Cell and Tissue Research

Cell Tiss. Res. 174, 55-67 (1976)

9 by Springer-Verlag 1976

The Fine Structure of Polychaete Septate Junctions * Denis G. Baskin** Department of Zoology, Pomona College Claremont, California, USA

Epidermal septate junctions of Nereis sp. and Cirriformia sp. fixed with OsO 4 or glutaraldehyde/OsO4 display variable structure in electron micrographs. In transverse section the septa are often indistinct and obscured by opaque material that fills the junctional cleft. Septa (spaced at 180-280 .~) are more clearly defined in slightly oblique transverse section; they exhibit an electron lucent center and appear to be linked by arms. En face views of the junction show a honeycomb pattern. Cytoplasmic faces of junctional membranes are backed with plaques opposite the septa. Lanthanum used as a tracer delineates junctional structure in negative contrast. In transverse section a chain-like lattice is present in the junctional cleft. En face views show parallel rows of pleated elements often linked by arms into honeycomb arrays. Oblique sections demonstrate that these pleated elements are continuous with the chain-like lattice seen in transverse sections. Lanthanum does not pass entirely through the junction. Lanthanum reveals that the septa have a very intricate substructure, but it is difficult to visualize the architecture that could generate the various images presented by these junctions when seen in different orientations. However, it is clear that these junctions possess some features that are diagnostic of several supposedly different types of septate junctions in invertebrates. Summary.

Septate junctions - Electron microscopy.

Key words:

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Polychaeta

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Fine structure

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Cell junctions

S e n d offprint requests to : Dr. Denis G. Baskin, Department of Zoology, Pomona College, Claremont,

California 91711, USA * Supported by USPHS grants NIH 5 P01 NS-07512, NIH 2701 GM-00102, and NB-00840, and by a grant from the Pomona College Research Committee ** I thank Sarah Wurzelmann, Stanley Brown, Nancy Kelly, and Gerhard Ott for excellent technical assistance. Portions of this study were carried out while I was a Postdoctoral Fellow in the Department of Anatomy, Albert Einstein College of Medicine. I dedicate this article to Berta Scharrer as a token of appreciation and affection for her guidance, encouragement, inspiration, and example of excellence

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Introduction This article describes the j u n c t i o n a l structures o f epithelial cells f r o m the e p i d e r m i s o f several p o l y c h a e t e s , with p a r t i c u l a r focus o n septate j u n c t i o n s . S e p t a t e j u n c t i o n s h a v e b e e n n o t i c e d b y investigators interested in the fine structure o f the p o l y c h a e t e e p i d e r m i s ( S t o r c h a n d Welsch, 1970, 1972; B o i l l y - M a r e r , 1972) b u t relatively little a t t e n t i o n has b e e n d i r e c t e d t o w a r d s cell j u n c t i o n s in these organisms. S e p t a t e j u n c t i o n s , o r " s e p t a t e d e s m o s o m e s " ( W o o d , 1959), are z o n u l a r belts at the a p i c o l a t e r a l m a r g i n s o f m a n y i n v e r t e b r a t e epithelial cells. A l t h o u g h this t e r m o r i g i n a l l y referred to a j u n c t i o n with electron o p a q u e b a r s (the " s e p t a " ) s p a n n i n g the i n t e r c e l l u l a r cleft, recent studies i n d i c a t e t h a t it has been a p p l i e d to at least two, a n d p o s s i b l y three, distinctive types o f cellular j u n c t i o n s (reviews b y Satir a n d G i l u l a , 1973; Staehelin, 1974; Gilula, 1974): (a) the p l e a t e d o r h o n e y c o m b type, in w h i c h t a n g e n t i a l sections reveal p a r a l l e l rows o f p l e a t e d s e p t a c o n n e c t e d b y crosslinks (Locke, 1965; D a n i l o v a et al., 1969; G i l u l a et al., 1970; N o i r o t - T i m o t h 6 e a n d N o i r o t , 1973); (b) the c o n t i n u o u s o r s m o o t h type, in w h i c h the s e p t a a r e either t r a n s p a r e n t ( H u d s p e t h a n d Revel, 1971) o r o b s c u r e d b y e l e c t r o n o p a q u e m a t e r i a l in the intercellular cleft ( N o i r o t a n d N o i r o t - T i m o t h 6 e , 1967; F l o w e r a n d Filshie, 1975); (c) the t y p e f o u n d in H y d r a , in w h i c h the s e p t a a p p e a r to be a c o m p l e x h e x a g o n a l lattice ( H a n d a n d G o b e l , 1972). T h e p r e s e n t s t u d y , w h i c h used l a n t h a n u m to d e l i n e a t e septal fine structure, reveals t h a t these p o l y c h a e t e septate j u n c t i o n s have a n intricate s u b s t r u c t u r e and, a l t h o u g h they r e s e m b l e the p l e a t e d type, they also show features t h a t are d i a g n o s t i c o f o t h e r types. A p r e l i m i n a r y r e p o r t o f these results has a p p e a r e d elsewhere (Baskin, 1976).

Materials and Methods Animals. Nereis limnicola, Nereis succinea (Nereidae); Cirriformia luxuriosa (Cirratulidae). Conventional Fixation. Small pieces of body wall (nereids) and segmental cirri (nereids, cirratulids) were fixed with 2-2.5~ glutaraldehyde in 0.1 M buffer (phosphate or cacodylate), pH 7.2-7.5, 1-2 h, either at room temperature or 4~C, followed by an overnight wash in the same buffer at 4~C. CaC12 (0.001 M) was added to the cacodylate solutions. Postfixation was with 1~ OsO4 in 0.1 M buffer (veronal acetate or cacodylate), pH 7.4, 1 h, 4~C. Some specimens of N. limnicola were fixed only with 1~ OsO 4 in 0.1 M phosphate buffer, pH 7.2, 2h, room temperature. Lanthanum Fixation. Fixative solutions containing colloidal lanthanum hydroxide were prepared

according to Revel and Karnovsky (1967). The final solutions contained 2-2.7~ glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3 7.5, 0.2 M sucrose, 0.001 M CaC12. Fixation was at room temperature for two h followed by an overnight wash at 4~ in buffer with 0.2 M sucrose. Postfixation was with 1~ OsO4 in the same buffer with 0.2 M sucrose at 4~C, 1 h. Lanthanum was added to the buffer wash and osmium solutions. Microscopy. Tissues fixed in glutaraldehyde and osmium were dehydrated in ethanol/propylene

oxide and embedded in Epon or an Epon-Araldite mixture. Tissues fixed in osmium alone were dehydrated in acetone and embedded in Maraglas. Sections cut with diamond knives and poststained with uranyl acetate and lead citrate (lanthanum-infiltrated specimens were stained only in lead) were photographed in an RCA EMU 3 G or Siemens Elmiskop ! A.

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Results

The fine structure of the epidermis is fundamentally similar in all polychaete groups that have been examined (Br6kelmann and Fischer, 1966; Storch and Welsch, 1970, 1972; Michel, 1972; Boilly-Marer, 1972). Epidermal cells are columnar epithelial elements that rest on a basement membrane and are covered by a cuticle of varying thickness. The cells interdigitate extensively, particularly in the apical regions occupied by junctional complexes (Fig. 1). The junctional complex consists of a zonula adhaerens and a septate junction. The zonula adhaerens is subjacent to the opening of the intercellular cleft to the cuticle; the cytoplasmic surfaces of its junctional membranes are coated with an

Fig. 1. Epidermis of N. limnicola, showing cuticle (C) and junctional complex (JC). x 17,000 Fig. 2. Junctional region of epidermal cells, showing zonula adhaerens (ZA) and septate junction (S J) with indistinct septa. N. limnicola x 90,000 Fig. 3. Junctional region of epidermal cells, showing zonula adhaerens (ZA) and septate junction (S J) with distinct septa. N. limnicola, x 120,000 Fig. 4. Septate junction (SJ) with opaque intercellular material, and desmosome-like junction (D). N. limnicola x 100,000 Fig. 5. Close membrane appositions resembling gap junctions. N. limnicola x 90,000

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Fig. 6. Plaques of electron opaque cytoplasmic substance (arrows) opposite septa revealed in obliquely sectioned junction. Cirriformia x 120,000 Fig. 7. Oblique section of septate junction fixed in OsO4 alone. Note electron lucent "cores" of septa (opposite arrows) and electron opaque strands connecting septa. N. limnicola x 165,000 Fig. 8. En face view of septate junction, showing honeycomb pattern of electron opaque septa. N. limnicola x 90,000

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Polychaete Septate Junctions

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electron opaque material (Figs. 2, 3). The most prominent feature of the junctional complex is a septate junction, which is located below the zonula adhaerens. The septate junction extends to a depth of several microns and exhibits a variable morphology. The epidermal cells also display a desmosome-like junction (Fig. 4). The latter contacts lack the laminated intercellular matrix that is typical of desmosomes in other polychaete tissues such as the glial cells of the nereid central nervous system (Baskin, 1971). Membrane appositions that resemble nexuses are abundant (Fig. 5), but further study with electron opaque tracers and with freeze-fracturingis required to confirm the status of these contacts as gap junctions. The septate junction presents variable images in electron micrographs. The septa are often indistinct, particularly in very thin (silver-gray) sections (Fig. 2), whereas thicker sections (light gold) frequently display electron opaque septa spanning the 180-200 A junctional cleft (Fig. 3). The septa are usually spaced at regular intervals. Although the periodicity varies from 180-200A, it is quite constant within a single junction. In some specimens the junctional cleft is uniformly filled with a fine-grained electron opaque substance and no septa are visible (Fig. 4). Junctions that are apparently devoid of intercellular material often show septa when sectioned slightly oblique to a transverse plane. These oblique sections frequently reveal electron opaque material on the cytoplasmic surface of the junctional membranes; this substance sometimes appears to be organized into plaques opposite the septa (Fig. 6). Favorable oblique sections show an electron lucent "core" in the septa, which also appear to be connected by strands that are coplanar with the junctional membranes (Fig. 7). Tangential sections of the uranyl-lead stained junction reveal faint, parallel pleated elements linked into a honeycomb pattern (Fig. 8). Colloidal lanthanum penetrates the cuticle and enters the septate junction to a depth of several microns (Fig. 9); it does not, however, pass completely through the junction to the basement membrane. The lanthanum delineates junctional substructure in negative contrast. In transverse sections of the septate junction, the lanthanum reveals images of unstained bands that span the intercellular cleft and are confluent with the electron lucent portion of the junctional membranes (Fig. 10). In the cleft region the tracer outlines delicate unstained strands that are roughly parallel to the junctional membranes and sometimes have the configuration of a chain-like lattice (Fig. 11). En face views of the lanthanum-infiltrated junction show transparent pleated elements (Fig. 12). Some tangential sections clearly show that these pleated elements are confluent with the electron lucent lattice observed in Fig. 9. Lanthanum-infiltrated junctional complex, showing cuticle (C) and deep penetration of lanthanum. Note substructure (arrows'). Cirriformia x 40,000 Fig. 10. Detail of lanthanum-infiltrated junction, showing septa in negative contrast (arrows), and electron lucent strands in junctional interspace oriented parallel to plasma membranes. N. succinea x 120,000 Fig. 11. Chain-like substructure (arrows) revealed in negative contrast in lanthanum-infiltrated septate junction. Note microtubule (M). N. succinea x 195,000

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Fig. 15. Patterns presented by septa revealed in lanthanum-infiltrated junction viewed en face. Cirriformia x 180,000 Fig. 16. Structure of septa revealed in lanthanum-infiltrated junction sectioned en face. Cirriformia x 215,000

Fig. 12. En face view o f lanthanum-infiltrated septate junction. N. succinea x 60,000 Fig. 13. Lanthanum-infiltrated junction rotating in plane of section, from transverse (t) to frontal (/). N. succinea x 1 |0,000 Fig. 14. Oblique section of lanthanum-infiltrated septate junction, showing complex chain-like substructure of septa. Continuity between septa seen in transverse section and pleated elements seen in frontal section of junction shown clearly at arrow. Cirriformia x 180,000

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transversely sectioned junctions (Fig. 13). These electron lucent structures, seen both in transverse and tangential section, seem to have a delicate latticelike substructure (Fig. 14). Furthermore, the pleated elements vary in width in electron micrographs. Some are tenuous, thorny strands with arms projecting from alternate vertices; these arms appear to link the pleated elements into an hexagonal lattice (Figs. 12, 14, 15). Others are wider and possess a chain-like substructure (Figs. 15, 16). Although most of these pleated strands are organized into gently curving, parallel arrays, some are isolated and some curve sharply (Fig. 16).

Discussion

This study suggests that septate junctions possess a substructure that is perhaps more intricate than previously appreciated. In most details, the polychaete septate junction conforms to the pleated type, although certain features of the other types are present: (a) images presented by lanthanum-infiltrated junctions are similar to those characteristic of the Hydra type; (b) the lack of distinct septa in some specimens is characteristic of the continuous type. It is difficult to visualize the architecture that could generate the various images presented by these junctions in this study. The variations in thickness presumably result from the projection of images presented by a thin, fencelike structure, depending upon the plane of section. Since the junctional cleft is 180-200 A wide, and the septal spacing is of similar dimensions, superimposition of structures is also a serious obstacle to interpreting these images, even when the sections are only 400-500 A thick. No previously published model of septate junction structure (Locke, 1965; Bullivant and Lowenstein, 1968; Leik and Kelly, 1970; Hand and Gobel, 1972; Flower, 1971; Gilula et al., 1970; Satir and Gilula, 1973; Staehelin, 1974; NoirotTimoth6e and Noirot, 1973; Flower and Filshie, 1975; Caveney and Podgorski, 1975) is completely satisfactory in this respect, although each has some features that are consistent with the images presented by these polychaete septate junctions. Perhaps most of these models are oversimplifications, particularly in that they view the septal elements as solid structures. The present study suggests that the septa are probably a delicate lattice of fine, filamentous components. However, it is difficult to understand how an open, chain-like structure could be seen in negative contrast if it is embedded in opaque material such as lanthanum. Although most models of the pleated type septate junction view the septa as partitions, there is in fact little firm evidence for that idea. An alternative hypothesis is that the "septa" seen in uranyl-lead stained sections represent the stained edges of surface particles that are arranged in an hexagonal lattice. In this respect it may be useful to compare septate junction membranes with the luminal membranes of urinary bladder epithelium, which exhibit arrays of hexagonally arranged surface particles that are of the same dimensions as the hexagonal subunits in these polychaete junctions, and are believed to provide a permeability barrier and structural rigidity (Hicks et al., 1974). The pleated type septate junction may represent complementary plaques of similar surface particles. Since it seems likely that lanthanum may stain surface components rather than fill extracellular

PolychaeteSeptate Junctions

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void (Doggenweiler and Frenk, 1965; Lesseps, 1967; Overton, 1968; Behnke, 1968), the pleated elements seen in lanthanum-infiltrated junctions may represent unstained spaces between hexag0nally arranged subunits. Furthermore, in their dimensions and arrangement, the hexagonal elements stained by the lanthanum correspond to the arrays of membrane particles seen in freeze-fracture replicas of septate junctions from molluscan epithelia (Gilula et al., 1970; Flower, 1971 ; Gilula, 1974). Presumably; the lanthanum-stained elements and the membrane particles are integral components of the junctional membranes, but our understanding of their relationship to each other, and to the "septa" seen in uranyllead stained sections, is vague. The problem of septate junction structure might benefit from examining isolated, negatively-stained septate junctions with optical filtering and linear integration techniques. The functions of septate junctions are not well understood. It is now generally believed that septate junctions probably are not low resistance pathways (Gilula, 1974; Staehelin, 1974; Caveney and Podgorski, 1975). Whatever their principal function may be, however, these junctions undoubtedly provide mechanical attachment and cell to cell :adhesion. Although the polychaete septate junction is partially permeable to lanthanum, the tracer does not pass entirely through it; this suggests that the septate junction may have a barrier function analagous to a zonula occludens. However, since the lanthanum is applied with the fixative, it does not reflect junctional permeability under physiological conditions. It would be useful to examine the permeability of these junctions to less toxic tracers such as horseradish peroxidase, particularly since septate junctions of the kidney sac epithelium of Helix apparently restrict the passage of this molecule (Newell and Skelding, 1973). Further, Berridge and Oschman (1969) suggested that in the Malpighian tubules of certain insects, septate junctions may restrict the movement of substances in the intercellular space. The nereids offer good models to study possible functions of septate junctions in epthelial permeability to water and ions. Physiological studies indicate that the permeability of the body wall of estuarine nereids can be altered by changing the ionic composition and concentration of the medium (Smith, 1970; Oglesby, 1972; Fletcher, 1974). Therefore, changes in fine structure of a septate junction and its permeability to an electron opaque tracer such as peroxidase could be investigated under controlled physiological conditions. The structural basis for a barrier function is not known. Certainly, the long, tortuous pathway between cells could permit access by tracer molecules but restrict the passage of small molecules over a distance. Since they can be penetrated by tracers, the junctions probably provide only a partial, perhaps leaky, barrier. Hand and Gobel (1972) suggest that if the septa bear a weak electrical charge, a junction with thirty or more of these elements would present a considerable barrier to the passage of many substances. If the septa represent glycoprotein, as has been suggested (Noirot-Timoth6e and Noirot, 1973), the carbohydrate components, or perhaps certain amino acids such as proline, if oriented suitably, could order water molecules in such a way that junctional permeability is considerably reduced. In summary, the function of septate junctions in polychaete epidermis and elsewhere will not be satisfactorily understood until the structure is resolved in more detail.

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References Baskin, D.G.: The fine structure of neuroglia in the central nervous system of nereid polychaetes. Z. Zellforsch. 119, 295-308 (1971) Baskin, D.G.: Fine structure of polychaete septate junctions. (Abstract.) J. Ultrastruct. Res. 55, 294 (1976) Behnke, O.: Electron microscopic observations on the surface coating of human blood platelets. J. Ultrastruct. Res. 24, 51-69 (1968) Berridge, M.J., Oschman, J.: A structural basis for fluid secretion by Malpighian tubules. Tissue & Cell 1, 247-272 (1969) Boilly-Marer, Y.: Etude ultrastructurale des cirres parapodiaux de N6r6idiens atoques (Ann61ides Polych6tes). Z. Zellforsch. 131, 309-327 (1972) Br6kelmann, Y., Fischer, A.: Uber die Cuticula von Platynereis dumerilii (Polychaeta). Z. Zellforsch. 70, 131-135 (1966) Bullivant, S., Loewenstein, W.R.: Structure of coupled and uncoupled cell junctions. J. Cell Biol. 37, 621-632 (1968) Caveney, S., Podgorski, C.: Intercellular communication in a positional field. Ultrastructural correlates and tracer analysis of communication between insect epidermal cells. Tissue &Cell 7, 559-574 (1975) Danilova, L., Rokhlenko, K.D., Bodryagina, A.V.: Electron microscopic study on the structure of septate and comb desmosomes. Z. Zellforsch. 100, 101 117 (1969) Doggenweiler, C.F., Frenk, S.: Staining properties of lanthanum on cell membranes. Proc. nat. Acad. Sci. (Wash.) 53, 425-430 (1965) Fletcher, C.R.: Volume regulation in Nereis diversicolor. III. Adaptation to a reduced salinity. Comp. Biochem. Physiol. 47A, 1221-1234 (1974) Flower, N.E.: Septate and gap junctions between the epithelial cells of an invertebrate, the mollusc Cominella maculosa. J. Ultrastruct. Res. 37, 259--268 (1971) Flower, N.E., Filshie, B.K.: Junctional structures in the midgut cells of lepidopteran caterpillars. J. Cell Sci. 17, 221-239 (1975) Gilula, N.B.: Junctions between cells. In: Cell communication (R. Cox, ed.), pp. 1-29. New York: Wiley 1974 Gilula, N.B., Branton, D., Satir, P.: The septate junction: a structural basis for intercellular coupling. Proc. nat. Acad. Sci. (Wash.) 67, 213-220 (1970) Hand, A.R., Gobel, S.: The structural organization of the septate and gap junctions of Hydra. J. Cell Biol. 52, 397-408 (1972) Hicks, R.M., Kettere, B., Warren, R.C.: The ultrastructure of the luminal plasma membrane of the mammalian urinary bladder: A structure with low permeability to water and ions. Phil. Trans. B 268, 23-38 (1974) Hudspeth, A.J., Revel, J.P.: Co-existence of gap and septate junctions in an invertebrate epithelium. J. Cell Biol. 50, 92-101 (1971) Leik, J., Kelly, D.E.: Septate junctions in the gastrodermal epithelium of Phialidium: a fine structural study utilizing ruthenium red. Tissue & Cell 2, 435-442 (1970) Lesseps, R.J. : The removal by phospholipase C of a layer of lanthanum-staining material external to the cell membrane in embryonic chick cells. J. Cell Biol. 34, 173-183 (1967) Locke, M.: The structure of septate desmosomes. J. Cell Biol. 25, 166-169 (1965) Michel, C.: Etude ultrastructurale et histochimique des papilles de la gaine de la trompe de Notomastus latericeus Sars (Ann61ide Polych+te S6dentaire). Z. Zellforsch. 128, 482-503 (1972) Newell, P.F., Skelding, J.M.: Structure and permeability of the septate junction in the kidney sac of Helix pomatia L. Z. Zellforsch. 147, 31-39 (1973) Noirot, C., Noirot-Timoth6e, C.: Un nouveau type de jonction intercellulaire (zonula continua) dans l'intestin moyen des insectes. C.R. Acad. Sci. (Paris) 264, 2796-2798 (1967) Noirot-Timoth6e, C., Noirot, C.: Jonctions et contacts intercellulaires chez les insectes. I. Les jonctions sept+es. J. Microscopie 17, 169-184 (1973) Oglesby, L.C.: Studies on the salt and water balance of Nereis diversicolor. II. Components of total sodium efflux. Comp. Biochem. Physiol. 41A, 765-790 (1972) Overton, J.: Localized lanthanum staining of the intestinal brush border. J. Cell Biol. 38, 447-452 (1968)

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Revel, J.P., Karnovsky, M.J.: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33, C7-12 (1967) Satir, P., Gilula, N.B.: The fine structure of membranes and intercellular communication in insects. Ann. Rev. Entomol. 18, 143-166 (1973) Smith, R.I., Chloride regulation at low salinities by Nereis diversicolor (Annelida, Polychaeta). lI. Water fluxes and apparent permeability to water. J. exp. Biol. 53, 93-100 (1970) Staehelin, L.A.:Structure and function of intercellular junctions. Int. Rev. Cytol. 39, 191 283 (1974) Storch, V., Welsch, U. : I~ber die Feinstruktur der Polychaeten-Epidermis (Annelida). Z. Morph. Tiere 66, 310-322 (1970) Storch, V., Welsch, U.: Ultrastructure and histochemistry of the integument of air-breathing polychaetes from mangrove swamps of Sumatra. Mar. Biol. 17, 13%144 (1972) Wood, R.L.: Intercellular attachment in the epithelium of Hydra as revealed by electron microscopy. J. biophys, biochem. Cytol. 6, 343-352 (1959)

Accepted June 2, 1976

The fine structure of polychaete septate junctions.

Cell and Tissue Research Cell Tiss. Res. 174, 55-67 (1976) 9 by Springer-Verlag 1976 The Fine Structure of Polychaete Septate Junctions * Denis G...
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