which physiological and biochemical studies have implicated Ca2+

in the control of cell coupling (Larsen, 1974), excitation-contraction coupling at thetransverse tubules of muscle cells (Heuser et al. 1971; Politoff et al. 1974), excitation-secretion coupling at the synapse (Baker et al. 1971; Hillman & Llinas, 1974; Oschmanet al. 1974; Pappas & Rose, 1976; Babel-Guerin et al. 1977), platelet release reaction(Skaer, Peters & Emmines, 1974), etc.

In the present work deposits were seen associated with the plasma membrane ofeither the innervated or the non-innervated face, which raises the question of thefunction of calcium in the electrocyte. It is possible that calcium deposits may bedrawn upon as needed, depending upon the Ca2+ concentration. Although theelectric organ originated from muscle cells (Esquibel et al. 1971) it does not contain astructure similar to the sarcoplasmic reticulum which, in muscle cells, plays a funda-mental role in the control of the concentration of Ca2+ in the cytoplasm. In thesarcoplasmic reticulum we can see such electron-dense deposits (Politoff et al. 1974;Pappas & Rose, 1976) and this suggests an analogy between this structure and theplasma membrane invaginations of the electrocyte. The simplified interpretation ofthe invagination as serving only to increase the surface has to be reviewed andclarified by new studies.

The presence of deposits associated with the synaptic vesicles of the nerve-electrocyte synapses has also been observed in other synapses (Heuser et al. 1971;Boyne, Bohan & Williams, 1974; Politoff et al. 1974; Pappas & Rose, 1976). Thedeposits appear to be associated with the membranes of the vesicles. Probably theyrepresent Ca2+-binding sites which play a role in the release of acetylcholine (Politoffet al. 1974; Pappas & Rose, 1976). Pappas & Rose (1976) did not observe Ca2+-binding sites in the synaptic vesicle membrane following intense stimulation of thenerve. However, the deposits seen in the post-synaptic membrane were still present.

As described in other tissues, deposits were also seen in the matrix of mitochondria,a structure which plays a fundamental role in the process of intracellular accumulationof Ca2+ (Carafoli & Lehninger, 1971).

Several mechanisms were discussed by Oschman et al. (1972, 1974) as beingpossibly implicated in the formation of Ca2+ deposits. Some of them, such as exposureof acidic groups of proteins, tri-complex flocculation of Ca2+, osmium and phospho-lipid, and coincidence with sites of ATPase activity, seem unlikely in the electrocytesince cytochemical localization of anionic sites and ATPase activity show a uniformdistribution of these components throughout the plasma membrane (Benchimol, DeSouza & Machado, 1977; Somlo, De Souza, Machado & Hasson-Voloch, 1977),while Ca2+-binding sites are not similarly localized. Moreover, in several tissuespostfixation with OsO4 is not necessary for visualization of the electron-dense deposits(Oschman & Wall, 1972; Oschman et al. 1974; Hillman & Llinas, 1974; Politoff et al.1974; Plattner, 1975; Fisher et al. 1976).

Calcium-binding sites in E. electricus 103

Recently Oliveira et al. (1978) observed binding of Ca2+ to isolated membranes ofthe electrocyte of Electrophorus electricus. Although a Ca2+-activated, magnesium-dependent ATPase was detected, experiments with several membrane fractionsisolated in a sucrose gradient show that the fraction corresponding to Ca2+-activated,magnesium-dependent ATPase was not the same as that showing Ca2+-bindingactivity. Childers & Siegel (1975) isolated a Ca2+-binding protein from the electricorgan of Electrophorus electricus, which was considered to be a major protein com-ponent of the electrocyte. Studies with the electric organ of Torpedo californica,Torpedo ocellata and Electrophorus electricus show that Ca2+ has high affinity foracetylcholine receptor proteins (Chang & Neuman, 1976; Riibsamen et al. 1976). Ourelectron-dense deposits may be the ultrastructural demonstration of this calcium-binding protein.

In view of these results showing the presence of Ca2+-binding sites in the post-synaptic structures of the neuro-electric junction, new studies are necessary toclarify the role of calcium in the electrocyte's physiology.

We are most grateful to Drs Maria Apparecida Esquibel and Mecia M. Oliveira for readingthe manuscript and making helpful suggestions. We are also indebted to Mr D. Harling ofJEOL (Boston) for the X-ray microanalysis.

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{Received 6 November 1978)