Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (cnidaria: anthozoa)

TISSUE & CELL 1985 17 (2) 199-213 @ 1985 Longman Group Ltd

GLEN M. WATSON* and RICHARD N. MARISCAL

ULTRASTRUCTURE OF NEMATOCYST DISCHARGE IN CATCH TENTACLES OF THE SEA ANEMONE HALIPLANELLA LUCIAE (CNIDARIA: ANTHOZOA) Key words: microtubule. nematocyst, calcium. sea anemone. Cnidaria, Anthozoa ABSTRACT. The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate. indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.

Introduction The nematocyst or stinging organelle of cnidarians consists of a capsule containing a folded thread. The nematocyst functions by the forceful eversion of the thread out of the capsule into contact with the target organism (reviewed by Mariscal, 1974). Nematocyst discharge begins with the opening of the sealed capsule tip. Hydrozoan and scyphozoan nematocysts are sealed by opercula. In contrast, anthozoan nematocysts are sealed by a tripartite series of apical flaps (Westfall, 1965). At the moment of nematocyst discharge, the operculum or apical flaps open to permit thread eversion. The mechanism for this opening of the capsule seal is not known. One interesting hypothesis is that a conformaDepartment of Biological Science, Florida State University, Tallahassee, Florida 32306, U.S.A. *Present address: Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda. California 92350. U.S.A. Received 28 February 1984. Revised 4 January 1985.

tional change occurs in the molecules in the operculum (or apical flaps) that weakens the seal and leads to capsule opening (Blanquet, 1970). Prior to nematocyst discharge, the uneverted thread is folded and helically pleated inside the capsule. After discharge, the now everted thread is completely unfolded and no longer pleated (Skaer and Picken, 1965); thus the thread everts and unfolds at nematocyst discharge. The basis for nematocyst thread unfolding at discharge is incompletely understood. Some data are available which suggest that a hydration of the thread might be involved. Skaer and Picken (1965) reported that isolated, dried segments of uneverted nematocyst thread unfold when exposed to water vapor, and then become folded again when desiccated. More recently, CarrC (1980) confirmed that isolated, uneverted nematocyst threads unfold when hydrated. The motive force that causes the nematocyst thread to evert at discharge remains unknown. However, several hypotheses have been suggested, including the contrac199

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tion hypothesis, the constant pressure hypothesis and the osmotic hypothesis (cf. Mariscal, 1974, 1984). In the contraction hypothesis, the nematocyst capsule is thought to become compressed (Cormier and Hessinger, 1980a, b), or to contract to cause the thread to evert. The constant pressure hypothesis suggests that thread eversion results from the sudden release of an existing hydrostatic pressure wlthin the undischarged capsule. Eversion supposedly follows a solubilization or weakening of the capsule seal (Blanquet, 1970; Salleo et al.. 1983). The osmotic hypothesis states that the permeability of the capsule wall to water or ions increases just prior to nematocyst discharge. A sudden influx of fluid would be expected to increase intracapsular hydrostatic pressure. The increased hydrostatic pressure, evidenced by capsule swelling (Robson, 1973), is thought to cause thread eversion (Weill, 1934; Picken, 1953; Robson, 1953, 1973; Picken and Skaer, 1966; Mariscal, 1974; Carre, 1980; Holstein and Tardent, 1984). Lubbock and Amos (1981) and Lubbock et al. (1981) introduced a modified version of the osmotic hypothesis following their discovery of substantial quantities of calcium within some undischarged nematocysts. Lubbock and his colleagues suggested that the nematocyst capsule is always permeable to water and that the calcium is removed from the nematocyst contents just prior to discharge. Supposedly, this removal of calcium results in an increase in osmotic pressure that leads to thread eversion. It is evident from the above discussion that the mechanism of nematocyst discharge is incompletely understood. In the present study, we have attempted to reconstruct how nematocyst discharge might normally occur in situ by studying the ultrastructure of undischarged, partially discharged and fully discharged nematocysts in catch tentacles of the sea anemone Haliplanella luciae. Because recent studies have indicated that calcium might play an important role in nematocyst discharge (e.g. Lubbock et al. 1981; Lubbock and Amos, 1981), we have included some observations employing calcium cytochemistry at the electron microscopic level.

Materials and Methods Induced catch tentacle formation

Unlike sea anemone feeding tentacles. catch tentacles (specialized for aggression, Williams. 1975; Purcell, 1977) have a maturity gradient along the tentacle length such that mature nematocysts occur at the tentacle tip, while immature nematocysts occur at the tentacle base (Watson and Mariscal, 1983b). Because large numbers of mature nematocysts of a single type (identified as holotrichous isorhizas using Mariscal. 1974) are present at the tentacle tip. catch tentacles are particularly suited for a study of nematocyst discharge. Catch tentacles develop from feeding tentacles after non-clonemate sea anemones are placed together in crowded anemone cultures (Purcell. 1977; Watson and Mariscal, 1983a). For purposes of catch tentacle induction. specimens of H. luciae (= H. lineata, Williams. 1978) were collected along with specimens of the sea anemone Diadumene gracillima (= Actinothoe gracillima, McMurrich, 1887) from separate oyster clumps near the Florida State University Marine Laboratory. Turkey Point, Florida. The anemones were crowded together in culture dishes filled with natural sea water (2%30%). which was changed daily and held at 17-19°C. Newly developed catch tentacles (= stage 3 catch tentacles. Watson and Mariscal, 1983a) formed in the H. fuciae after the first week. Such catch tentacles were removed from the anemones and processed for electron microscopy by the methods described below. Transmission electron microscopy

Catch tentacles were fixed both by classical methods (i.e. glutaraldehyde and osmium, protocol no. 1) and by other methods in which a second reagent was used in combination with the glutaraldehyde (protocol no. 2) or the osmium tetroxide (protocol no. 3). Classical fixation methods may be adequate for preserving most cellular structures but can inadequately stabilize some cellular structures. Glutaraldehyde fixation does not prevent the extraction of certain proteins during subsequent tissue processing steps (i.e. washing steps in buffered saline) (Hayat, 1981). Proteins fixed by glutaraldehyde are thought to be further

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stabilized by osmium post-fixation. However, a substantial amount of cellular protein in glutaraldehyde-fixed tissue can be extracted following post-fixation with 0~04 (Hayat, 1981). The loss of protein from erythrocyte ghosts following osmication was quantified at about 70% of the total membrane complement of protein (Luftig and McMillan, 1981). Considering the possibility that some nematocyst protein or proteinaceous structure might be inadequately preserved by classical fixation methods, we supplemented the classical fixatives with two chemicals that might enhance their functioning, tannic acid and ruthenium red. Tannic acid is known to help stabilize certain cellular structures (e.g. microtubules and microfilaments) when used in combination with glutaraldehyde and followed by osmium post-fixation (Hayat, 1981). Ruthenium red is best known as a cytochemical stain for cell-surface mucopolysaccharides (e.g. Luft, 1971). However, when ruthenium red is used in conjunction with osmium tetroxide, it acts as a fixative and can improve the preservation of certain cellular structures (e.g. membranes and myofilaments in muscle cells, Hayat, 1975). Specimens of H. luciae were anesthetized in their culture dishes using a 7.5% solution of MgClz-6H20 mixed with an equal volume of sea water. Catch tentacles were removed from the animals using fine scissors and drawn by suction into 100~1 micropipettes. The tentacles were dropped into the primary fixative and further processed according to one of the three protocols given below. 1. Primary fixation in 2.5% glutaraldehyde in 0.45pm Millipore filtered sea water (FSW) followed by post-fixation in 1% 0~0~ in glass distilled water (GDW). 2. Primary fixation in 2.5% glutaraldehyde and 0.8% tannic acid in FSW followed by post-fixation in 1% 0~0~ in GDW. 3. Primary fixation in 2.5% glutaraldehyde in FSW followed by post-fixation in 1% 0~0~ amd 0.15% ruthenium red in GDW. In all instances, primary and post-fixation were carried out at room temperature for

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2 hr. Tentacles were dehydrated in a graded acetone series, then embedded in Spurr Low-Viscosity Embedding Medium (Polysciences, Inc.). Thin sections were obtained using fresh glass knives and then post-stained in 2% uranyl acetate in 50% methanol for 5min, followed by 0.2% lead citrate for 1 min. Sections were viewed with a Philips 201 transmission electron microscope. Measurements were taken from micrographs using a x 20 measuring microscope (Ted Pella, Inc.). Calcium cytochemistry

Calcium was localized in cnidocytes using the potassium pyroantimonate method modified from that of Cardasis et al. (1978). Isolated catch tentacles were dropped in 2.5% glutaraldehyde in FSW and fixed for 45 min at room temperature. The tissue was rinsed in FSW and GDW, then placed in 1% 0~0~ and 2% potassium pyroantimonate (lot no. 3-1863, Polysciences, Inc.) in GDW for 1 hr at room temperature. Tentacles were either washed for 1 hr in GDW, or treated in 0.2M EGTA (ethyleneglycoltetraacetic acid) for 1 hr, and then dehydrated in acetone and embedded in Spurr. EGTA destaining is specific for calcium-antimonate precipitates and does not remove magnesium-antimonate or sodium-antimonate precipitates from cells (Wick and Hepler, 1982). Thus, specific calcium localization is possible using the antimonate technique provided destaining follows EGTA treatment. Thin sections were obtained and post-stained in uranyl acetate followed by lead citrate. Discharged nematocysts

Catch tentacles were removed from anesthetized H. luciae and placed in fresh sea water. Once the tentacles showed signs of recovery from the anesthetic (i.e. exhibited muscle contraction at the tentacle base) they were drawn into suction electrodes and stimulated with a single pulse from a Grass S-5 stimulator (Grass Instruments, Inc.). Electrical stimulation caused massive nematocyst discharge at the catch tentacle tip. The tentacles were processed for electron microscopy using one of the protocols described above.

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Results

The nematocyst capsule is an intracellular structure that lies at the cnidocyte cell surface (Figs. 1, 2, 4). Several structures are present in the cnidocyte apex that appear to contact the capsule near the capsule tip. For example, microtubules (0.>0.5prn long) extend from amorphous dense bodies at the plasma membrane to an area of the capsule just beneath the capsule tip (Figs. 14). The microtubules appear to be interconnected by 1Onm (diameter) filaments (Fig. 2). When viewed in cross section, numerous dense bodies are visible surrounding the nematocyst capsule (Fig. 3) (as many as 50 dense bodies were observed in a single cnidocyte). Thus, a sort of scaffolding surrounds the nematocyst capsule tip, formed by the microtubules emanating from the dense bodies and interconnected by the 10 nm filaments.

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In addition to the microtubules and 10 nm filaments, small intermembrane particles (6nm wide x 12nm long) are visible at the capsule tip between the plasma membrane and the membrane that covers the nematocyst capsule (Fig. 4). An extracellular fibrous network is present over the cnidocyte cell surface above the capsule tip. In longitudinal section. the fibrous network has an arborescent appearance with a distinct stalk (3-4 nm wide X SC 150nm long) and crown (SO-75nm across). together giving the fibrous network a total height of 13522Snm above the cnidocyte cell surface (Fig. 4). Portions of the fibrous network appear to contact the cilium and microvilli that together make up the ciliary cone receptor apparatus for the nematocyst (Fig. 4). When viewed in a cross section of the nematocyst tip (Fig. 5). the fibrous network appears as a dense mat covering the cell surface over the capsule tip (shown

Fig. 1. Longitudinal sectron of a mature. undischarged nematocyst at the cnrdocyte cell surface. The nematocyst capsule (c) lies at the plasma membrane. surrounded hy a microtubule array (m) that appears to contact an area of the capsule wall (arrow) lust beneath the capsule tip. The microtubules appear to originate from a dense body (dh) located at the cell surface. X 21,Otkl. Fig. 2. Longitudinal section of a mature nematocy\t at the cmdocytc cell surface. The microtubule array apparently consists of microtubules (m) and filaments (f). The apical flaps (a) and uneverted thread (ut) are visible. X 49.fMk). Fig. 3. Cross section of a cnidocyte containing an undischarged nematocyst. This section was taken just beneath the cell surface. Numerous dense bodies (db) are visible in the cytoplasm surrounding the nematocyst capsule (c). Microtubules (m) originating from the dense bodies. extend toward the nematocyst capsule. as well as in other directions. x 60,500. Fig. 4. Longitudinal section of a nematocyat at the cnidocyte cell surface Intermemhrane particles (between the converging arrowheads) apparently interconnect the plasma membrane and the nematocyst limiting membrane (nm). The extracellular fibrous network (fn) occur\ over the cell surface above the capsule tip. Portions of the fibrous network appear to contact a microvillus (arrow no. 1) and cilium (arrow no. 2). Microtubules (m) extend from a dew body (dh) to the nernatocyst capsule (c) The apical flaps (a) are visrble in thuspreparatron. x 68,000. Fig. 5. Cross section of a nematocyst capsule tip showing the tips of the three apical flaps (a) and the extracellular fibrous network (fn) that covers the cell surface. X 68,tXK). Fig. 6. Cross section of a nematocyst capsule tip. Thts section was taken directly through the apical flaps (a). The apical flaps are joined together along radial seams (rs) that meet at the centrally located apical tube (at). Radial laminae (rl) occur laterally on each srde of the radial seams. Note that subunits fill the total radtal seam width. An electron-dense band (dbd) and an electron-lucent hand (Ibd) occur exterior to the radial laminae. Granular material (pm) fills the remainmg area of the apical flaps. x 6X.500.

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exterior to the exposed apical flaps in Figure 5). The apical flaps are joined together along three complex radial seams (Figs. 5, 6). A structure which we call the apical tube is present in the center of the nematocyst tip where the three apical flaps meet (Fig. 6). Serial sections reveal that the apical tube extends through the entire capsule tip to the capsule interior (not shown). The apical tube outside diameter is 50nm, while the tube wall measures 11-13 nm thick and the tube interior measures 25-30 nm across. The electron-lucent apical tube wall is morphologically similar to the inner wall of the everted nematocyst thread (to be described later). The radial seams (25-30nm across) contain numerous electron-lucent subunits that measure 12nm across (Fig. 6). Three electron-dense radial laminae, measuring 9nm in width, occur on either side of each radial seam (Fig. 6). Exterior to the laminae are two additional bands. The inner band is an electron-dense structure that measures 20nm across, while the outer band is electron-lucent and measures 35 nm across (Fig. 6). Finely granular electron-

dense material fills the remaining area of the apical flaps (Fig. 6). Upon nematocyst discharge, the three apical flaps separate along the radial seams so that the subunits mentioned above are now clearly visible (Fig. 7). Following the opening of the apical flaps, the thread everts out of the capsule. Both the outer and inner walls of the everted thread are covered with repeating subunits. Subunits on the outer wall of the everted thread are designated OE subunits (taken from the first letters of the words. ‘outer’ and ‘everted’). whereas subunits on the inner wall of the everted thread are called IE subunits. The OE subunits are not visible in nematocyst preparations fixed according to classical fixation methods. However. the OE subunits are clearly visible in tannic acid nematocyst preparations, in which they appear to be electron-lucent structures (15 nm across) lying beneath a layer of electron-dense, tannic acid precipitate (Fig. 8). It is interesting that the OE subunits are apparently unstained by the tannic acid (and hence appear electron-lucent) but are somehow stabilized by its presence, possibly

Frg. 7. Ruthenium red prepared, longitudinally sectioned ncmatocyst fixed dung discharge. The capsule interior (c) and a single apical flap (a) are shown. The aprcal flaps have separated along the radial seams so that the subunits (normally located within the radial seams) are now exposed (arrows). The radial laminae (rl) are also clearly visible as are the electron-dense and electron-lucent bands (dbd and Ibd. respectively). x 116,500. Fig. 8. Tannic acid prepared, longitudinally sectioned, fully everted nematocyst thread (et). Spines (sp) and OE subunits (OE, arrows) are visible on the outer thread wall. The OE subunits appear moderately electron-lucent beneath a layer of electron-dense tannic acrd precipitate. X 76,500. Fig. 9. Longitudinal section of a ruthenium red prepared cverting thread (et) containmg a segment of uneverted thread (ut). The electron-dense outer wall (ow) and the electron-lucent inner wall (iw) are visible in the everted thread segment. The OE subunits shown in Frg. 8 are not clearly visible in this preparation. X 58,ooO. Fig. 10. Cross section of a ruthenium red prepared everting thread (et) containmg a segment of uneverted thread (ut). OE subunits (OE, arrows) are present on the outer wall of the everted thread. The electron-dense outer wall (ow) and the electron-lucent. inner wall (iw) are visible in the everted thread. x 49,CKIO. Fig. II. Potassium antimonate stained, fully everted thread (et) shown m cross section. Spines (sp) are vtsible on the outer wall, and electron-dense. antimonate stained IE subumta (IE, arrows) arc visible on the inner wall of the everted thread. x 49.ooO.

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because of the overlying precipitate. In contrast, the OE subunits are stained by ruthenium red (Fig. 10) and also by antimonate (Fig. 12) and therefore appear electron-dense in these two preparations. However, it should be pointed out that the OE subunits are not always adequately preserved by the ruthenium red protocol (Fig. 9) or by the antimonate fixation protocol (not shown) and thus are not visible in some preparations. The OE subunits are not clearly visible in antimonate preparations subsequently treated with EGTA (Fig. 13). The IE subunits, also 15 nm across, appear electron dense in antimonate preparations of nematocyst threads (Figs. 11, 12), and can appear electron-lucent following EGTA treatment (Fig. 13), indicating that the IE subunits bind calcium. The everted thread is composed of a double wall consisting of an outer electrondense wall (9-11 nm thick) and an inner electron-lucent wall measuring 1l-13 nm thick (Figs. 9, 10). Spines that measure 0.4pm (length) by 0.3pm (base width) are also present on the outer wall of the everted thread (Figs. 8, 11). Recall that the uneverted nematocyst thread is complexly folded into a triplypleated, helical arrangement (Skaer and Picken, 1965). According to Skaer and Picken’s terminology, a region of the thread which is folded is called a ‘fold’, whereas two areas of the thread surface brought together by folding are called ‘pleats’. The holotrich nematocyst thread in the present study, like most nematocyst threads, is triply pleated (Figs. 12, 13). In antimonate preparations, the region separating the thread walls of a pleat (1.5nm across) is made up of electron-dense material (Fig. 12) that appears less electron-dense following EGTA treatment (Fig. 13). An opening in one pleat (i.e. a slight separation of the opposing thread walls) in Fig. 12 reveals that particles are present in this area between the walls of a pleat. The three pleats are coiled around the long axis of the thread (i.e. the axis perpendicular to the plane of the sectioning in Figs. 12 and 13) so that the end of one pleat is in contact with the middle region of another pleat. Subunits on the outer wall of the uneverted thread, including those at

points of contact between overlapping pleats, are stained by antimonate (Fig. 12), and destained by EGTA treatment (Fig. 13). Previous studies employing antimonate cytochemistry and X-ray microanalysis have shown that the H. luciae catch tentacle holotrich nematocyst contains a large quantity of calcium (Watson and Mariscal, 1984). The fate of the nematocyst calcium at discharge is incompletely understood. Examination of an antimonate preparation of a partially everted thread (i.e. a portion of everted nematocyst thread containing a portion of uneverted nematocyst thread) reveals electron-dense material in the thread interior (i.e. the space between the everted and uneverted segments) (Fig. 12). EGTA treatment causes a decrease in the electron density of this material, suggesting that it contains calcium (Fig. 13). In contrast, the fully everted nematocyst thread interior and capsule interior lack electrondense material (Figs. 11 and 14, respectively). Likewise, the area of the cnidocyte immediately outside of, and surrounding, the capsule of the discharged nematocyst lacks electron-dense material (Fig. 14). Thus, at least some nematocyst calcium is present within the nematocyst interior during discharge, but apparently is absent from the cnidocyte, capsule interior or thread interior following discharge.

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Discussion The mature, undischarged nematocyst is a large, intracellular structure that normally occurs at the cell surface with the capsule tip orientated outward. The means by which the nematocyst remains at the cnidocyte cell surface until discharge is poorly understood. In the present study, we described a microtubule array in the cytoplasm surrounding the capsule tip. The array consists of microtubules that extend to the capsule wall from amorphous, dense bodies located at the plasma membrane. The dense bodies are morphologically similar to known cytoplasmic microtubule organizing centers (Schliwa, 1978), and are also morphologically similar to microtubule termination sites in the tips of cilia (Dentler, 1981). The microtubules appear to be inter-

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connected by 10 nm (diameter) filaments that are morphologically similar to the intermediate filaments in other cells that provide a supportive function (Lazarides. 1980). We suggest that the microtubule array helps to hold the nematocyst capsule at the cell surface until discharge. Small structures which we call intermembrane particles occur at the capsule tip between the plasma membrane and the membrane that covers the nematocyst capsule. The intermembrane particles might be involved in forming attachments between the nematocyst limiting membrane and the cnidocyte plasma membrane, and therefore could also help to hold the nematocyst at the cell surface until discharge. It is interesting to consider these ideas with respect to the Lubbock et al. (1981) hypothesis on nematocyst discharge. According to this hypothesis, the nematocyst is ‘relatively deeply embedded’ in the cnidocyte and moves to the cell surface just prior to discharge, where it undergoes membrane fusion and thread eversion. The results of the present study show in H. luciae. at least, the undischarged nematocyst already occurs at the plasma membrane, indicating that no significant movement of the nematocyst at the moment of discharge is necessary. Furthermore, we suggest that the nematocyst is held at the plasma membrane until discharge by several different cytoplasmic structures. Discharge might follow membrane fusion as was previously suggested (Lubbock et al.. 1981) or. alternatively, discharge might follow membrane deformation or disruption. We envision a possible triggering mechanism in which the elements of the ciliary cone (i.e. the cilium of microvilli) are displaced by contact with the target organism. Because of the extracellular fibrous network extending from the cilium and microvilli to the cell surface over the capsule tip, ciliary cone displacement might cause the fibrous network to pull on the plasma membrane over the capsule tip. It is conceivable that such a pulling force might deform (or even rupture) the plasma membrane so that it becomes more permeable to sea water. Sea water could then enter the cnidocyte and contact the membrane covering the nematocyst capsule. The sea water could then pass through the nematocyst

AND MAKIX‘AL.

limiting membrane to contact the nematocyst capsule tip. However. it is also possible that the nematocyst limiting membrane is impermeable to sea water. As was previously mentioned, the intermembrane particles might form attachment points between this membrane and the plasma membrane. Thus. any deformation (or rupturing) of the plasma membrane caused by ciliary cone displacement might also deform (or rupture) the nematocyst limiting membrane so that it becomes more permeable to sea water. again permitting sea water to contact the capsule tip. The undischarged capsule tip is sealed by three apical flaps joined together along complex radial seams. The total seam width is 25-30 nm. Upon nematocyst discharge. the apical flaps separate to reveal l2nm subunits on each radial seam. Hence, in the undischarged condition. a subunit from one apical flap is probably opposite a similar subunit from the adjacent apical flap (i.e. two subunits normally span the total seam width). We speculate that the subunits bind the apical flaps together until nematocyst discharge. at which point they uncouple to allow the flaps to separate. The presumed mechanism of subunit binding or uncoupling is not known. One possibility is that hydrogen bonding is involved. Hence. increased hydration caused by an influx of sea water would cause the subunits to uncouple and therefore allow the apical flaps to separate. The nematocyst thread is lined by two different types of subunits. One type of subunit is called the OE subunit because it occurs on the outer wall of the everted thread. Because the thread everts at discharge, we suggest that the OE subunit is probably also present on the inner wall of the uneverted thread. The particles observed between the walls of a single pleat of uneverted thread (see Fig. 12) might be equivalent to the OE subunits. The total distance separating opposing walls of a pleat is approximately 1Snm. the same as the measured width of an OE subunit. Hence. the subunits on the opposing walls of a pleat probably alternate like the teeth of a zipper in order to fit into the 1Snm space separating the two thread walls. We suggest that the OE subunits bind the

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Fig. 15. Diagrammatic interpretation of an everting nematocyst thread showmg a hypothetical mechanism for subunit regulation of discharge. The OE subunits (OE) and spines (SP) occw on the outer wall of the everted thread (ET) and the inner wall of the uneverted thread (UT). The IE subunits have the opposite orientation. occurring on the inner wall of the everted thread and the outer wall of the uneverted thread. Top: The uneverted thread is shown unfolding and uncoiling at, and slightly inside. the everting tip. Bottom: (a) The OE and IE subunits bind the uneverted thread in its folded and coiled configuration. then uncouple (h) to allow the uneverted thread to unfold and uncoil (c and d). The unfolded, uncoiled thread segment then ever&. presumably because of intracapsular hydrostatic pressure.

halves of the pleat together. Upon nematocyst discharge, these subunits would then uncouple to permit pleat opening (diagrammatically shown in Fig. 15). The second type of subunit is called the IE subunit because it occurs on the inner wall of the everted thread. This subunit is probably also present on the outer wall of the uneverted thread and apparently corresponds to the electron-dense subunits observed on the outer wall of the uneverted thread (see Fig. 12). The uneverted thread is helically coiled around its long axis in addition to being

pleated (Skaer and Picken, 1965). Thus, the pleats are wrapped around the thread so that the end of one pleat contacts another pleat (i.e. the pleats overlap). The IE subunits appear to be present in the space between overlapping pleats, and might bind the pleats in their coiled configuration. Uncoupling of these subunits at discharge would allow the overlapping pleats to separate and thus allow the thread to uncoil (diagrammatically shown in Fig. 15). A model of nematocyst thread unfolding at discharge based on subunit uncoupling must include the assumption that the un-

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coupling of the two subunit types on opposite sides of a segment of uneverted thread is coordinated in some fashion so that the segment of thread unfolds (i.e. the pleats open) and uncoils as eversion proceeds. This is because only an unfolded and uncoiled segment could evert. The presumed mechanism of thread subunit binding and uncoupling is not presently known. One possibility is that hydrogen bonding is involved. Increased hydration of the thread caused by an influx of sea water might cause the subunits to uncouple and allow the thread to unfold and uncoil. This is an attractive idea because the sea water would contact the capsule tip, cause the apical flaps to separate and then contact the inner wall of the thread base. Assuming that the thread wall is permeable to sea water, the OE subunits would be hydrated at about the same time as the IE subunits located on the outer wall of the same segment of uneverted thread. The high concentration of proteins and other molecules in the capsule interior would help to pull the sea water through the thread wall (by osmosis) and at the same time, increase the intracapsular hydrostatic pressure. The increased hydrostatic pressure would initiate discharge. As the thread subunits became hydrated, the thread would unfold and uncoil along its length to permit eversion to continue to completion. A second possibility is that the influx of sea water might cause calcium to dissociate from the thread subunits and therefore cause the subunits to uncouple. Both the OE and IE subunits are stained by antimonate. The IE subunits appears less electrondense following EGTA treatment, indicating that the IE subunit binds calcium. The OE subunit is not clearly visible on the outer wall of the everted thread following EGTA treatment. Considering the difficulty with which the OE subunit is preserved (i.e. it is visible in tannic acid preparations and in some ruthenium red and some antimonate preparations), its extremely faint following EGTA treatment appearance could indicate that the calcium-antimonate is removed from the subunit or, alternatively, that the subunit is not adequately preserved by the antimonate fixation protocol to withstand the exposure to EGTA. Thus, we cannot say with certainty that the OE

WATSON

AND MARISCAL

subunit binds calcium. However, the particulate material between the walls of a pleat of uneverted thread (which is probably made up of the OE subunits) is stained by antimonate and destained by EGTA. it appears that both subunit Therefore. types might bind calcium. Some published reports suggest that a removal of calcium from the nematocyst contents causes an increase in intracapsular hydrostatic pressure that in turn causes thread eversion (Lubbock and Amos, 1981: Lubbock et al., 1981). In the present study. using antimonate cytochemistry. we were unable to confirm the Lubbock etal. observation that calcium moves out of the capsule into an area surrounding the capsule prior to discharge. Instead we observed, as did Mariscal(l984) who used X-ray microanalysis, the presence of large amounts of calcium in the capsule and thread interior of partially discharged nematocysts, but not in fully discharged nematocysts. These results suggest that a significant amount of calcium is injected into the target tissue along with the nematocyst venom. The function of such nematocyst calcium is not known at the present time. However, some data are available that indicate that calcium augments the activity of certain toxins. Schanne et (11. (1979) found that a variety of cell lytic toxins are totally ineffective in killing cultured cells unless calcium is included in the culture medium. Schanne et al. concluded that a disruption of intracellular calcium homeostasis could be responsible for the increased cell death. We speculate that nematocyst calcium injected into the target aids the nematocyst venom in causing cell death in the target tissue. Another possible explanation for our failure to observe calcium outside of the discharging nematocyst is that such calcium might be lost from our preparations during tissue processing steps (e.g. dehydration and embedding) and therefore go undetected by our methods. On the other hand, the observation of extracapsular nematocyst calcium as reported by Lubbock et al. (1981) might be an artifact of tissue preparation. Lubbock and colleagues preserved anemone samples by immersing them in liquid Freon. Using this technique, the freezing rate is virtually instantaneous at the sample surface but considerably slower

211

NEMATOCYST DISCHARGE IN A SEA ANEMONE

deeper in the sample (Mazur, 1970). Generally speaking, rapidly frozen samples can he artifact-free in the first 1525pm of tissue depth (Biihler, 1979). The occurrence of freezing artifacts increases with depth into the tissue. Some common freezing artifacts include a concentration and translocation of solutes due to a removal of water during ice crystal formation (Bohler, 1979; Meryman, 1971; Mazur, 1970). Considering the large size (60-80pm long) of the holotrich nematocysts used in the Lubbock et al. (1981) study, the possibility that artifacts might have accompanied freezing cannot be dismissed. Therefore, we suggest that the role of calcium in nematocyst discharge be considered an open question until more data are available. At the present time, there are at least three possible functions for the nematocyst calcium that might not be mutually exclusive .

1. The nematocyst calcium might be injected into the target tissue to aid nematocyst venom in causing cell death (Mariscal, 1984, and the present study 2. The calcium might help stabilize subunit binding on the uneverted nematocyst thread. A dissociation of this calcium from the subunits would cause subunit uncoupling and thereby permit the uneverted thread to unfold and uncoil, a normal process in nematocyst discharge (the present study). 3. The calcium might help stabilize aggregates of large molecules presumed to be present in the capsule interior (Lubbock et al., 1981; Lubbock and Amos, 1981). A dissociation of the calcium from the aggregates would cause them to separate and thereby increase osmo-

14

tic pressure. The increased osmotic pressure would cause an influx of fluid that would lead to thread eversion (Lubbock et al., 1981; Lubbock and Amos, 1981). Finally, it should be pointed out that not all nematocysts studied to date appear to contain calcium. Using X-ray microanalysis, Mariscal (1984) found that undischarged holotrichous isorhiza nematocysts isolated from H. luciae feeding tentacles lack calcium and instead contain a large amount of phosphorus. In addition, the adhesive spirocysts of H. luciae feeding tentacles appear to lack both calcium and phosphorus. Spirocysts are the most abundant type of cnida (everting organelle) in the feeding tentacles of a variety of sea anemones (e.g. Schmidt, 1982; Purcell, 1977; Bigger, 1982; Watson and Mariscal , 1983a). In view of the fact that only a few nematocysts and spirocysts from a very few species have been examined using X-ray microanalysis and cytochemical procedures, perhaps it is not surprising that the distribution of calcium is irregular among the cnidae so far investigated. Clearly, more work in nematocysts (and spirocysts) is needed before it will be possible to determine the function of calcium in nematocysts and to develop a more comprehensive hypothesis dealing with the mechanism of nematocyst discharge.

Acknowledgements

The authors thank Robert Seaton for the identification of Diadumene gracillima, Tom Fellers and Bill Miller for technical assistance in electron microscopy and Heidi Watson for typing the revised manuscript.

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