Evidence for two separate categories of junctional particle during the concurrent formation of tight and gap junctions

JOURNAL OF ULTRASTRUCTURE RESEARCH 77, 54--65 (1981)

Evidence for Two Separate Categories of Junctional Particle during the Concurrent Formation of Tight and Gap Junctions NANCY J. L A N E

ARC Unit of lnvertebrate Chemistry & Physiology, Department of Zoology, Cambridge University, Downing Street, Cambridge CB2 3EJ, United Kingdom Received September 5, 1980, and in revised form April 24, 1981 The stages in the formation of vertebrate-like tight junctions and gap junctions, which coexist in tissues of the adult, have been followed in the CNS of young hatchling spiders. These junctions develop concurrently between the cells of the outer glial layer, or perineurium, that ensheathes the main ganglionic mass. In the mature state the two junctional types can be distinguished not only on the basis of the way in which their component intramembranous particles (IMPs) are arranged, but also by the size and fracturing characteristics of their respective IMPs. Hence they can be identified accurately during the initial and subsequent stages of their formation. The fully formed gap junctions, composed of clusters of 13-nm E-face (EF) IMPs, are found in the membranes between EF grooves that are complementary to a network of P-face (PF) ridges of 8- to 10-nm IMPs that compose the tight junctions. In hatchlings, the latter appear first as individual particles which become aligned initially in rows of 2 to 3. With time, the IMPs seemingly become fused together to form ridges, and the length of these particle rows is increased until they are arrayed as discontinuous strands scattered over the presumptive junctional area. The t3-nm EF IMPs, which are inserted between these tight junctional structures, are apparently restricted in their translateral movement by the strands of the latter which pinch the,adjacent membranes together. As the ridge-groove system becomes interconnected into a reticular network, the gap junctions coalesce into linear, then macular aggregates, always intercalated between the strands of the network. It is clear that these two junctions are composed of IMPs which do not share a common precursor particle as has been suggested for some vertebrate tissues, but which arise as two quite distinct particle populations. The mode of formation of these anastomosing tight junctions, hitherto not explored in invertebrate tissues, seems very similar to the general pattern of events reported previously for vertebrate zonulae occludentes. It therefore seems likely that, in chordates, these tight junctional particles also arise separately from those forming the gap junctions.

In recent years there have been a number of studies on the development and mode of formation of intercellular junctions. These have primarily been concerned with junctional formation in vertebrate tissues, often in cultured cells, and frequently have involved situations in which fight and gap junctions coexist and therefore develop simultaneously (Decker and Friend, 1974; Revel, 1974; Elias and Friend, 1976; Montesano et al., 1975; Decker, 1976a,b; Revel and Brown, 1976). In freeze-fractured preparations both these junctional types are characterised by clustering of the intramembranous particles (IMPs) into characteristic formations. However, in vertebrate tissues, with only a few exceptions such as

the Sertoli cells of the testis (Nagano and Suzuki, 1976a,b) and endothelial cells (Simionescu et al., 1978), the individual tight junctional and gap junctional IMPs, visible by freeze-cleaving, are indistinguishable; that is, both are within the same size range, and both fracture onto the same fracture face, the P face (PF) of the cell membrane. During the early stages of junction formation therefore, when the junctional IMPs are dispersed over the membrane P faces, they may all be described as particles of "ambiguous identity" (Larsen, 1977). It is, consequently, a matter of debate as to whether or not gap and tight junctions arise from a common intramembranous precursor particle. It seems that the vertebrate 54

0022-5320/81/100054-12502.0010 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

C O E X I S T I N G Y E T D I S T I N C T G A P A N D T I G H T J U N C T I O N A L IMPs

systems thus far studied cannot resolve the issue of the origin, distinct or common, of these two categories of junctional particles, in spite of the fact that in mature systems they appear to be involved in quite distinct physiological activities. It has recently come to light that vertebrate-like tight junctions exist in certain invertebrate tissues (Lane and Chandler, 1980; Lane, 1980a, 1981; Lane et al., 1981) although earlier investigators had commented on their apparent absence since no tight junctions had till then been reported in nonchordate organisms (Satir and Gilula, 1973; Noirot and Noirot-Timoth6e, 1976; Green et al., 1979; Noirot-Timoth6e et al., 1978). The convenience of such tight junction-possessing systems for studies on junctional formation is immediately apparent when the features of these invertebrate junctions are analysed. The tight junctions found in some arthropods are like those of vertebrates, in that they have been found to consist of a network of 8- to 10-nm PF particles, arranged as a circumferential occluding belt (Lane and Chandler, 1980). The arthropod gap junctions, however, are characterised by E-face (EF) particles which are aggregated into macular plaques and which are rather larger, about 13 nm in diameter (Flower, 1972, 1977; Lane and Skaer, 1980), than the tight junctional particles. Since the component particles of these two junctional types differ in both size and in fracturing characteristics in arthropods it seems possible that one could follow the joint paths of their formation in an unequivocal way in tissues where they coexist. Each junctional particle can be defined as being of one type or the other with almost complete certainty. An analysis of the development of the nervous system of several genera of hatchling arachnids has therefore been undertaken since the outer glial layer, the perineurium, in spiders, consists of cells associated with one another by both vertebrate-like tight junctions and intercalated gap junctions (Lane and Chandler, 1980). The various stages in the con-

55

current formation of these two junctions have been followed in these tissues and it is here shown that it is possible to distinguish them throughout their development as two quite distinct categories of IMP. A preliminary abstract of these results has been published elsewhere (Lane, 1980b). MATERIALS AND METHODS The central n e r v o u s s y s t e m or f u s e d ganglionic m a s s from newly h a t c h e d spiders (Arachnida: Araneida) of the genera A r a n a e u s and Tegenaria was dissected out after flooding with fixative. In s o m e cases the tissue was not fixed but placed directly in spider Ringer (Lane and Chandler, 1980), to which cryoprotectant was added, before freezing. More conventionally the tissue was fixed in one of a variety of fixatives such as 2 to 2.5% glutaraldehyde in 0.1 M cacodylate or p h o s p h a t e buffer, p H 7.4, plus 0 . l - 0 . 1 5 M NaC1 and a few drops of a 1% CaC12 solution so that the osmolarity was adjusted to about 350 m O s m . Fixation proceeded for 15-30 rain at r o o m t e m p e r a t u r e (RT) and then the material was w a s h e d in buffer plus sucrose or NaC1, either osmicated, en bloc stained with uranyl acetate, d e h y d r a t e d and e m b e d d e d in Araldite for sectioning, or treated for 15-30 min in 25% glycerol in buffer, and m o u n t e d in a yeast p a s t e on copper holders. T h e s e were then rapidly frozen in F r e o n 22 cooled by liquid nitrogen (N2) and stored in liquid N2containing holders until required. T h e tissues were fractured at - 1 0 0 ° C a n d in a v a c u u m of 1.5 x 10 -6 Torr (1.33 × 10-4 N m -z) in a Balzers B A 360M freezecleaving device, w h e r e shadowing by t u n g s t e n - t a n talum and backing by carbon then took place. The material was brought to RT, the metallic surfaces were coated with a drop of celloidin, and the adherent tissue was r e m o v e d from the replicas by t r e a t m e n t with hypochlorite and detergent. The cleaned replicas were washed in several c h a n g e s of distilled water, m o u n t e d , and dried down on a finder grid, arid celloidin was r e m o v e d by amyl acetate before examination in a Philips EM300 at 60-80 kV.

OBSERVATIONS

The glial cells that surround the spider CNS in the form of a thin perineurial layer appear to be in the process of forming junctional complexes in the period immediately after hatching. This would seem to be so by virtue of comparing the perineurium of adults, characterised by both mature tight and gap junctions (Lane and Chandler, 1980) with that of hatchlings. In thin sections, at these early hatchling stages, the

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NANCY J. LANE

p e r i n e u r i u m is e x t r e m e l y attenuated, but the intercellular b o r d e r s already interdigitate e x t e n s i v e l y (Figs. 1 and 2). T h e neural lamella is a l m o s t negligible with but a few strands o f collagen (Fig. 2). T h e r e are a n u m b e r of areas o f close c o n t a c t s b e t w e e n the a d j a c e n t perineurial cell m e m b r a n e s (Figs. 1 and 2) and these c o n t a c t regions a p p e a r short and gap-like or punctate. T h e y do not s h o w the extensive gap j u n c t i o n s or n u m e r o u s points o f distinct m e m b r a n e fusion that are so characteristic of the adult spider p e r i n e u r i u m ( L a n e and C h a n d l e r , 1980). T h e y a p p e a r to be early stages in j u n c t i o n f o r m a t i o n since there are m a n y s p o t s w h e r e the m e m b r a n e s are d r a w n close t o g e t h e r but do n o t actually fuse or f o r m a distinct 2- to 3-nm " g a p . " S u c h images tie in with the f r e e z e - f r a c t u r e data, and with the latter the areas o f cell c o n t a c t can be m o r e decisively divided into t h o s e regions pertaining to tight junctional or t h o s e relating to gap j u n c t i o n a l f o r m a t i o n . Stages in the d e v e l o p m e n t o f t h e s e c o e x i s t i n g j u n c t i o n s m a y be f o l l o w e d b y examining f r e e z e - c l e a v e d p r e p a r a t i o n s f r o m animals o f a r a n g e o f sizes. F r e q u e n t l y s e v e r a l stages in d e v e l o p m e n t m a y o c c u r within the s a m e p r e p a r a t i o n and in all the hatchlings e x a m i n e d , b o t h tight a n d gap j u n c t i o n s could be seen to be at various stages in the p r o c e s s of f o r m a t i o n .

1. Forming Tight Junctions as Seen in Freeze-Fractured Replicas I n the p r e s u m p t i v e j u n c t i o n a l perineurial m e m b r a n e the first indication of j u n c t i o n f o r m a t i o n is the a p p e a r a n c e o f " p a t c h e s " of i n t r a m e m b r a n o u s particles (IMPs) of a range of diameters on the P face. At such stages, the I M P s are scattered o v e r the P F with no a p p a r e n t o r d e r within the p a t c h e s . In slightly older animals, these I M P s begin to b e c o m e aligned into short ridges c o m p o s e d of three to four particles (Fig. 3). T h e s e P F particles m e a s u r e a b o u t 8-10 n m in diameter, although t h e y exhibit a range f r o m 6-12 nm. A t such stages, the E face reveals short g r o o v e s (Fig. 3) w h i c h w o u l d a p p e a r to be c o m p l e m e n t a r y to the beadlike P F ridges. T h e s e ridges a p p e a r to incorporate m o r e I M P s which b e c o m e aligned at the ends o f particle r o w s already there (Fig. 4). T h e particle r o w s ultimately bec o m e fused into m o r e solid ridges or fibrils (Fig. 5). A t the s a m e time, the c o m p l e m e n t a r y E face exhibits short f u r r o w s w h i c h gradually b e c o m e e x t e n d e d into a reticulum of g r o o v e s (Fig. 6) w h i c h s p r e a d s across the perineurial m e m b r a n e face. A t this stage, the gap j u n c t i o n s h a v e n o t yet f o r m e d , and individual particles are still f o u n d (Fig. 6). Ultimately a large n u m b e r o f P F ridges and E F g r o o v e s a p p e a r and t h e y gradually s e e m

FIG. 1. Developingjunctions in the perineurium from young spiders in thin-sections consist of a few close membrane appositions (arrows) between the lateral cell borders, x 64 200. FIG. 2. Thin section through the attenuated perineurium (PN) of a slightly older spider showing that the adjacent cells interdigitate extensively even in these early stages. The overlying neural lamella (NL) is almost negligible with only a few collagen fibrils..The lateral membranes are frequently in close apposition (at arrows) which in some cases could be forming tight junctions and a few regions occur which might represent short gap junctions (GJ). x 50 160. FIG. 3. Freeze-fracture replica from an early stage in junctional development in the perineurium where the P face (PF) possesses scattered intramembranous particles, presumably the precursors of tight junctional ridges, which are beginning to become aligned into rows of 2 or 3 (arrows). The attenuated cytoplasm lies between this and the underlying EF face (EF) which exhibits short grooves, probably the complementary structures to the particle rows. × 44 150. FIG. 4. Perineurial PF at a somewhat later stage in junctional development when the short particle rows are being extended and are beginning to become fused laterally into ridges (arrows). x 65 200. FIG. 5. Perineurial membranes after the particle rows have fused into a number of discontinuous PF ridges (arrows) of varying length. On the overlying EF the 13-nm gap junctional particles (GJP) are at this stage seen as free IMPs. C, cytoplasm, x 61 200.

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58

NANCYJ. LANE

to b e c o m e longer and i n t e r c o n n e c t e d (Fig. 7). T h e latter finally are c o n t i n u o u s , alt h o u g h offset, with the P F ridges a c r o s s m e m b r a n e face transitions (Fig. 8); at this stage, therefore, the a d j a c e n t ridges s e e m to be beginning to fuse a c r o s s the intercellular space to p r o d u c e the occlusions or seals characteristic of these tight j u n c t i o n s .

2. Forming Gap Junctions Seen in FreezeFractured Replicas As the tight j u n c t i o n a l P F ridge and E F g r o o v e s y s t e m b e c o m e s i n c o r p o r a t e d into an e v e r - i n c r e a s i n g n e t w o r k o f ridges or f u r r o w s i n t e r c o n n e c t e d in one b r o a d circ u m f e r e n t i a l n e t w o r k o f fibrils, the gap j u n c t i o n s b e g i n t o d e v e l o p . T h e s e are c h a r a c t e r i s e d b y larger I M P s w h i c h average 13 n m in diameter, although t h e y exhibit a range f r o m 12 to 14 nm. T h e s e app e a r at first as f r e e , q u i t e s e p a r a t e , individual 13-nm particles scattered at rand o m o v e r the perineurial m e m b r a n e E face (Fig. 5). E v e n at this early stage these j u n c tional particles m a y display w h a t could be a central p o r e (Fig. 6, insert) t h r o u g h w h i c h the cells w h e n c o u p l e d might e x c h a n g e ions and molecules. At this stage the putative c h a n n e l is p r e s u m e d to be n o n f u n c t i o n a l , p e r h a p s due to a configurational c h a n g e o f the c o m p o n e n t p r o t e i n subunits ( U n w i n and Zampighi, 1980). T h e s e free 13-nm E F particles c o m e to lie in b e t w e e n the g r o o v e s of the f o r m i n g tight j u n c t i o n s (Figs. 6 and 9); once there, t h e y are clearly p r e v e n t e d

f r o m migrating translaterally v e r y far afield by the fused tight junctional elements which act as a kind o f scaffolding b e t w e e n w h i c h the E F gap junctional particles are aggregated. With time, these E F particles f o r m loose linear arrays or clusters aligned alongside the tight j u n c t i o n a l g r o o v e s (Fig. 9). T h e s e ultimately c o a l e s c e into loose aggregates (Fig. 10) and t h e n E F m a c u l a e (Fig. 11) w h i c h lie in intimate association with, but quite distinct f r o m , the tight j u n c t i o n a l g r o o v e s ( F i g . 11). W h e n f u l l y f o r m e d , the tight j u n c t i o n s are c h a r a c t e r ised b y p u n c t a t e appositions, seen clearly in c r o s s - f r a c t u r e d replicas (Fig. 12) w h e r e P F ridges and E F g r o o v e s are coincident a c r o s s face transitions, as well as in thin sections (Fig. 13). T h e gap j u n c t i o n s lie bet w e e n these exhibiting their characteristic r e d u c e d intercellular clefts (Figs. 12 and 13); as m a t u r e plaques, their c o m p o n e n t particles are relatively loosely p a c k e d but still p o s s e s s central channels w h i c h are distinct after l a n t h a n u m infiltration (Fig. 14, insert); these n o w m a y be c o m p l e t e l y functional with r e s p e c t to cell-to-cell coupling and the e x c h a n g e of ions and molecules, although as y e t no u n e q u i v o c a l e v i d e n c e for the transfer o f physiologically significant molecules is available. T h e extensive nature o f the circumferential tight j u n c t i o n s m a y be seen ultimately in adult tissues (Fig. 14) w h e r e the gap j u n c t i o n a l particle clusters are intercalated at intervals b e t w e e n the n e t w o r k o f i n t r a m e m b r a n e ridges and

FIG. 6. Replica illustrating the way a "wave" of junctional development may occur across a membrane face. On the left-hand side, gap junctional particles are still scattered free over the membrane E face (EF), and the discontinuous tight junctional ridge precursors are randomly arranged over the P face (PF). On the righthand side the latter are beginning to form a network of EF grooves or furrows, while the gap junctional particles (GJP) are starting to show signs of clustering into linear arrays or loose aggregates. Insert shows that the free gap junctional EF particles may exhibit a central pit which could represent the putative channel or pore through which molecules are thought to be exchanged when cells are coupled, x 46 000; insert, x 150 500. FIG. 7. Tight junctional ridges beginning to lengthen and interconnect into a PF network. Large arrow indicates where the PF ridge is coincident with an EF groove over a face transition. C, cytoplasm, x 100 900. FIG. 8. Cross-fracturing through perineurial membranes showing that although many ridges and grooves are coincident across face transitions (arrows), not all (*) are at this early stage. This may mean that the cell-to-cell membrane fusion via the ridges has not yet occurred at all tight junctional sites or that some ridges and grooves are still discontinuous. Gap junctional (GJP) particles are still free here and not yet clustered, x 90 300.

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COEXISTING YET DISTINCT GAP AND TIGHT JUNCTIONAL IMPs g r o o v e s w h i c h r e p r e s e n t the o c c l u d i n g junctions. DISCUSSION The b l o o d - b r a i n barrier in the spiders studied here seems not to develop completely during e m b r y o n i c life in that stages in the formation of the occluding junctional complexes, which a p p e a r to f o r m the morphological basis of this outer glial permeability barrier (Lane and Chandler, 1980), are found in the tissues of early hatchlings. This is supported by the data f r o m young spiders in both the thin-section evidence, wherein the numbers of punctate tight junctions b e t w e e n these v e r y attenuated glial cells are not as great as the close contacts, and by the freeze-fracture data, where the r i d g e - g r o o v e s y s t e m that c o m p o s e s the tight junctions m a y be in various stages of development. A similar p h e n o m e n o n occurs in certain insects, for example, the blowfly Calliphora, which also completes the formation of its b l o o d - b r a i n barrier after hatching (Lane and Swales, 1978a). It seems clear that the various changes in I M P configuration during the early stages of d e v e l o p m e n t in the hatchling spider perineurium r e p r e s e n t different stages in junctional f o r m a t i o n . The free i n t r a m e m b r a nous particles o b s e r v e d in the v e r y young animals, which fall into two categories, 8 10 nm PF and 13 n m EF, p r e s u m a b l y are newly inserted junctional particles. There seem to be no larger p r e c u r s o r particles on either i n t r a m e m b r a n o u s face as h a v e been reported in v e r t e b r a t e tissues (Decker and Friend, 1974; Revel, 1974; Johnson et al.,

61

1974; Revel et al., 1978) b e c a u s e there is no apparent change in diameter in either the P F or E F junctional I M P s between the free and the aggregated state. Both junctional types a p p e a r to mature b y translateral migration of I M P s f r o m the free state into one of two final states of aggregation. In the case of the tight junctions, the IMPs coalesce into ridges, while in the case of the gap junctions the IMPs aggregate into clusters or plaques. S o m e aspects of these phen o m e n a a p p e a r to h a p p e n c o n c u r r e n t l y o v e r the s a m e time period although the tight junctions s e e m to be initiated j u s t slightly earlier; short PF ridges m a y be seen while the 13-nm E F GJ particles are still almost completely disaggregated. However, even within the same m e m b r a n e face, several stages i n j u n c t i o n d e v e l o p m e n t m a y be occurring simultaneously (for, e.g., see Figs. 6 and 9). The stages in the formation of the tight junctions of the cells of the spider perineurium are s o m e w h a t similar to those described in a variety of v e r t e b r a t e tissues f r o m foetal organs (Revel et al., 1973; D e c k e r and Friend, 1974; Ducibella et al., 1974; M o n t e s a n o et al., 1975; H u m b e r t et al., 1976; L u c i a n o et al., 1976; Magnuson et al., 1977; Tice et al., 1977; S c h n e e b e r g e r et al., 1978), and m a t u r e cultured cells (Johnson et al., 1974; P o r v a z n i k et al., 1976; Elias and Friend, 1976; Dermietzel et al., 1977). The free junctional particles in these v e r t e b r a t e tissues are reported to bec o m e lined up into short rows which fuse into ridges; these then b e c o m e interconnected and coalesce into the confluent net-

FIG. 9, Forming gap junctions on the perineurial E face (EF) show areas with the 13-nmparticles beginning to become aligned into rows or elongated clusters (arrows) which lie between the grooves of the forming tight junctions, x 59 200. FIG. 10. Later stage in perineurial junctional development showing the gap junctional clusters (GJ) becoming more plaque-like although the particles are still relatively loosely packed, x 67 200. Fro. 11. Still later stage in formation of gap junctions from the perineurium in a young spider, exhibiting macular plaques of EF gap junctional particles (GJ), intercalated between the tight junctional furrows. These grooves may not necessarily extend all around the plaques, and may terminate at the plaque periphery (arrows). x 50 200.

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COEXISTING YET DISTINCT GAP AND TIGHT JUNCTIONAL IMPs work characteristic of mature tight junctions. In e p i d i d y m a l epithelium, the mature f o r m is that of alignments of parallel ridges, rather than a ridge network, but here too, discontinuous short i n t r a m e m b r a n o u s segments b e c o m e t r a n s f o r m e d into extensive rows (Nagano and Suzuki, 1976a; Gilula et al., 1976; M e y e r et al., 1977; Suzuki and Nagano, 1978a, 1979). The m e c h a n i s m of tight j u n c t i o n f o r m a t i o n in t e r m s of the stages that occur in i n t r a m e m b r a n o u s particle translateral migration and rearrangem e n t s can t h e r e f o r e be s e e n f r o m the above, c o m p a r e d with the spider data, to be similar in certain ways in v e r t e b r a t e and in invertebrate tissues. H o w e v e r , clear formation areas are not apparent in the latter, nor does a " c o a t i n g " material always transform the aligned particles into ridges, as happens in some v e r t e b r a t e tissues (Montesano et al., 1975; Luciano et al., 1976; Yee and K a r n o v s k y , 1979; Porvaznik et al., 1979). Equally, the formation of the gap junctions described here is like that o b s e r v e d in other s y s t e m s , both i n v e r t e b r a t e (Lane, 1978; L a n e and S w a l e s , 1978a,b, 1979, 1980) and v e r t e b r a t e (Decker and Friend, 1974; Y a n c e y et al., 1979). In the latter, h o w e v e r , larger p r e c u r s o r particles occur (Revel et al., 1978); these have not been seen in a r t h r o p o d tissues (Lane, 1978; L a n e and Skaer, 1980). In addition, formation plaque areas are not found in these spider replicas of forming gap junctions, as they

63

are in v e r t e b r a t e tissues (Johnson et al., 1974; D e c k e r , 1976a). This a p p a r e n t absence m a y be a function of the junctional particles fracturing onto the E F which in any case lacks a v e r y substantial I M P population (Lane and Skaer, 1980; L a n e and Swales, 1980); h o w e v e r , not all v e r t e b r a t e systems display formation plaques either (Yancey et al., 1979). Frequently, especially in epithelial tissues, tight and gap junctions coexist (Farquhar and Palade, 1965; Elias and Friend, 1976; Revel and Brown, 1976) and it m a y be difficult during formation or disassembly to determine whether both junctional types originate f r o m a c o m m o n p r e c u r s o r particle, in that the junctional particles are indistinguishable ( M o n t e s a n o et al., 1975; Revel and Brown, 1976; Elias and Friend, 1976; P o r v a z n i k et al., 1979; Wynograd and Nicolas, 1980; Decker, 1981). This is particularly so since the vertebrate gap junctions are c o m p o s e d of IMPs which fracture onto the PF and which, in these systems, are a p p r o x i m a t e l y the same diameter as the PF particles that comprise the tight junctions. (Exceptions to this include the tight junctions found in oligodendrocytes (Dermietzel et al., 1978), Sertoli cells (Nagano and Suzuki, 1976b), and endothelial ceils (Simionescu et al., 1978), but these need not coexist with gap junctions (Nagano and Suzuki, 1976a,b).) U n d e r conditions where tight and gap junctions coexist in v e r t e b r a t e tissues, with

FIG. 12. Replica from an older spider showing cross-fractured perineurial membranes. The punctate fused membrane appositions of the tight junctions are apparent at points (arrows) across which the PF ridges and EF grooves are coincident. The gap junctions, intercalated between the tight junctions, show their characteristically reduced intercellular cleft as the fracture plane shifts from 13-nm particle-laden EF (GJ) to the macular arrays of pits in the P face (PF). x 60 700. FI~. 13. Thin sections of adult Aranaeus showing the punctate fusions typical of fully formed tight junctions (arrows) and the 2- to 3-nm intercellular cleft characteristic of mature gap junctions (GJ). At higher magnification (insert) these features are very readily apparent. Fig. 13, x 79 200; insert, x 99 000. Fie. 14. Freeze-fracture replica of an adult spider revealing the extensive, belt-like, network of tight junctional ridges (on the PF) and grooves (on the EF) between which are intercalated the mature gap junctional plaques (GJ). The insert shows one of these mature plaques at higher power cut en face after lanthanum incubation, demonstrating the central channel present in each of the component particles; in comparison with vertebrate tissues these particles are relatively loosely packed even in adult tissues, x 22 800; insert, z 229 000.

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their c o m p o n e n t particles fracturing on the same face, particles of " a m b i g u o u s identity" (Larsen, 1977) m a y therefore occur, particularly during the stages in junction formation. The spider tissue under investigation in this report does not suffer f r o m this disadvantage, in that the two types of junctional particle fracture onto different faces and are also of very different diameter. M o r e o v e r , in f a v o u r a b l y s h a d o w e d replicas, it is possible to o b s e r v e what m a y be gap junctional central channels in the free 13-nm E F particles prior to aggregation, as a further aid in identification. It must, h o w e v e r , be borne in mind that this apparent central p o r e m a y actually be an artefact of preparation and that, m o r e o v e r , no evidence is currently at hand to support the contention that ions and molecules are in fact exchanged via the central channel. In arachnid tissues, the two kinds of IMPs are immediately recognisable as seemingly belonging to one or other of the two junctional types. It is therefore likely f r o m the data p r o d u c e d here, that the two junctions m a y arise quite separately, from two distinct i n t r a m e m b r a n o u s p a r t i c l e p o p u l a tions. By analogy, it is reasonable to supp o s e t h a t in v e r t e b r a t e s y s t e m s , the particles comprising the two types of junction are also different even though they cannot be distinguished by conventional freeze-fracture criteria. This would suggest that they do not arise from a c o m m o n precursor particle, as might otherwise have been supposed. Such a situation would be more tenable physiologically since the gap junctional particles are thought to be very specifically structured for their function, consisting, at least in vertebrate tissues, of six h e x a m e r s that surround a central p o r e (Makowski e t a l . , 1977; Casper et a l . , 1977) through which the exchange of ions and molecules is thought to take place during cell-to-cell coupling (Loewenstein e t al., 1978). Less is k n o w n about the tight junctional particles since they h a v e not yet been isolated nor, consequently, have their structural proteins been characterised, but

they do not require a central channel since their role is to fuse the m e m b r a n e s of adjacent cells together. H e n c e gap and tight junctions a p p e a r to f o r m f r o m two distinct populations of i n t r a m e m b r a n o u s particles which are p r e s u m a b l y synthesised and inserted into the p l a s m a l e m m a quite separately. Studies using inhibitors of protein synthesis might support this contention by interfering with the synthesis of only one junctional type, should a sufficiently large pool of the other type, for example gap junctional p r e c u r s o r s , already exist; such precursors h a v e b e e n reported to be present in certain systems during gap junctional formation (Cox e t a l . , 1976; Epstein e t a l . , 1977). I should like to express my gratitude to Mr. William M. Lee for his technical assistance with both the freeze-fracturing and photographic processing and to Mr. J. Barrie Harrison for the thin-sectioning. REFERENCES CASPER, D. L. D., GOODENOUGH,D. A., MAKOWSKI, L., AND PHILLIPS,W. C. (1977) J. Cell Biol. 74, 605-628. Cox, R. P., KRAUSS,M. R., BALIS,M. E., AND DANCIS, J. (1976) J. Cell Biol. 71, 693-703. DECKER, R. S. (1976a) J. Cell Biol. 69, 669-685. DECKER, R. S, (1976b) J. Cell Biol. 70, 412A. DECKER, R. S. (1981) Develop. Biol. 81, 12-22. DECKER, R.S., AND FRIEND, D. (1974) J. Cell Biol. 62, 32-47. DERMIETZEL, K., MILLER, K., TETYLAFF, W., AND WAELSCH, M. (1977) Cell Tissue Res. 181, 427-441. DERMIETZEL, R., SCHUNKE, D., AND LEIBSTEIN, A. (1978) Cell Tissue Res. 193, 61-72. DUCIBELLA, T., ALBERTINI, D. F., ANDERSON, E., AND BIGGERS,J. D. (1974) J. Cell Biol. 63, 89a. ELIAS, P. M., AND FRIEND, D. S. (1976) J. Cell Biol.

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