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SCALARIFORM JUNCTIONS: A REVISED MODEL
Cell Biology International, 1997, Vol. 21, No. 1, 23–34
SCALARIFORM JUNCTIONS: A REVISED MODEL R. DALLAI1, P. BURIGHEL2, G. B. MARTINUCCI2 and N. J. LANE3* 1
Dip. Biologia Evolutiva, via Mattioli 4, Università di Siena, Siena, Italy, 2Dip. Biologia, via Trieste 75, Università di Padova, Padova, Italy, and 3Department of Zoology, Downing Street, University of Cambridge, Cambridge CB2 3EJ, U.K. Accepted 26 September 1996
The structure of scalariform junctions has been analysed in a number of different animal groups by a variety of techniques. These junctions generally display a highly regular intercellular cleft straddled by indistinct cross-striations. They are often in intimate spatial association with mitochondria which lie in parallel to the leaflets of their junctional membranes. Both intercellular and intramembranous features of these junctions have been studied in this report and certain common characteristics are revealed in all species examined. The intercellular cross-striations seen in this junctional type are found to be actually hollow columns or pillars, of hour-glass shape, regularly spaced in rhomboidal, or hexagonal arrays which may be anchored in place by certain of the intramembranous particles (IMPs) that occur in abundance on the membrane Pface. The density of the pillars is less than that of the IMPs. The other particles in the junctional area on both P and E faces may therefore be involved in ion transport, since the particular tissues in which these junctions are found are always involved in fluid flow. Those organisms which exhibit scalariform junctions have no closed circulatory system and thus may require some specialized osmoregulatory control in their excretory tissues. Those tissues which exhibit these junctions include organs such as rectal pads, rectal papillae, gills and nephridia, all of which share a similar physiological function even though they are found in organisms which occupy very different habitats. Such organisms include arthropods of different classes, as well as tunicates, where the presence of the scalariform junctions is restricted to a very specialized gut region. A revised model of scalariform junctions is presented here, which encompasses our new observations of their features. These include intercellular columns which are hollow and ‘hour-glass’, rather than straight sided, in side view, together with their spacing and orderly hexagonal or rhomboidal pattern of distribution; their possible tethering by only a certain percentage of the IMPs in the junctional area is also incorporated in this model. ? 1997 Academic Press Limited
K: intercellular junctions; IMPs; ion transport; scalariform junctions
INTRODUCTION Scalariform junctions are intercellular associations restricted to the arthropods and a few other animal groups. They are considered to be important in tissues that are involved in fluid flow and transmembrane ion transport. They appear to function as devices that both hold cells together during rapid fluid transfer yet also serve to maintain a distinct cleft between cells, so that it does not collapse *To whom correspondence should be addressed. 1065–6995/97/010023+12 $25.00/0/cb960112
during the rigours of massive fluid movements. They were first described as scalariform junctions by Fain-Maurel and Cassier (1972) in thin sections of Malpighian tubules and the labial nephridia of the apterigotan wingless insect, Petrobius. In 1979, freeze–fracture studies were made on the scalariform junctions in the rectal pads of cockroach, termite and fire brat (Noirot-Timothée et al., 1979) and in cockroach rectal pads and blow-fly rectal papillae (Lane, 1979). Earlier workers (Oschman and Wall, 1969) had examined cockroach rectal pads, and blow-fly rectal papillae (Gupta and ? 1997 Academic Press Limited
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Berridge, 1966; Berridge and Gupta, 1968) and reported structures which were subsequently to be termed scalariform junctions (Fain-Maurel and Cassier, 1972). These junctions were considered in reviews in 1980 (Noirot-Timothée and Noirot, 1980; Lane and Skaer, 1980) when it was assumed they were unique to the arthropods and closely related groups. However, in 1985, studies on other organisms (Burighel et al., 1985) indicated that comparable scalariform junctions were also to be found in a specialized region of the gut of certain tunicates, namely the ascidian Botryllus, in the plicated cells of this animal’s pyloric caecum. A further more recent review article (Lane et al., 1994) summarized the position at that time which revealed that little further information had been forthcoming on scalariform junctions for over a decade, except that comparable junctions appeared to occur in the nephridia of the primitive onychophoran Peripatus (Lane et al., 1992). In the same year that scalariform junctions were originally definitively described, another, apparently similar, junctional type, the ‘spacing junction’ was reported by Friend and Gilula (1972) as a distinctive cell contact in the rat adrenal cortex, forming channels which opened directly into the blood stream. However, this contact had no typical freeze–fracture profile, nor did it reveal intercellular cross-striations, but instead possessed discrete intercellular densities, unattached to the adjacent membranes. This cannot, therefore, be considered to be a scalariform junction, even though, like the scalariform junctions, it was deemed to function in maintaining a consistent width of the intercellular cleft in order to provide channels between cell borders in the canaliculus and blood stream, as well as to act as a cation depot or reservoir. In this paper we report, for the first time in some systems, that scalariform junctions are to be found in a number of groups of organisms. We assess their features, after new methods of preparation and in tissues not previously examined. In addition,
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we present a revised interpretation via a model of the possible fine structural organization of these hitherto little understood junctions. MATERIALS AND METHODS The tissues studied for this report were the rectal papillae of the thrips, Frankliniella occidentalis, the medfly, Ceratitis capitata and the blowfly, Calliphora erythrocephala; the nephridia of the onychophoran, Peripatus sp., the posterior gills of the freshwater crab, Austropotamobius pallipes; the rectal pads of the cockroach, Periplaneta americana and the caddis fly, Limnephilus bipunctatus; and the gut of the tunicates, Botryllus schlosseri, Diplosoma listerianum and Doliolum nationalis. The tissues were flooded with fixative in situ or dissected out and fixed immediately. Conventional fixation, embedding and sectioning Most of the tissues were fixed in 2.5% glutaraldehyde in a 0.1 phosphate buffer, pH 7.2, with added 0.15 NaCl or 0.18 sucrose. Tissues of tunicates were fixed in 2% glutaraldehyde in a 0.2 cacodylate buffer, pH 7.2, with added 0.27 NaCl. After fixation at room temperature (RT) for 1–2 h, the tissues were washed in three changes of buffer. Treatment with 1% osmium tetroxide in buffer followed for 1 h at RT. Some preparations of thrips gut were treated with 1% tannic acid added to the fixative, for one to three days, subsequently omitting osmium tetroxide treatment (Dallai and Afzelius, 1990). After osmication, tissues were rinsed in buffer and stained en bloc with 1% aqueous uranyl acetate for 1 h at RT. Dehydration through an ascending series of ethanols and propylene oxide ensued, followed by embedding inAraldite. Sections were cut on a LKB Ultrotome III. Sections 1 ìm thick, were stained for examination under the light microscope. Ultrathin sections
Figs 1–4. These are all thin sections of the rectal papillae from the thrips, Frankliniella occidentalis, after conventional glutaraldehyde fixation, followed by osmium and en bloc uranyl acetate treatment. (1) Frontal view of a rectal papilla which demonstrates the existence of scalariform junctions (ScJs) between the adjacent cell borders. They are characterized by an orderly array of pillars observed en face in the intercellular space (arrow). These pillars can be seen to be hollow tubes or columns which link the neighbouring cell membranes to produce, in longitudinal section, a ladder-like striated appearance (arrowheads). Mitochondria (M) are found in close spatial association. #91,800. (2) Scalariform junctions between adjacent cells, cut in longitudinal section, can be seen to produce extremely regular intercellular clefts, which measure around 15 to 20 nm across. The columns themselves are arrayed in fairly regular fashion, being aligned parallel to one another and separated one from the other by about 10 nm. M, mitochondria. #57,800. (3) Higher magnification of a longitudinal section of the ladder-like scalariform junctions, illustrating the hollow columns as they straddle the regular intercellular cleft. #83,300. (4) Higher magnification of an en-face view of ScJs showing the regular arrangement of their component columns. These measure about 11 nm in diameter as seen in this view and are arranged in regular rhomboidal or hexagonal patterns (circles indicate where these are most clearly evident). The number of pillars per ìm2 is 3500 to 3600. #153,000.
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were stained with uranyl acetate and lead citrate and examined in a Philips EM 300, 420 or CM 10, Hitachi H 600 or Joel 200 CX at 80-100 kV. Freeze–fracture For the most part, the various tissues were dissected out and prepared for freeze–fracturing after brief fixation with 2.5% glutaraldehyde in 0.1 phosphate buffer, pH 7.2, with 0.18 sucrose for 20 min at RT. Tissues from tunicates were briefly fixed as reported above. All tissues were then washed in several changes of the same buffer and incubated for 15 to 20 min at RT in 20% glycerol made up in buffer or in an ascending series from 10% to 30% glycerol. Tissues were frozen in Freon 22 cooled in liquid nitrogen (N2) and stored in N2 until use. Material was fractured in a Balzer’s freeze cleaving device (BA360M) at "100)C and at a pressure of 1.33#10 "4 Nm "2 (1.5# 10 "6 Torr). Shadowing was carried out using tungsten-tantalum or carbon-platinum followed by backing with carbon. The freeze–fractured replicas were cleaned with sodium hypochlorite or sulphuric acid, picked up on coated grids and examined in a Philips EM 300, 420 or CM 10 and Hitachi H 600 or Joel 200 CX electron microscopes. The micrographs are mounted so that the direction of shadow is either from the bottom or side. OBSERVATIONS Scalariform junctions are characterized by a regular intercellular cleft of about 15 to 20 nm in thrips and the other species (Fig. 1), except for Doliolum where it measures 25 nm or more across (Fig. 14). This is straddled by thin cross-striations or columns; in some cases these may be relatively indistinct. One organism in which the columns of the scalariform junctions are particularly well resolved with traditional methods or with methods avoiding
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osmium fixation and using tannic acid, is the thrips, Frankliniella; this is an example of the Thysanoptera, which are relatively primitive insects. The tissues which exhibit this junctional type are the rectal papillae. The regular intercellular cleft can be seen with the characteristic cross-striations (Figs 1–3). In these preparations they can be seen to be hollow columns, aligned in parallel linear arrays or rows and spaced about 18 nm centre-to-centre (Fig. 4). A typical image shows that these hollow columns exhibit a regular rhomboidal pattern in enface reviews (Figs 1, 4 and in the model in Fig. 18). When measuring these columns in sections, where they can be perceived to be the rungs of ‘ladders’ in the cleft, the spacing of the ‘rungs’ or columns ranges from 7 to 10 nm. This inconsistency can be explained by the possibility that they are, in side view, actually like hour-glasses, rather than columns of constant diameter. Such an interpretation is supported by their appearance in favourable sections cut to reveal them side on (Figs 1–3). When measured close to the membrane, they are only 7 nm apart, but when measured in the midcleft region, when the ‘waist’ of the hour-glass is to be found, so that the inter-columnar space would be wider, they are ca 10 nm apart (see model in Fig. 18a). The density of the pillars is 3500 to 3600/ìm2. The pattern generated by the rows of columns in en-face views, is usually that of a rhomboid, but when the columns are viewed in areas where the membrane is undulating, the pattern then may appear diamond-shaped or hexagonal (see circles in Fig. 4 and Fig. 18b). When tissues are treated with tannic acid for short periods of time, the details of the scalariform junctional intercellular columns are enhanced (Fig. 5). This is more effective than treatment with lanthanum (see Lane, 1979). When treated with tannic acid for longer periods of time, such as 3 days, many of the structural features are obliterated as if destroyed by the extensive acidic treatment even though the plasma membranes and associated glycocalyx appear well preserved (Fig. 6). The deposition of tannic acid in the clefts of the scalariform junction appears to differ depending on
Figs 5 and 6. Thin sections of the rectal papillae of the thysanopteran Frankliniella occidentalis after impregnation with tannic acid, but without osmium. The tissue in Fig. 5 was incubated for 1 day, but that in Fig. 6 was subjected to prolonged treatment for 3 days. Fig. 5 shows a tangential section illustrating the regular rhomboidal (R) patterns produced by the columns. #82,000. Fig. 6 demonstrates that the prolonged treatment with tannic acid leads to collapse of the intercellular columns, which cannot now be seen with clarity. M, mitochondrion. #76,500. Fig. 7. Thin section from the nephridia of the primitive onychophoran, Peripatus sp., briefly fixed in conventional buffered glutaraldehyde. Note the regular intercellular clefts between adjacent cells and the suggestion of the cross-striations which would be indicative of the presence of scalariform junctions. HD, hemi-desmosomes. #37,400. Fig. 8. Thin section of the posterior gills from the crab, Austropotamobius pallipes, after fixation without tannic acid. Note the suggestion of intercellular cross-striations indicating scalariform junctions (arrows). M, mitochondrion. #46,750.
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the length of the time of incubation. When the columns are still clearly present, after short periods of tannic acid treatment, then the deposition seems to be on the outer surface of the columns, in that they appear as dark hollow circles in en-face views (Fig. 5). When the columns have collapsed, the density of the tannic acid is associated with the true outer surface of the membrane, as if on a little hillock of collapsed columnar material (Fig. 6). After ruthenium red treatment, the columns are also stained on their periphery (see Fain-Maurel and Cassier, 1972). Scalariform junctions are almost always associated with mitochondria which lie in the cytoplasm close to them as seen in sections (Figs 1, 2, 13 and 14) or replicas (Figs 10, 11, 16 and 17). Frequently the mitochondrial membrane is aligned precisely in parallel with the plasma membrane of the junctions. The distance between the outer mitochondrial membrane and the scalariform junction is in the region of 14 nm. This space is frequently also straddled by cross-striations (Figs 13 and 14). In certain tissues which possess them, such as the rectal papillae of Diptera (Figs 11 and 12), the stacks of scalariform junctional membrane occur between thin leaflets without associated mitochondria, but mitochondria are to be found in abundance in the nearby cytoplasm. The scalariform junctions may also co-exist with other junctional types such as hemi-desmosomes (Fig. 7), gap junctions, reticular septate junctions or desmosomes. The scalariform junctions themselves tend to be restricted to the lower region of the lateral border of the component cells. On the upper border are found tight junctions in tunicates, and septate junctions in the arthropods. On the lower border, too, are sometimes found gap junctions, and zonulae adhaerentes. The reticular septate junctions are confined to the trichopteran rectal pads (Dallai et al., 1985) and to the dipteran rectal papillae (Lane, 1979), near the middle regions of the lateral borders. Another feature of the rectal papillae cells is that they are associated with numerous intercellular infoldings, which greatly increase the surface area of the cell surface. This
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would further enhance the possibilities for ionic exchange. After freeze-fracturing, the Pface of the scalariform junctional membrane is characterized by numerous intramembranous particles (IMPs) (Figs 9–11 and 15–17) in these fixed tissues. The Eface features only a very small number of IMPs, with no obvious complementary pits to the IMPs (Figs 9–11 and 15–17). The Pface IMPs exhibit a range of diameter, with a mean size of 8–9 nm (Figs 10 and 11). Analyses of these sizes indicate that they cover a spectrum rather than representing distinct categories. The Eface particles are in general of the larger size, around 9 nm, and exhibit no apparent fixed pattern. On the Pface, the IMPs form a thick carpet; in some cases, these appear aligned in short rows, but generally no regular pattern is immediately obvious. The density of IMPs/ìm2 on the Pface is about 4000 (Fig. 9), 4000 to 4900 (Fig. 10), 4200 to 4300 (Fig. 11). The tissues in which we have observed scalariform junctions include the arthropods, both insects and crustaceans. The former include the Dictyoptera (Fig. 9), Thysanoptera (Figs 1–6), Trichoptera (Fig. 10) and Diptera (Figs 11 and 12). The crustaceans include the Decapoda (Fig. 8). The primitive Onychophora, sometimes considered closely related to arthropods, exhibit junctional areas in some areas with striations that resemble scalariform junctions (Fig. 7). Tunicates of various different classes, also feature what appear to be scalariform junctions. These include ascidians of both the enterogonid (Fig. 17) and the pleurogonid groups (Figs 13 and 16), as well as the thaliacean doliolids (Figs 14 and 15). In some cases these junctions are characterized only by intercellular cross-striations. The tissues in which scalariform junctions have been observed are all ones concerned with ion transport (see Berridge and Oschmann, 1972). They include rectal pads and papillae, which are excretory structures additional to the lower end of the insect gut, specialized for fluid readsorption, the nephridia, which have a similar excretory function, gills, which either absorb or eliminate salt through
Figs 9–12. These figures are all replicas made by freeze-fracturing tissues from areas of scalariform junctions in different insects. Shadowing was with C-Pt (Figs 10 and 11) in both or with tungsten-tantallum (Figs 9 and 12). (9) Replica from the rectal pad of the orthopteran cockroach, Periplaneta americana, (Dictyoptera) showing the numerous IMPs in the Pface (PF) of the membranes of the ScJs. Note that the Eface (EF) possesses only a few IMPs. The insert shows that there is a suggestion of columns (arrowheads) in the intercellular cleft between P and Eface. #33,150; Insert, #68,400. (10) Tissue from the rectal pad of a trichopteran caddis fly, Limnephilus bipunctatus. The replica shows the IMPs in the Pface (PF) and relative dearth of IMPs on the Eface (EF). M, mitochondria. #42,500. (11, 12). Rectal papillae from the dipteran medfly, Ceratitis capitata (Fig. 11) and blow fly, Calliphora erythrocephala (Fig. 12) showing the prepoderance of IMPs on the Pface (PF) and the scalariform junctional membranes. The distribution of the IMPs at times appears to exhibit a linear alignment, but at others seems without any clear regular arrangement. M, mitochondria. Fig. 11, #29,750; Fig. 12, #31,500.
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the plasma membrane, (depending on whether a freshwater or marine environment is at hand), and specialized parts of the tunicate gut, which are also possibly involved in the ionic modification of fluids. Taking all the factors and measurements of scalariform junctions into account, we have constructed a revised model of a generalized scalariform junction (Fig. 18) (based on that presented in 1980 by Lane and Skaer). It takes into account the possible hour-glass shape of the columns, their regular pattern and spacing, and the fact that only a certain percentage of the Pface IMPs would be used to act as anchors for the hollow columns, given that IMPs outnumber the columns. The observation that the Pface IMPs are indistinguishable into separate categories by size and frequency, makes it impossible to determine which of the IMP population would be the contenders for the role of the anchoring sites of the intercellular columns. The model (Fig. 18) shows the spacing of the pillars in lateral view (Fig. 18a), in en-face view (Fig. 18b), and their distribution in a three-dimensional scheme (Fig. 18c). DISCUSSION The columns in the scalariform junctions are clearly less substantial than comparable intercellular structures in cell–cell contacts such as the septate junctions, where extensive intercellular septal ribbons are found. Moreover the columns of the scalariform junctions seem readily destroyed by harsh regimes such as prolonged tannic acid treatment. This suggests that the columns are fragile and have collapsed under such treatment; they may therefore be composed of some modification of the glycocalyx, rather than a more rigid protein structure. Fixation by glutaraldehyde concurrent with tannic acid treatment would be expected to crosslink and preserve protein, but not necessarily carbohydrates. In addition, treatment with pronase
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does not destroy the columns (Fain-Maurel and Cassier, 1972), which corroborates the theory that they are not primarily proteinaceous in nature. Since all the tissue for freeze–fracture replication was first briefly glutaraldehyde-fixed before cryoprotection, freezing and fracturing, then the columns in the clefts might not be expected to be perfectly maintained and hence visible in replicas. This is corroborated by the appearance of the clefts in freeze–fracture replicas (for example, insert in Fig. 9), where little clear indication of columns can be resolved. It seems most likely that the position of the columns is relatively rigorously regulated, to judge by their fairly regular centre-to-centre spacing. This suggests that some transmembrane moiety, such as an intramembranous particle, could be responsible for tethering them in position. Although it has been postulated (Noirot-Timothée and Noirot, 1980) that there can be no correspondence between the IMPs and the intercellular columns in scalariform junctions, because of the higher density of IMPs, than columns, nevertheless there could be a correlation, hinted at earlier (Lane, 1979) that at least some of the IMPs have this function. Thus there may be a correlation in position between certain select IMPs and the columns, which is rendered obscure by the overall large number of Pface IMPs. This could mean that only certain IMPs actually hold the columns in place. It is possible that a proportion of the IMPs are functioning in this way, whilst others are involved in different functional roles, such as transport and ion pumping events. The pattern of packing of the columns (Fig. 18) may be significant as reflecting the minimum spacing of columns necessary to maintain the regularity of the intercellular space. This intercellular cleft, interestingly, is roughly the same as the centre-to-centre spacing of the columns (i.e. 18 nm). This suggests that a regular and consistent intercellular cleft is required for the functioning of the tissues with which scalariform junctions are associated.
Figs 13–17. These figures are all from the gut of tunicates. (13) This tissue is from the plicated cells of the pyloric caecum of the Ascidian Botryllus schlosseri, a stolidobranchian pleurogonid. This reveals, in thin sections, columns of apparent ScJs in the intercellular cleft between adjacent cells (arrows). Mitochondria (M) lie near these junctions, with a regular distance between their outer membrane and the plasma membrane; this region may also feature cross-striations (arrowheads). #76,500. (14) Proximal intestine from the doliolid (thaliacean) Doliolum nationalis. This tissue shows the suggestion of cross-striations (arrows) between adjacent plasmalemmae, with mitochondria (M) prominent in the underlying cytoplasm. #76,500. (15) As in Fig. 14, this preparation is from the intestine of Doliolum nationalis but here it has been frozen and shadowed to produce a freeze-fractured replica. The numerous IMPs are concentrated on the Pface (PF) whilst they are scarce on the Eface (EF). Mitochondria, M. #25,500. (16, 17) These are replicas of tissues from the ascidian Botryllus schlosseri (Fig. 16) (stolidobranchian pleurogonid) and Diplosoma listerianum (aplousobranchian enterogonid). Fig. 16 shows pyloric caecum and Fig. 17, proximal intestine. Both reveal areas of ScJs characterized by numerous Pface IMPs (PF). The junction-associated mitochondria (M) are clearly seen within the cytoplasmic processes. EF, Eface. Fig. 16, #40,800; Fig. 17, #44,200.
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(a)
(c)
(b)
Cytoplasm
E-Face
P-Face
12 cm base
Fig. 18. Model of a generalized scalariform junction, based on that presented previously (Lane and Skaer, 1980), with revisions that indicate: (a) the hour-glass, rather than straight-sided, shape of the intercellular columns in side view, (b) the regular spacing of the hollow columns in the form of a rhomboid, irregular hexagon or a non-equilateral triangle, in en-face view, (c) their anchoring to only a certain percentage of the Pface IMPs. These latter appear to be more numerous than the columns and are of variable size, so that some could be insertion sites for the columns, while others could be involved in the ion transport which is thought to take place across these junctional membranes as indicated by the presence of ATPase. This model suggests that only some of the columns are directly associated with Pface IMPs, but it is possible that all of them are, in one way or another. In (c) the columns are shown as slimmer than they actually are (such as in (a)); this is to show more clearly the regular and orderly packing of the columns.
As indicated in the observations, the tissues in which these junctions are found, rectal pads, rectal papillae, nephridia and gills are all excretory or regulatory organs, associated with the transport of ions across membranes. They have been shown to have this function in various earlier studies on rectal pads (Noirot-Timothée et al., 1979; Dallai et al., 1985), rectal papillae (Oschmann and Wall, 1969; Berridge and Gupta, 1968; Noirot-Timothée and Noirot, 1980), nephridia (Fain-Maurel and Cassier, 1972), and gills (Copeland, 1968; Bielawski, 1971). Interestingly, collapse of the scalariform junctions occurs under conditions of starvation and hence fluid deprivation (Berridge and Gupta, 1968); this has been interpreted by the authors as a cellular device to prevent fluid flow through intercellular spaces during times of dessication. The pyloric caecum and the proximal intestine of the tunicates also exhibit some physiological similarities. The latter is thought to assume the role of osmoregulating body fluids, in the absence of an excretory organ. The former is an evagination of the normal stomach, and its role has been considered variously to be modifying the secretion of the
pyloric gland or changing the characteristics of the internal fluids (Burighel and Milanesi, 1975; Burighel et al., 1985). As with the other tissues in which scalariform junctions are found, these activities also require an immediate energy source, which the closely associated mitochondria could supply. In both of these systems, although the blood fluid may be the same osmolarity as the sea water, the cells are able to regulate the ionic composition of the circulating fluid, so that it is in certain ways distinct from that of sea water (Goodbody, 1974; Robertson, 1987). All of the tissues in which scalariform junctions occur, then, are involved in some kind of ionic homeostasis. This is irrespective of the evolutionary position of the organism in which these tissues are found. Hence, they occur in both primitive insects such as the Dictyoptera and Apterygota, but also in highly evolved groups such as the dipteran insects. In the tunicates, which are primitive chordates, they occur as shown in a spectrum of different groups. However, they have not been described in the other chordates—the cephalochordates or vertebrates. Different mechanisms have evolved in
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vertebrate tissues such as the kidney, avian salt glands and teleost gills for example. All these have intercellular clefts closely associated with energyproducing mitochondria, but definitive scalariform junctions have not been reported. The virtue of the scalariform junctions in the tissues described here is that they would hold the cells’ plasma membranes a very precise distance apart, preventing collapse of the intercellular cleft, during crucial fluid flow. That these spaces retain their integrity during fluid transport has been demonstrated by Oschmann and Wall (1969), indicating that it is the columns which serve in the maintenance of this space which appears to be so important for the adequate functioning of the cells that possess them. When bulk fluid flow out of a tissue lumen produces so rapid a reduction in luminal volume that the intercellular spaces are subjected to pressures tending to collapse them, it could be argued that the contractile muscular tissues that invest them will act to maintain normal pressure and hence tissue and cleft integrity. However, in the tunicates, the tissues involved in fluid transport with scalariform junctions are not invested with muscular tissue. This is absent all along the gut and the scalariform junctions could be assumed to be required to prevent collapse of the intercellular cleft. In an attempt to speculate as to why the scalariform junctions are not found in vertebrate tissues, one might suggest that perhaps two different systems have evolved to perform a similar function. Both arthropods and tunicates possess barriers against the external world, as their epithelia are sealed by means of septate and tight junctions respectively. Their blood flows in lacunae and/or vessels which are not sealed and hence lack a true endothelium, so that blood and the other fluids move freely around most of their bodily organs. This is different in vertebrates, which possess barriers against the external world; the blood flows inside a closed circulatory system, the vasculature, which is provided with its own endothelia sealed by tight junctions. In vertebrates, regulation of body fluids occurs through a more complex type of excretory organ (the kidney) whose activity is more finely controlled by the endocrine system. This kidney establishes a special relation with the inner closed system, the circulatory system, at the level of glomeruli and renal tubules. This different organization and the acquisition of more efficient systems over time could explain why the vertebrates have lost the scalariform junctions, which were presumably present in their ancestors and are maintained in tunicates which are chordates yet
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have tight junctions as well as scalariform junctions. Of course limited closed systems can also be present in particular organs of both invertebrates (such as the nervous system of arthropods and the cardio-pericardium vesicle of tunicates) and in vertebrates (blood–brain, blood–testis barriers, etc.). However, arthropods lack tight junctions except in their CNS and testis where blood–brain barriers and blood–testis barriers exist (Lane, 1981). The blood–brain barrier appears to be necessary for the efficient functioning of the CNS in insects and arachnids; these relatively sophisticated brains require an extracellular fluid in which the proportions of ions are carefully regulated to ensure the proper functioning of the nerve cells (Lane and Treherne, 1980; Lane et al., 1982). These speculations concerning scalariform junctions will be tested by immunocytochemical approaches in the future to test for the presence of enzymes such as ATPase. ACKNOWLEDGEMENTS We wish to acknowledge Mr C. Friso for the drawings in Fig. 18. R.D. and P.B. are grateful to Murst (40%) and CNR for financial support during the course of this work. N.J.L. is indebted to the Wellcome Trust, for support in the form of grants (# 032970/1.4U and 032970/Z/90/B). REFERENCES B MJ, G BL, 1968. Fine structural localization of adenosine triphosphate in the rectum of Calliphora. J Cell Sci 3: 17–32. B MJ, O JL, 1972. Transporting Epithelia. Academic Press. New York, London. B J, 1971. Ultrastructure and ion transport in gill epithelia of the crayfish, Astacus leptodactylus. Esch. Protoplasma 73: 177–190. B P, M C, 1975. Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous, endocrine and plicated cells. Z. Zellforsch. 158: 481–496. B P, D R, M GB, 1985. Scalariform junctions in the gut of tunicates. Biol Cell 54: 171–176. C DE, 1968. Fine structure of salt and water uptake in the land-crab Carcinus laberalis. Am Zool 8: 417–432. D R, A BA, 1990. Microtubular diversity in insect spermatozoa: Results obtained with a new fixative. J Struct Biol 103: 164–179. D R, M G, C F, C S C, 1985. Freeze-fracture study of the rectal pads in Stenophylax permistus McL. (Trichoptera). Boll Zool 52: 407– 420. F-M MA, C P, 1972. Un nouveau type de jonction: les jonctions scalariformes. Etude ultrastructurale et cytochimique. J Ultrastruct Res 39: 222–238.
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F DS, G NB, 1972. A distinctive cell contact in the rat adrenal cortex. J Cell Biol 53: 148–163. G I, 1974. The physiology of ascidians. Adv Mar Biol 12: 1–149. G BL, B MJ, 1966. Fine structural organization of the rectum in blowfly Calliphora erythrocephala (Meig.) with special reference to connective tissue, tracheae and neurosecretory innervation in the rectal papillae. J Morphol 120: 23–82. L NJ, 1979. Freeze–fracture and tracer studies on the intercellular junctions of insect rectal tissues. Tissue Cell 11: 481–506. L NJ, 1981. Tight junctions in arthropod tissues. Int Rev Cytol 73: 243–318. L NJ, H JB, B RF, 1982. A vertebratelike blood–brain barrier with interganglionic blood channels and occluding junctions, in the scorpion. Tissue Cell 13: 557–576. L NJ, S H B, 1980. Intercellular junctions in insect tissues. Adv Insect Physiol 15: 35–213. L NJ, T JE, 1980. Functional organization of arthropod neuroglia. In: Locke M, Smith DS, eds. Insect Biology in the Future- VBW 80. Academic Press. London, 765–795.
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L NJ, C SS, L WM, 121992. Junctional types in the tissues of an Onychophora; the apparent lack of gap and tight junctions in Peripatus. Tissue Cell 26: 143–154. L NJ, D R, M GB, B P,1994. Electron microscopic structure and evolution of epithelial junctions. In: Citi S., ed. Molecular Mechanisms of Epithelial Cell Junctions: from Development to Disease. R.G. Landes Company, Austin, Texas, 23–44. N-T´ C, N C, 1980. Septate and scalariform junctions in arthropods. Int Rev Cytol 63: 97–140. N-T´ C, N C, S DS, C ML, 1979. Jonctions et contacts intercellulaires chez les insectes. II Jonctions scalariformes et complexes formés avec les mitochondries: Etude par coupes fines et cryofractures. Biol Cell 34: 137–136. O JL, W BL, 1969. The structure of the rectal pads of Periplaneta americana L. with regard to fluid transport. J Morphol 127: 475–510. R JD, 1987. Osmotic constituents of the bodywall muscles of the hemichordate Balanoglossus clavigerus, the tunicate Ciona intestinalis, and the cephalochordate Branchiostoma lanceolatum. Comp Biochem Physiol 87A: 363–373.
SCALARIFORM JUNCTIONS: A REVISED MODEL R. DALLAI1, P. BURIGHEL2, G. B. MARTINUCCI2 and N. J. LANE3* 1
Dip. Biologia Evolutiva, via Mattioli 4, Università di Siena, Siena, Italy, 2Dip. Biologia, via Trieste 75, Università di Padova, Padova, Italy, and 3Department of Zoology, Downing Street, University of Cambridge, Cambridge CB2 3EJ, U.K. Accepted 26 September 1996
The structure of scalariform junctions has been analysed in a number of different animal groups by a variety of techniques. These junctions generally display a highly regular intercellular cleft straddled by indistinct cross-striations. They are often in intimate spatial association with mitochondria which lie in parallel to the leaflets of their junctional membranes. Both intercellular and intramembranous features of these junctions have been studied in this report and certain common characteristics are revealed in all species examined. The intercellular cross-striations seen in this junctional type are found to be actually hollow columns or pillars, of hour-glass shape, regularly spaced in rhomboidal, or hexagonal arrays which may be anchored in place by certain of the intramembranous particles (IMPs) that occur in abundance on the membrane Pface. The density of the pillars is less than that of the IMPs. The other particles in the junctional area on both P and E faces may therefore be involved in ion transport, since the particular tissues in which these junctions are found are always involved in fluid flow. Those organisms which exhibit scalariform junctions have no closed circulatory system and thus may require some specialized osmoregulatory control in their excretory tissues. Those tissues which exhibit these junctions include organs such as rectal pads, rectal papillae, gills and nephridia, all of which share a similar physiological function even though they are found in organisms which occupy very different habitats. Such organisms include arthropods of different classes, as well as tunicates, where the presence of the scalariform junctions is restricted to a very specialized gut region. A revised model of scalariform junctions is presented here, which encompasses our new observations of their features. These include intercellular columns which are hollow and ‘hour-glass’, rather than straight sided, in side view, together with their spacing and orderly hexagonal or rhomboidal pattern of distribution; their possible tethering by only a certain percentage of the IMPs in the junctional area is also incorporated in this model. ? 1997 Academic Press Limited
K: intercellular junctions; IMPs; ion transport; scalariform junctions
INTRODUCTION Scalariform junctions are intercellular associations restricted to the arthropods and a few other animal groups. They are considered to be important in tissues that are involved in fluid flow and transmembrane ion transport. They appear to function as devices that both hold cells together during rapid fluid transfer yet also serve to maintain a distinct cleft between cells, so that it does not collapse *To whom correspondence should be addressed. 1065–6995/97/010023+12 $25.00/0/cb960112
during the rigours of massive fluid movements. They were first described as scalariform junctions by Fain-Maurel and Cassier (1972) in thin sections of Malpighian tubules and the labial nephridia of the apterigotan wingless insect, Petrobius. In 1979, freeze–fracture studies were made on the scalariform junctions in the rectal pads of cockroach, termite and fire brat (Noirot-Timothée et al., 1979) and in cockroach rectal pads and blow-fly rectal papillae (Lane, 1979). Earlier workers (Oschman and Wall, 1969) had examined cockroach rectal pads, and blow-fly rectal papillae (Gupta and ? 1997 Academic Press Limited
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Berridge, 1966; Berridge and Gupta, 1968) and reported structures which were subsequently to be termed scalariform junctions (Fain-Maurel and Cassier, 1972). These junctions were considered in reviews in 1980 (Noirot-Timothée and Noirot, 1980; Lane and Skaer, 1980) when it was assumed they were unique to the arthropods and closely related groups. However, in 1985, studies on other organisms (Burighel et al., 1985) indicated that comparable scalariform junctions were also to be found in a specialized region of the gut of certain tunicates, namely the ascidian Botryllus, in the plicated cells of this animal’s pyloric caecum. A further more recent review article (Lane et al., 1994) summarized the position at that time which revealed that little further information had been forthcoming on scalariform junctions for over a decade, except that comparable junctions appeared to occur in the nephridia of the primitive onychophoran Peripatus (Lane et al., 1992). In the same year that scalariform junctions were originally definitively described, another, apparently similar, junctional type, the ‘spacing junction’ was reported by Friend and Gilula (1972) as a distinctive cell contact in the rat adrenal cortex, forming channels which opened directly into the blood stream. However, this contact had no typical freeze–fracture profile, nor did it reveal intercellular cross-striations, but instead possessed discrete intercellular densities, unattached to the adjacent membranes. This cannot, therefore, be considered to be a scalariform junction, even though, like the scalariform junctions, it was deemed to function in maintaining a consistent width of the intercellular cleft in order to provide channels between cell borders in the canaliculus and blood stream, as well as to act as a cation depot or reservoir. In this paper we report, for the first time in some systems, that scalariform junctions are to be found in a number of groups of organisms. We assess their features, after new methods of preparation and in tissues not previously examined. In addition,
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we present a revised interpretation via a model of the possible fine structural organization of these hitherto little understood junctions. MATERIALS AND METHODS The tissues studied for this report were the rectal papillae of the thrips, Frankliniella occidentalis, the medfly, Ceratitis capitata and the blowfly, Calliphora erythrocephala; the nephridia of the onychophoran, Peripatus sp., the posterior gills of the freshwater crab, Austropotamobius pallipes; the rectal pads of the cockroach, Periplaneta americana and the caddis fly, Limnephilus bipunctatus; and the gut of the tunicates, Botryllus schlosseri, Diplosoma listerianum and Doliolum nationalis. The tissues were flooded with fixative in situ or dissected out and fixed immediately. Conventional fixation, embedding and sectioning Most of the tissues were fixed in 2.5% glutaraldehyde in a 0.1 phosphate buffer, pH 7.2, with added 0.15 NaCl or 0.18 sucrose. Tissues of tunicates were fixed in 2% glutaraldehyde in a 0.2 cacodylate buffer, pH 7.2, with added 0.27 NaCl. After fixation at room temperature (RT) for 1–2 h, the tissues were washed in three changes of buffer. Treatment with 1% osmium tetroxide in buffer followed for 1 h at RT. Some preparations of thrips gut were treated with 1% tannic acid added to the fixative, for one to three days, subsequently omitting osmium tetroxide treatment (Dallai and Afzelius, 1990). After osmication, tissues were rinsed in buffer and stained en bloc with 1% aqueous uranyl acetate for 1 h at RT. Dehydration through an ascending series of ethanols and propylene oxide ensued, followed by embedding inAraldite. Sections were cut on a LKB Ultrotome III. Sections 1 ìm thick, were stained for examination under the light microscope. Ultrathin sections
Figs 1–4. These are all thin sections of the rectal papillae from the thrips, Frankliniella occidentalis, after conventional glutaraldehyde fixation, followed by osmium and en bloc uranyl acetate treatment. (1) Frontal view of a rectal papilla which demonstrates the existence of scalariform junctions (ScJs) between the adjacent cell borders. They are characterized by an orderly array of pillars observed en face in the intercellular space (arrow). These pillars can be seen to be hollow tubes or columns which link the neighbouring cell membranes to produce, in longitudinal section, a ladder-like striated appearance (arrowheads). Mitochondria (M) are found in close spatial association. #91,800. (2) Scalariform junctions between adjacent cells, cut in longitudinal section, can be seen to produce extremely regular intercellular clefts, which measure around 15 to 20 nm across. The columns themselves are arrayed in fairly regular fashion, being aligned parallel to one another and separated one from the other by about 10 nm. M, mitochondria. #57,800. (3) Higher magnification of a longitudinal section of the ladder-like scalariform junctions, illustrating the hollow columns as they straddle the regular intercellular cleft. #83,300. (4) Higher magnification of an en-face view of ScJs showing the regular arrangement of their component columns. These measure about 11 nm in diameter as seen in this view and are arranged in regular rhomboidal or hexagonal patterns (circles indicate where these are most clearly evident). The number of pillars per ìm2 is 3500 to 3600. #153,000.
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were stained with uranyl acetate and lead citrate and examined in a Philips EM 300, 420 or CM 10, Hitachi H 600 or Joel 200 CX at 80-100 kV. Freeze–fracture For the most part, the various tissues were dissected out and prepared for freeze–fracturing after brief fixation with 2.5% glutaraldehyde in 0.1 phosphate buffer, pH 7.2, with 0.18 sucrose for 20 min at RT. Tissues from tunicates were briefly fixed as reported above. All tissues were then washed in several changes of the same buffer and incubated for 15 to 20 min at RT in 20% glycerol made up in buffer or in an ascending series from 10% to 30% glycerol. Tissues were frozen in Freon 22 cooled in liquid nitrogen (N2) and stored in N2 until use. Material was fractured in a Balzer’s freeze cleaving device (BA360M) at "100)C and at a pressure of 1.33#10 "4 Nm "2 (1.5# 10 "6 Torr). Shadowing was carried out using tungsten-tantalum or carbon-platinum followed by backing with carbon. The freeze–fractured replicas were cleaned with sodium hypochlorite or sulphuric acid, picked up on coated grids and examined in a Philips EM 300, 420 or CM 10 and Hitachi H 600 or Joel 200 CX electron microscopes. The micrographs are mounted so that the direction of shadow is either from the bottom or side. OBSERVATIONS Scalariform junctions are characterized by a regular intercellular cleft of about 15 to 20 nm in thrips and the other species (Fig. 1), except for Doliolum where it measures 25 nm or more across (Fig. 14). This is straddled by thin cross-striations or columns; in some cases these may be relatively indistinct. One organism in which the columns of the scalariform junctions are particularly well resolved with traditional methods or with methods avoiding
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osmium fixation and using tannic acid, is the thrips, Frankliniella; this is an example of the Thysanoptera, which are relatively primitive insects. The tissues which exhibit this junctional type are the rectal papillae. The regular intercellular cleft can be seen with the characteristic cross-striations (Figs 1–3). In these preparations they can be seen to be hollow columns, aligned in parallel linear arrays or rows and spaced about 18 nm centre-to-centre (Fig. 4). A typical image shows that these hollow columns exhibit a regular rhomboidal pattern in enface reviews (Figs 1, 4 and in the model in Fig. 18). When measuring these columns in sections, where they can be perceived to be the rungs of ‘ladders’ in the cleft, the spacing of the ‘rungs’ or columns ranges from 7 to 10 nm. This inconsistency can be explained by the possibility that they are, in side view, actually like hour-glasses, rather than columns of constant diameter. Such an interpretation is supported by their appearance in favourable sections cut to reveal them side on (Figs 1–3). When measured close to the membrane, they are only 7 nm apart, but when measured in the midcleft region, when the ‘waist’ of the hour-glass is to be found, so that the inter-columnar space would be wider, they are ca 10 nm apart (see model in Fig. 18a). The density of the pillars is 3500 to 3600/ìm2. The pattern generated by the rows of columns in en-face views, is usually that of a rhomboid, but when the columns are viewed in areas where the membrane is undulating, the pattern then may appear diamond-shaped or hexagonal (see circles in Fig. 4 and Fig. 18b). When tissues are treated with tannic acid for short periods of time, the details of the scalariform junctional intercellular columns are enhanced (Fig. 5). This is more effective than treatment with lanthanum (see Lane, 1979). When treated with tannic acid for longer periods of time, such as 3 days, many of the structural features are obliterated as if destroyed by the extensive acidic treatment even though the plasma membranes and associated glycocalyx appear well preserved (Fig. 6). The deposition of tannic acid in the clefts of the scalariform junction appears to differ depending on
Figs 5 and 6. Thin sections of the rectal papillae of the thysanopteran Frankliniella occidentalis after impregnation with tannic acid, but without osmium. The tissue in Fig. 5 was incubated for 1 day, but that in Fig. 6 was subjected to prolonged treatment for 3 days. Fig. 5 shows a tangential section illustrating the regular rhomboidal (R) patterns produced by the columns. #82,000. Fig. 6 demonstrates that the prolonged treatment with tannic acid leads to collapse of the intercellular columns, which cannot now be seen with clarity. M, mitochondrion. #76,500. Fig. 7. Thin section from the nephridia of the primitive onychophoran, Peripatus sp., briefly fixed in conventional buffered glutaraldehyde. Note the regular intercellular clefts between adjacent cells and the suggestion of the cross-striations which would be indicative of the presence of scalariform junctions. HD, hemi-desmosomes. #37,400. Fig. 8. Thin section of the posterior gills from the crab, Austropotamobius pallipes, after fixation without tannic acid. Note the suggestion of intercellular cross-striations indicating scalariform junctions (arrows). M, mitochondrion. #46,750.
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the length of the time of incubation. When the columns are still clearly present, after short periods of tannic acid treatment, then the deposition seems to be on the outer surface of the columns, in that they appear as dark hollow circles in en-face views (Fig. 5). When the columns have collapsed, the density of the tannic acid is associated with the true outer surface of the membrane, as if on a little hillock of collapsed columnar material (Fig. 6). After ruthenium red treatment, the columns are also stained on their periphery (see Fain-Maurel and Cassier, 1972). Scalariform junctions are almost always associated with mitochondria which lie in the cytoplasm close to them as seen in sections (Figs 1, 2, 13 and 14) or replicas (Figs 10, 11, 16 and 17). Frequently the mitochondrial membrane is aligned precisely in parallel with the plasma membrane of the junctions. The distance between the outer mitochondrial membrane and the scalariform junction is in the region of 14 nm. This space is frequently also straddled by cross-striations (Figs 13 and 14). In certain tissues which possess them, such as the rectal papillae of Diptera (Figs 11 and 12), the stacks of scalariform junctional membrane occur between thin leaflets without associated mitochondria, but mitochondria are to be found in abundance in the nearby cytoplasm. The scalariform junctions may also co-exist with other junctional types such as hemi-desmosomes (Fig. 7), gap junctions, reticular septate junctions or desmosomes. The scalariform junctions themselves tend to be restricted to the lower region of the lateral border of the component cells. On the upper border are found tight junctions in tunicates, and septate junctions in the arthropods. On the lower border, too, are sometimes found gap junctions, and zonulae adhaerentes. The reticular septate junctions are confined to the trichopteran rectal pads (Dallai et al., 1985) and to the dipteran rectal papillae (Lane, 1979), near the middle regions of the lateral borders. Another feature of the rectal papillae cells is that they are associated with numerous intercellular infoldings, which greatly increase the surface area of the cell surface. This
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would further enhance the possibilities for ionic exchange. After freeze-fracturing, the Pface of the scalariform junctional membrane is characterized by numerous intramembranous particles (IMPs) (Figs 9–11 and 15–17) in these fixed tissues. The Eface features only a very small number of IMPs, with no obvious complementary pits to the IMPs (Figs 9–11 and 15–17). The Pface IMPs exhibit a range of diameter, with a mean size of 8–9 nm (Figs 10 and 11). Analyses of these sizes indicate that they cover a spectrum rather than representing distinct categories. The Eface particles are in general of the larger size, around 9 nm, and exhibit no apparent fixed pattern. On the Pface, the IMPs form a thick carpet; in some cases, these appear aligned in short rows, but generally no regular pattern is immediately obvious. The density of IMPs/ìm2 on the Pface is about 4000 (Fig. 9), 4000 to 4900 (Fig. 10), 4200 to 4300 (Fig. 11). The tissues in which we have observed scalariform junctions include the arthropods, both insects and crustaceans. The former include the Dictyoptera (Fig. 9), Thysanoptera (Figs 1–6), Trichoptera (Fig. 10) and Diptera (Figs 11 and 12). The crustaceans include the Decapoda (Fig. 8). The primitive Onychophora, sometimes considered closely related to arthropods, exhibit junctional areas in some areas with striations that resemble scalariform junctions (Fig. 7). Tunicates of various different classes, also feature what appear to be scalariform junctions. These include ascidians of both the enterogonid (Fig. 17) and the pleurogonid groups (Figs 13 and 16), as well as the thaliacean doliolids (Figs 14 and 15). In some cases these junctions are characterized only by intercellular cross-striations. The tissues in which scalariform junctions have been observed are all ones concerned with ion transport (see Berridge and Oschmann, 1972). They include rectal pads and papillae, which are excretory structures additional to the lower end of the insect gut, specialized for fluid readsorption, the nephridia, which have a similar excretory function, gills, which either absorb or eliminate salt through
Figs 9–12. These figures are all replicas made by freeze-fracturing tissues from areas of scalariform junctions in different insects. Shadowing was with C-Pt (Figs 10 and 11) in both or with tungsten-tantallum (Figs 9 and 12). (9) Replica from the rectal pad of the orthopteran cockroach, Periplaneta americana, (Dictyoptera) showing the numerous IMPs in the Pface (PF) of the membranes of the ScJs. Note that the Eface (EF) possesses only a few IMPs. The insert shows that there is a suggestion of columns (arrowheads) in the intercellular cleft between P and Eface. #33,150; Insert, #68,400. (10) Tissue from the rectal pad of a trichopteran caddis fly, Limnephilus bipunctatus. The replica shows the IMPs in the Pface (PF) and relative dearth of IMPs on the Eface (EF). M, mitochondria. #42,500. (11, 12). Rectal papillae from the dipteran medfly, Ceratitis capitata (Fig. 11) and blow fly, Calliphora erythrocephala (Fig. 12) showing the prepoderance of IMPs on the Pface (PF) and the scalariform junctional membranes. The distribution of the IMPs at times appears to exhibit a linear alignment, but at others seems without any clear regular arrangement. M, mitochondria. Fig. 11, #29,750; Fig. 12, #31,500.
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the plasma membrane, (depending on whether a freshwater or marine environment is at hand), and specialized parts of the tunicate gut, which are also possibly involved in the ionic modification of fluids. Taking all the factors and measurements of scalariform junctions into account, we have constructed a revised model of a generalized scalariform junction (Fig. 18) (based on that presented in 1980 by Lane and Skaer). It takes into account the possible hour-glass shape of the columns, their regular pattern and spacing, and the fact that only a certain percentage of the Pface IMPs would be used to act as anchors for the hollow columns, given that IMPs outnumber the columns. The observation that the Pface IMPs are indistinguishable into separate categories by size and frequency, makes it impossible to determine which of the IMP population would be the contenders for the role of the anchoring sites of the intercellular columns. The model (Fig. 18) shows the spacing of the pillars in lateral view (Fig. 18a), in en-face view (Fig. 18b), and their distribution in a three-dimensional scheme (Fig. 18c). DISCUSSION The columns in the scalariform junctions are clearly less substantial than comparable intercellular structures in cell–cell contacts such as the septate junctions, where extensive intercellular septal ribbons are found. Moreover the columns of the scalariform junctions seem readily destroyed by harsh regimes such as prolonged tannic acid treatment. This suggests that the columns are fragile and have collapsed under such treatment; they may therefore be composed of some modification of the glycocalyx, rather than a more rigid protein structure. Fixation by glutaraldehyde concurrent with tannic acid treatment would be expected to crosslink and preserve protein, but not necessarily carbohydrates. In addition, treatment with pronase
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does not destroy the columns (Fain-Maurel and Cassier, 1972), which corroborates the theory that they are not primarily proteinaceous in nature. Since all the tissue for freeze–fracture replication was first briefly glutaraldehyde-fixed before cryoprotection, freezing and fracturing, then the columns in the clefts might not be expected to be perfectly maintained and hence visible in replicas. This is corroborated by the appearance of the clefts in freeze–fracture replicas (for example, insert in Fig. 9), where little clear indication of columns can be resolved. It seems most likely that the position of the columns is relatively rigorously regulated, to judge by their fairly regular centre-to-centre spacing. This suggests that some transmembrane moiety, such as an intramembranous particle, could be responsible for tethering them in position. Although it has been postulated (Noirot-Timothée and Noirot, 1980) that there can be no correspondence between the IMPs and the intercellular columns in scalariform junctions, because of the higher density of IMPs, than columns, nevertheless there could be a correlation, hinted at earlier (Lane, 1979) that at least some of the IMPs have this function. Thus there may be a correlation in position between certain select IMPs and the columns, which is rendered obscure by the overall large number of Pface IMPs. This could mean that only certain IMPs actually hold the columns in place. It is possible that a proportion of the IMPs are functioning in this way, whilst others are involved in different functional roles, such as transport and ion pumping events. The pattern of packing of the columns (Fig. 18) may be significant as reflecting the minimum spacing of columns necessary to maintain the regularity of the intercellular space. This intercellular cleft, interestingly, is roughly the same as the centre-to-centre spacing of the columns (i.e. 18 nm). This suggests that a regular and consistent intercellular cleft is required for the functioning of the tissues with which scalariform junctions are associated.
Figs 13–17. These figures are all from the gut of tunicates. (13) This tissue is from the plicated cells of the pyloric caecum of the Ascidian Botryllus schlosseri, a stolidobranchian pleurogonid. This reveals, in thin sections, columns of apparent ScJs in the intercellular cleft between adjacent cells (arrows). Mitochondria (M) lie near these junctions, with a regular distance between their outer membrane and the plasma membrane; this region may also feature cross-striations (arrowheads). #76,500. (14) Proximal intestine from the doliolid (thaliacean) Doliolum nationalis. This tissue shows the suggestion of cross-striations (arrows) between adjacent plasmalemmae, with mitochondria (M) prominent in the underlying cytoplasm. #76,500. (15) As in Fig. 14, this preparation is from the intestine of Doliolum nationalis but here it has been frozen and shadowed to produce a freeze-fractured replica. The numerous IMPs are concentrated on the Pface (PF) whilst they are scarce on the Eface (EF). Mitochondria, M. #25,500. (16, 17) These are replicas of tissues from the ascidian Botryllus schlosseri (Fig. 16) (stolidobranchian pleurogonid) and Diplosoma listerianum (aplousobranchian enterogonid). Fig. 16 shows pyloric caecum and Fig. 17, proximal intestine. Both reveal areas of ScJs characterized by numerous Pface IMPs (PF). The junction-associated mitochondria (M) are clearly seen within the cytoplasmic processes. EF, Eface. Fig. 16, #40,800; Fig. 17, #44,200.
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(a)
(c)
(b)
Cytoplasm
E-Face
P-Face
12 cm base
Fig. 18. Model of a generalized scalariform junction, based on that presented previously (Lane and Skaer, 1980), with revisions that indicate: (a) the hour-glass, rather than straight-sided, shape of the intercellular columns in side view, (b) the regular spacing of the hollow columns in the form of a rhomboid, irregular hexagon or a non-equilateral triangle, in en-face view, (c) their anchoring to only a certain percentage of the Pface IMPs. These latter appear to be more numerous than the columns and are of variable size, so that some could be insertion sites for the columns, while others could be involved in the ion transport which is thought to take place across these junctional membranes as indicated by the presence of ATPase. This model suggests that only some of the columns are directly associated with Pface IMPs, but it is possible that all of them are, in one way or another. In (c) the columns are shown as slimmer than they actually are (such as in (a)); this is to show more clearly the regular and orderly packing of the columns.
As indicated in the observations, the tissues in which these junctions are found, rectal pads, rectal papillae, nephridia and gills are all excretory or regulatory organs, associated with the transport of ions across membranes. They have been shown to have this function in various earlier studies on rectal pads (Noirot-Timothée et al., 1979; Dallai et al., 1985), rectal papillae (Oschmann and Wall, 1969; Berridge and Gupta, 1968; Noirot-Timothée and Noirot, 1980), nephridia (Fain-Maurel and Cassier, 1972), and gills (Copeland, 1968; Bielawski, 1971). Interestingly, collapse of the scalariform junctions occurs under conditions of starvation and hence fluid deprivation (Berridge and Gupta, 1968); this has been interpreted by the authors as a cellular device to prevent fluid flow through intercellular spaces during times of dessication. The pyloric caecum and the proximal intestine of the tunicates also exhibit some physiological similarities. The latter is thought to assume the role of osmoregulating body fluids, in the absence of an excretory organ. The former is an evagination of the normal stomach, and its role has been considered variously to be modifying the secretion of the
pyloric gland or changing the characteristics of the internal fluids (Burighel and Milanesi, 1975; Burighel et al., 1985). As with the other tissues in which scalariform junctions are found, these activities also require an immediate energy source, which the closely associated mitochondria could supply. In both of these systems, although the blood fluid may be the same osmolarity as the sea water, the cells are able to regulate the ionic composition of the circulating fluid, so that it is in certain ways distinct from that of sea water (Goodbody, 1974; Robertson, 1987). All of the tissues in which scalariform junctions occur, then, are involved in some kind of ionic homeostasis. This is irrespective of the evolutionary position of the organism in which these tissues are found. Hence, they occur in both primitive insects such as the Dictyoptera and Apterygota, but also in highly evolved groups such as the dipteran insects. In the tunicates, which are primitive chordates, they occur as shown in a spectrum of different groups. However, they have not been described in the other chordates—the cephalochordates or vertebrates. Different mechanisms have evolved in
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vertebrate tissues such as the kidney, avian salt glands and teleost gills for example. All these have intercellular clefts closely associated with energyproducing mitochondria, but definitive scalariform junctions have not been reported. The virtue of the scalariform junctions in the tissues described here is that they would hold the cells’ plasma membranes a very precise distance apart, preventing collapse of the intercellular cleft, during crucial fluid flow. That these spaces retain their integrity during fluid transport has been demonstrated by Oschmann and Wall (1969), indicating that it is the columns which serve in the maintenance of this space which appears to be so important for the adequate functioning of the cells that possess them. When bulk fluid flow out of a tissue lumen produces so rapid a reduction in luminal volume that the intercellular spaces are subjected to pressures tending to collapse them, it could be argued that the contractile muscular tissues that invest them will act to maintain normal pressure and hence tissue and cleft integrity. However, in the tunicates, the tissues involved in fluid transport with scalariform junctions are not invested with muscular tissue. This is absent all along the gut and the scalariform junctions could be assumed to be required to prevent collapse of the intercellular cleft. In an attempt to speculate as to why the scalariform junctions are not found in vertebrate tissues, one might suggest that perhaps two different systems have evolved to perform a similar function. Both arthropods and tunicates possess barriers against the external world, as their epithelia are sealed by means of septate and tight junctions respectively. Their blood flows in lacunae and/or vessels which are not sealed and hence lack a true endothelium, so that blood and the other fluids move freely around most of their bodily organs. This is different in vertebrates, which possess barriers against the external world; the blood flows inside a closed circulatory system, the vasculature, which is provided with its own endothelia sealed by tight junctions. In vertebrates, regulation of body fluids occurs through a more complex type of excretory organ (the kidney) whose activity is more finely controlled by the endocrine system. This kidney establishes a special relation with the inner closed system, the circulatory system, at the level of glomeruli and renal tubules. This different organization and the acquisition of more efficient systems over time could explain why the vertebrates have lost the scalariform junctions, which were presumably present in their ancestors and are maintained in tunicates which are chordates yet
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have tight junctions as well as scalariform junctions. Of course limited closed systems can also be present in particular organs of both invertebrates (such as the nervous system of arthropods and the cardio-pericardium vesicle of tunicates) and in vertebrates (blood–brain, blood–testis barriers, etc.). However, arthropods lack tight junctions except in their CNS and testis where blood–brain barriers and blood–testis barriers exist (Lane, 1981). The blood–brain barrier appears to be necessary for the efficient functioning of the CNS in insects and arachnids; these relatively sophisticated brains require an extracellular fluid in which the proportions of ions are carefully regulated to ensure the proper functioning of the nerve cells (Lane and Treherne, 1980; Lane et al., 1982). These speculations concerning scalariform junctions will be tested by immunocytochemical approaches in the future to test for the presence of enzymes such as ATPase. ACKNOWLEDGEMENTS We wish to acknowledge Mr C. Friso for the drawings in Fig. 18. R.D. and P.B. are grateful to Murst (40%) and CNR for financial support during the course of this work. N.J.L. is indebted to the Wellcome Trust, for support in the form of grants (# 032970/1.4U and 032970/Z/90/B). REFERENCES B MJ, G BL, 1968. Fine structural localization of adenosine triphosphate in the rectum of Calliphora. J Cell Sci 3: 17–32. B MJ, O JL, 1972. Transporting Epithelia. Academic Press. New York, London. B J, 1971. Ultrastructure and ion transport in gill epithelia of the crayfish, Astacus leptodactylus. Esch. Protoplasma 73: 177–190. B P, M C, 1975. Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous, endocrine and plicated cells. Z. Zellforsch. 158: 481–496. B P, D R, M GB, 1985. Scalariform junctions in the gut of tunicates. Biol Cell 54: 171–176. C DE, 1968. Fine structure of salt and water uptake in the land-crab Carcinus laberalis. Am Zool 8: 417–432. D R, A BA, 1990. Microtubular diversity in insect spermatozoa: Results obtained with a new fixative. J Struct Biol 103: 164–179. D R, M G, C F, C S C, 1985. Freeze-fracture study of the rectal pads in Stenophylax permistus McL. (Trichoptera). Boll Zool 52: 407– 420. F-M MA, C P, 1972. Un nouveau type de jonction: les jonctions scalariformes. Etude ultrastructurale et cytochimique. J Ultrastruct Res 39: 222–238.
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