Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates

Tissue & Ceil, 1995 27 (5) 545-553 © 1995 Pearson Professional Ltd.

Tissue&Cell

Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates N. J. Lane*, R. Dallait, G. B. Martinucci**, P. Burighel**

Abstract. The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role. Keywords; Gap junctions, intercellularjunctions, cytoskeleton, ultrastructure,freeze-fracture, Urochordata

Introduction In previous papers we have analyzed the intercellular junctions in a range of the lower chordate tunicates (Burighel et al., 1989, 1992; Lane et al., 1986, 1994; Martinucci et al., 1988, 1990, 1991). Tunicates are * Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK. 1 Department of Evolutionary Biology, University of Siena, Siena, 1-53100, Italy. ** Department of Biology, University of Padova, Padova, 1-35121, Italy. Received: 3 April 1995 Accepted: 25 May 1995 Correspondence to: Dr P. Burighe], Department of Biology, University of Padova, Via Trieste 75-35121 Padova, Italy. Tel: 0039 49 8276185; Telefax 0039 49 8276199.

marine filter-feeders, invertebrates which are phylogenetically close to vertebrates. With the latter they share a common pattern of intercellular junctions in epithelia, owing to the presence of tight junctions (TJ), gap junctions (GJ) and zonulae adhaerentes (ZA), although they also have associations, such as the scalariform junctions, considered typical of invertebrates (Burighel et al., 1985). T J, G J, and ZA are found in species representative of the three classes of tunicates, i.e. ascidians, thaliaceans and appendicularians. In the branchial basket of ascidians, ciliated cells of the fissures (stigmata) are seen to be joined by intercellular junctions arranged according to two different patterns. Both aplousobranch and 545

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phlebobranch ascidians have a typical apico-basal sequence of associated TJs, GJs and ZA, whereas the adjacent cells of stolidobranch ascidians lack ZA and have a relatively extended TJ (Martinucci et al., 1988). Moreover, in the branchial basket of all ascidians GJs are common. Every stigmatal cell has a single row of long cilia, whose rootlets insert on the lateral membrane both at the level of TJs and at ZA when present. In gut and branchial basket, GJs are characterized by a narrow 2 to 3 nm intercellular cleft and have in freeze-fracture replicas the characteristic appearance of vertebrate GJs, because their intramembranous particles (IMPs) fracture on the P-face with corresponding pits on the E-face; however, it is not uncommon for IMPs to fracture on the E-face with corresponding P-face pits, as occurs in arthropods (Lane, 1978). In addition, special characteristics are found in the GJs sited in close association with TJ and ZA on the lateral plasmalemma of ciliated stigmatal cells of aplousobranch and phlebobranch ascidians, as well as in the thaliaceans and pyrosomes (Burighel et al., 1992). These GJs exhibit a fuzzy mat on the cytoplasmic face of their junctional membranes, which seems to correspond to the 'close junction' described by Mackie et al. (1974) in the branchial basket of the ascidian Corella willmeriana. The authors have suggested a mechanical role for these junctions in addition to the more conventional one of intercellular communication. The main purpose of this paper is to better characterize these unusual GJs by means of thin sections and freeze-fracture replicas of fixed and unfixed tissues.

glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4, plus 1.5% NaC1 for 2 h at 4°C; in some cases, 1% colloidal lanthanum was added to the cacodylate buffer, using the tracer lanthanum as an extracellular negative stain and (2) 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, plus 2% sucrose, plus 1% tannic acid for 2 to 3 days at 4°C. Tissues were then embedded in Epon for thin-sectioning or frozen for the preparation of freezefracture replicas. For the former, tissues were washed, post-fixed in osmium tetroxide, stained en bloc in uranyl acetate, dehydrated through an ascending series of ethanols, and embedded in epoxy resin. For the latter, the tissues were washed and cryo-protected in glycerol at 10%, 20% and 30% in buffer for varying periods of time before freezing by plunging into Freon 22 cooled in liquid nitrogen. The material was then mounted in a Balzers freeze-fracturing device (BAF 301 or BA 360 M models) and fractured at -115°C and at 2 x 10 -6 Torr (1 Torr = 133.3 Pa). The specimens were shadowed with platinum-carbon or tungsten-tantalum and backed with carbon. The tissue was removed with sodium hypochlorite or biological detergent, the replicas washed in water and mounted on copper grids for examination in a Philips EM 300 or Hitachi H 600 electron microscope at 60 or 80 Kv. Micrographs are mounted with the shadow coming from the bottom or side. Some pieces of colony and tissues were rapidly frozen, without prior chemical fixation, by plunging directly into liquid propane (K.F80). These were transferred to liquid nitrogen and then fractured in the Balzers freezefracturing device as before.

Materials and methods

Results

The animals examined in our study belonged to the tunicates (phylum Chordata, subph. Urochordata or Tunicata) including the ascidians Ciona intestinalis, Ascidiella aspersa (Ascidiacea, Phlebobranchia), Diplosoma listerianum (Ascidiacea, Aplousobranchia) and Botryllus schlosseri (Ascidiacea, Stolidobranchia) as well as the thaliaceans Pyrosoma atlanticum (Thaliacea, Pyrosomatida) and Doliolum nationalis (Thaliacea, Doliolida). The specimens of ascidians were collected from the lagoon of Venice (Italy), those of thaliaceans were collected in the Tirrhenian Sea next to the bay of Villefranche-sur-mer (France). Live animals were fixed immediately after collection, or kept for 24 h in tanks of circulating sea-water before use. The tissues studied were the branchia and the gut; in the case of small colonial species, pieces of each colony, containing whole zooids, were processed for electron microscopy, while in forms with large zooids (such as Ciona intestinalis), pieces of the branchial basket and gut were selected and cut into small pieces with scissors. The zooids and tissues were treated with a variety of fixatives, of which the most successful were: (1) 1.5%

The GJs in the gut tract of a range of tunicates are conventional in appearance in both thin sections (Figs 1-4) and in freeze-fracture replicas (Figs 5-7). In the former, they exhibit the characteristic close apposition of the junctional membranes in two adjacent cells, with a gap of 2 to 3 nm which tends to stain only rather lightly (Figs 1 and 2) and is only slightly enhanced after staining with lanthanum (Fig. 4). The cytoplasmic surface may display some slight density (Figs 1 and 2) which is reinforced after tannic acid staining (Fig. 3). In replicas, they fracture to reveal typical plaques of P-face particles which may cleave to show the overlying E-face of the adjacent cell, with its complementary EF pits (Fig. 5). When frozen rapidly without fixation, the individual connexons in a plaque and the EF pits can be seen rather more clearly (Figs 6 and 7). In the branchial baskets of most tunicates, with the exception of stolidobranch ascidians such as Botryllus schlosseri, there are striking arrays of apical adhaering junctions beneath the cilia, to which the ciliary rootlets are attached (ZA in Fig. 8). Between these ZA are found GJs (arrows in Figs 8 and 9). These exhibit the

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Figs 1 to 4 Thin sections of normal gap junctions (GJs) from the gut of Tunicates. Fig. 1 Pyrosoma atlanticum (thaliacean) gut, fixed in the conventional way; x 120000. Fig. 2 Diplosoma listerianum (ascidian) gut, fixed in the conventional way; x 120000• Fig. 3 Diplosoma listerianum (ascidian) gut, fixed with tannic acid; x 120 000. Fig. 4 Botryllus schlosseri (ascidian) gut, infiltrated with colloidal lanthanum (La 3+) and fixative; x 150 000. Figs 5 to 7 Freeze-fracture replicas from tunicate tissue. Fig. 5 Ascidiella aspersa (ascidian) gut with usual macular GJs; these display P-face connexon particles (PF), E-face pits (EF) and the characteristic reduced intercellular cleft (arrow); x 95 000. Fig. 6 Botryllus schlosseri (ascidian) branchial basket showing cells from the outmost part of its component stigma when parietal cells abut against the ciliated stigmatal cells. Here there are only 'normal' GJs, as are present in the gut. This preparation was ClyO-fixed by fast freezing with propane, and shows the individual PF connexons on the P-face (PF). These GJs lie in intimate association with the strands of TJ with their E-face (EF) grooves. These organisms do not exhibit any ZA and lack the unusual GJs found in the other orders of tunicates; x 150 000. Fig. 7 Diplosoma listerianwn (ascidian) gut with macular GJs in intimate association with tight junctional strands on the periphery of each plaque; the former exhibit the conventional fracturing with EF pits (EF). The preparation was cryo-fixed by fast freezing with propane; x 120000.

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conventional 2 to 3 nm intercellular cleft, but feature unusual paramembranous dense mats on their cytoplasmic surfaces (arrows in Fig. 8). When examined after tannic acid staining, they show the fibrous nature of this detise mat and also reveal a density in their intercellular cleft (insert in Fig. 9 and Fig. 10). The paramembranous density suggests an association with cytoskeletal components, which is not normal for GJs, and the density of the intercellular cleft suggests an enhanced stickiness of the extracellular adhering molecules lying within the cleft. Also freeze-fracturing indicates the placement of these unusual apical GJs between the areas of ZA (Figs 11-17), and underneath the apical TJs (Figs 12, 13 and 17). The TJs exhibit the characteristic rows of P-face particles (Figs 13 and 17) or E-face grooves (Fig. 12), into which a few IMPs may cleave. The ZA feature no typical membrane profile, and display either many IMPs (Fig. 16), no IMPs (Fig. 13) or a few IMPs (Figs 11, 14, 15 and 17). This concurs with the suggestion that these junctions are unlikely to feature intramembranous insertion of the associated filaments which would result in a more consistent freeze-fracture appearance of many intramembranous particles. The unusual GJs, that occur on the apical border beneath the TJs and between the adhaering junctions, also exhibit unusual freeze-cleaving properties. Frequently their conjoined membranes adhere so firmly that the E-face of one plasma membrane remains attached to the P-face of the underlying cell's plasma membrane (Figs 13-16). This reflects the enhanced adherence between the adjacent junctional cell membranes as indicated by the enhanced density seen after tannic acid staining in sections. In some cases, remnants of the P-face of the half membrane leaflet above is also adherent to the E-face (Fig. 12) and in other cases the shared connexons of the junctions can be seen from the side, beneath the overlying E-face (arrows in Fig. 14). In yet other instances, there are fragments of cytoplasm (also containing, it is presumed, the E-face of the membrane) from the region adjacent to the junctional membrane, which continue to adhere to the P-face. It must be supposed that these cytoplasmic fragments (arrowheads in Figs 13, 15 and 16) contain the dense fibrous mat associated with the cytoplasmic surface of these unusual GJs, which serve to maintain the integrity of the cell-cell associations. Only rarely do GJs fracture

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apart to reveal the typical P-face connexons (as in Fig. 17).

Discussion and conclusions GJs with the typical appearance of junctional membranes of two adjacent cells with a gap of 2 to 3 nm have been detected in different tissues of all tunicates examined so far (Lorber and Rayns, 1972, 1977; Georges, 1979; Cavey and Wood, 1986; Lane et al., 1986; Martinucci et al., 1988, 1990; Burighel et al., 1992). These GJs have always been localized in the basal part of the stigmatal cells, a region probably less implicated in the mechanical stress of the ciliary beating. It is likely that these GJs serve mainly for cell-cell communication, as they share the morphological characteristics of those in other tissues (Zampighi and Simon, 1985; Musil, 1994). In the branchial basket of two ascidian orders, however, (namely Aplouso- and Phlebobranchia) stigmatal cells possess two kinds of GJs along the same cell surface: those located in the deepest part of the cell have the conventional appearance, while those just beneath the apical TJs and between the ZA show a dense mat of fibrous material on the cytoplasmic surfaces of the two opposing junctional membranes. The presence of two kinds of GJ in the branchial stigmatal cells seem to be the rule in the phylum, as all phlebobranch and aplousobranch ascidians, as well as pyrosomatid and doliolid thaliaceans, share this uncommon feature. Why the stolidobranch ascidians do not possess the unusual GJs present in the other two groups is still a matter for debate. We may speculate that this could depend on the presence in this group of a deeper and more elaborate TJ along the apical cell junctional contact; This could functionally substitute for the ZA and the fibrous GJ, both lacking in this order of ascidians (Martinucci et al., 1988). In the conventional gap junctional structure, lanthanum passes apparently freely, through the narrow intercellular cleft and no appreciable or consistent density is visible on the cytoplasmic faces of the two apposed membranes at the junctional level. In freeze-fracture replicas of ascidian tissues, the normal appearance of connexon particles is that typically described in many other vertebrates and invertebrates. The plaques of particles are associated with the P-face

Figs 8 to 10 Thin sections through the branchial basket of tunicates showing the area where the zonulae adhaerentes (ZA) alternate with GJs; both exhibit a paramembranous dense mat, but such a dense mat is not normally seen in typical GJs. Fig. 8 DipIosoma listerianum (ascidian). The ZA display a considerable cytoplasmic density where the ciliary rootlets (CR) insert; this paramembranous density is somewhat greater than that found associated with the GJs (arrows); x 84 000. Fig. 9 Pyrosoma atlantieum (thaliacean) also exhibits paramembranous densities at both the ZA and GJs (arrows). The density of the latter is well demonstrated by the addition of tannic acid to the fixative as shown in the insert (arrowhead). Here the adhering 'sticky' material in the intercellular cleft is also clearly shown. Fig. 9 x 105 000; insert Fig. 9 x 125 000. Fig. 10 A higher magnification of the branchial basket of Ciona intestinalis (ascidian) after tannic acid treatment, displaying both the paramembranous cytoplasmic density of the GJ as well as the dense intercellular cleft material; x 500 000.

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Figs 11-13 Freeze-fracture replicas of chemically-fixed branchial baskets from tunicates. Fig. 11 Pyrosoma atlanticum (thaliacean) showing the unusual GJs (arrows), in elongated arrays between the ZA. This is a region of the P-face but it displays plaques of E-face membrane half (EF) with associated pits, overlying the regions of P-face gap junctional particles, while the adjacent TJs are fractured to reveal the P-face (PF) only; x 78 000. Figs 12 and 13 Ciona intestinalis (ascidian) branchial basket showing the narrow elongated regions of the unusual GJs (arrows) alternating with the ZA. Fig. 12: here the TJs appear as particles on the P-face (PF) and grooves on the E-face (EF). GJs (arrows) are seen as EF pits except for a few regions (arrowhead) where the PF connexons of the neighbouring cell's GJs are adhering to this cell's junctional plaque. Fig. 13: here the P-face (PF) is revealed by particle rows of TJs and regions of GJ where fragments of overlying membrane and cytoplasm (arrowheads) are adhering to this underlying cell's P-face. The TJs are arranged in arch-like configurations with strands penetrating into the underlying region of ZA and GJs; Fig. 12. x 63 000; Fig. 13. x 110 000.

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and they often cleave to show the complementary pits on the overlying E-face of the adjacent cell membrane (Musil, 1994). This normal appearance of ascidian tissues does not change after rapid freezing with propane in the absence of any chemical fixative. On the other hand, the unusual GJs described here are distinct in several respects in comparison with the conventional GJs. Firstly, they exhibit a dense mat on their cytoplasmic surface which is enhanced in density after tannic acid treatment showing its fibrous nature. Secondly the intercellular cleft, after this treatment, becomes more electron opaque (Fig. 10). These two lines of evidence support the view that cytoskeletal elements are indeed associated with this kind of junction and that the two apposing membranes are somehow 'glued' via the intercellular cleft to maintain junctional integrity. The unusual presence of dense material on the cytoplasmic surface of the GJs was mentioned as early as 1974 (Mackie et al., 1974). These GJs also have uncommon features in freeze-fractured material. The two opposite membranes are so firmly adherent that the E-face of one plasma membrane remains attached to the P-face of the underlying one, a fact that has never been observed in the conventional GJs present in other neighbouring regions of the same cell. It may be that this 'stickiness' observed upon freeze-cleaving is due to the firm association of the two adjacent plasma membranes, presumably caused by the dense material in the intercellular cleft and the association of cytoskeletal elements with the cytoplasmic faces of the junctional plasma membranes. The mechanism of connection of the cytoskeleton to the membrane at the gap-junctional level is not yet understood. Previous work with deep-etching has shown that cytoplasmic surfaces of gap junctions are smooth (Hirokawa and Heuser, 1982). It has been suggested, however, that assembly of gap junctions may involve migration of molecules whose position within the membrane is controlled by perturbations of the cytoskeleton (Tadvalker and Pinto da Silva, 1983); after assembly, the GJs may become free from cytoskeletal control. Thus, connexon distribution and movement in the membrane could be mediated by cytoplasmic filaments as supported by the data obtained by Green and Severs (1984) in rat cardiac muscle after ultra-rapid freezefracture treatment. Therefore, it seems that, at least in conventional GJs, the apparent disagreement between the presence or absence of cytoskeletal structures associated with gap junctional membranes may depend on the time observations are made. Gap junctional formation or disassembly must be a fairly rapid process whose initiation may require the presence of cytoskeletal elements; once connexons are assembled in patches, however, perhaps the cytoskeleton is no longer associated, certainly it is no longer visible (Hirokawa and Heuser, 1982; Tadvalker and Pinto da Silva, 1983). In the unusual gap junctions of ascidians and thaliace-

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arts, filamentous material is permanently found associated with the junctional membrane. The data may be telling us that the turnover of association and dissociation of cytoskeletal elements, which perhaps occurs normally, does not take place in these types of GJs. One may speculate on this point and suggest a particular function of this kind of GJ, possibly involved in both intercellular communication and mechanical adhesion of adjacent membranes. The former function could be required for a rapid passage of the signal relating to the synchrony of ciliary beating and the type of innervation formed by a simple synaptic bouton in nearly all of the ciliated cell clusters of the branchial basket (Arkett et al., 1989). The mechanical function, due to the presence of the dense mat associated with the junctional membranes and the cytoskeleton, could be required to enable the cells to cope with the physical stress of the active ciliary beating. An interesting point to be verified would be whether all connexons constituting these unusual GJs have a similar structure and play the same function or, whether instead, a specialization occurs among them. Recent work on the molecular structure of connexons (Stevenson and Paul, 1989; Musil, 1994) and their variability in terms of the molecular weight of their connexin components (Finbow et al., 1983, 1984; Nicholson et al., 1987; Ryerse, 1989; Musil, 1994) raised the question whether different connexons, possibly with different functional significance, may occur within the same GJ. If so, it could be suggested that some connexons are involved only in the transitory attachment to the cytoskeleton. Under this hypothesis the GJs could assume a twofold role; they would serve not only to allow the exchange of ions and small molecules between cells, but also for the mechanical adhesion of the two opposite plasma membranes, through the participation of the cytoskeleton in order to avoid modification of the cell shape. This hypothesis is in line with the recent suggestion that cell-cell junctions in epithelia are not autonomous structures, but rather part of an interrelated dynamic network with both mechanical and signalling functions (Musil, 1994). For example, a similar function has been postulated for the follicle cells of the stick insect during the vitellogenetic growth phase, when coordination is required of cell differentiation at the same time as the mechanical control of cell-cell arrangements in the growing oocyte (Mazzini and Giorgi, 1985) is needed. ACKNOWLEDGEMENTS We would like to thank Dr R. Fenaux for help in furnishing some specimens of Doliolum and Pyrosoma, Mr P. Salvatici and Mr V. Miolo for skilful technical assistance, and MURST and CNR for grants to R.D. and P.B. during the course of this research. We are also indebted to the Wellcome Trust for grants to N.J.L. (032970/1.4U and 032970/z/90/B).

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Figs 14-17 Freeze-fracture replicas of branchial basket. Figs 14 and 15. Pyrosoma atlanticum (thaliacean) showing the region of unusual GJs and intervening ZA. These replicas are of the P-face (PF), except that in the gap junctional regions the E-face (EF) adheres to the junctional plaques owing to the enhanced adherence in these regions. To one side of the plaques of EF pits can be discerned the junctional PF connexons (arrows in Fig. 14). In some cases (Fig. 15) small areas of the P-face connexons (PF) are revealed as well as regions of cytoplasm (arrowheads in Fig. 15) where the overlying cell's membrane plus cytoplasmic mat remain adhaerent, sticking to the P-face; Fig. 14 x 132 000; Fig. 15 x 157 000. Fig. 16 Doliolum nationalis (thaliacean) branchial basket. Here the P-face (PF) is exposed with particle rows of TJ except that in the region of the unusual GJs, the E-face (EF) adheres, showing its junctional pits. In a few places (arrowheads) the overlying cell's membrane plus cytoplasmic mat also remain adherent to the P-face. Note the large number of P-face IMPs lying in the ZA sites, in contrast to the situation in Pyrosoma (Fig. 11 ) or Ciona (Fig. 13) where there are fewer or essentially no PF IMPs in the ZA region; x 65 000. Fig. 17 Ciona intestinalis (ascidian). The micrograph shows the tight junctional belt and associated GJs which lie between deeply invaginated areas of the ZA. In this preparation, unusually, the PF particles of the elongated GJs (arrows) are revealed; x 65 000.

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