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The specialization of septate junctions in regions of tricellular junctions
JOURNAL OF ULTRASTRUCTURE RESEARCH 78, 136-151
(1982)
The Specialization of Septate Junctions in Regions of Tricellular Junctions I. Smooth Septate Junctions (=Continuous Junctions) F . G R A F , * C . N O I R O T - T I M O T H I ~ E , t AND C H . NOIROT~
*Laboratoire de Biologie animale et gdndrale, and ?Laboratoire de Zoologie, Universit( de Dijon, Boulevard Gabriel, 21100 Dijon, France Received June 1l, 1980, and in revised form August 19, 1981 Some regions from the midgut of a crustacean and an insect were comparatively studied, both in sections of lanthanum-impregnated tissues and on freeze-fracture replicas. The epithelial cells a r e apically joined by zonular smooth septate junctions which are actually composed of alternating parts built and shared in common by either two cells (bicellular junctions) or three cells (tricellular junctions). The bicellular junctions are composed of strands which include a smooth extracellular septum stretched between two intramembrane particle rows. The strands run nearly or preferentially parallel to the apical surface, except near the cell corners where they become parallel to the axes of the tricellular junctions. This change in strand orientation characterizes the marginal regions of the bicellular junctions, which moreover are bordered by a limiting strand. At tricellular junctions each cell corner membrane displays a series of regularly spaced doublets of juncture particles, somewhat larger than the strand particles. The doublets are apparently the intramembrahe components of junctional units (partitions), the chief part of which is an extracellular diaphragm showing a central vesicular part and a thinner periphery. The diaphragms compartmentalize the extracellular space and are linked to the limiting strands of the bicellular junctions abutting onto a tricellular junction.
In arthropods, as in most other inverte- ber and pattern showing a good correlation brates, epithelial cells are joined by an api- with the extracellular septa. Thus it is incal junctional complex which commonly in- ferred that each septum is associated with cludes a zonular septate junction. There is two particle rows, one in each plasma memno general agreement about the functional brane, the whole constituting the structural significance of the septate junctions though unit of the junctions and called the juncvarious data suggest that besides their role tional strand (13). in cellular cohesion they probably restrict Several types of septate junctions are the paracellular movement of fluids and recognized, two of which commonly occur thus are functionally similar to the tight in arthropods (7, 13, 15). In pleated septate junctions of vertebrate epithelia (review in junctions (4, 13), the septa show regularly (13)). spaced (20-22 nm) fine pleats and the cenThe structural organization of the septate ter-to-center spacing of the particles along junctions has mainly been studied in re- a row ranges from 10 to 20 nm. In all degions where two cells are linked. Such bi- scribed cases most particles preferentially cellular junctions are first characterized on adhere to the protoplasmic cleavage face sections by ribbon-like septa which span (P face) (I) on freeze-fracture replicas. In the intercellular space and join the two ad- smooth septate or continuous junctions (4, jacent plasma membranes. Moreover, the 9, 13, 15), the septa are parallel-sided (not freeze-fracture technique allows recogni- pleated) and the particles of a row are very tion of particle rows which are in alignment close to each other, so that they frequently in both membranes, and which have a num- form ridges. Depending on the species, tis136 0022-5320/82/020136-16502.00/0 Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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sue, or preparative procedure, most parti- tacea, Amphipoda). Others were made on the midgut cles adhere either to the protoplasmic (P) and the anterior caeca of the midgut from several species of cockroaches, usually adults or last instar or to the exoplasmic (E) fracture face, or nymphs of Blaberus craniifer (Insecta, Dictyoptera). show no preferential binding. Moreover, small intermembrane pillars without intra- Techniques membrane counterparts are commonly ob1. Conventional sections. These were prepared as served in the interseptal spaces as soon as described elsewhere (6, 12). 2. Lanthanum impregnation. A lanthanum solution these are large enough to accommodate at was prepared according to the procedure of Revel and least one row of pillars. Karnovsky (14) and added to the fixation and washing A zonular junction also includes regions mixtures (1% final lanthanum concentration). Tissues where three cells are joined. Some features were fixed (18 hr) in a solution of 2% formaldehyde of these regions of tricellular junctions, (freshly prepared from paraformaldehyde) and 3% glustudied in sections, have been previously taraldehyde in 0.08 or 0.1 M cacodylate buffer. They were washed in the same buffer, postfixed (1 hr) in reported in the case of pleated septate junc- buffered 1% OSO4, then dehydrated in an ethanol setions (12). In these regions several septa of ries and embedded in an Araldite-Epon mixture. Secthe convergent bicellular junctions run in tions were examined without further contrast enparallel alongside the junction line between hancement. 3. Freeze-fracture replicas. Most specimens were the three cells and the most central septa briefly (about 0.5 hr) fixed in cacodylate-buffered 2% are joined by a series of regularly spaced glutaraldehyde and infiltrated with a buffered 40% sobridges, with each bridge showing a ring- lution of glycerol for at least 12 hr. Some of these shaped central part and thin lateral arms. specimens were frozen in Freon 21 and stored in liquid In order to obtain new and more precise nitrogen, then fractured and replicated in a BAF 301 information on the regions of tricellular (Balzers) apparatus. Other specimens were frozen in melting nitrogen before processing in a Cryofract 250 junctions, tissues have been selected where (Reichert) apparatus (3). For the Blaberus midgut the septate junctions are of either the pleat- some tissue specimens were not fixed and were either ed or the smooth type and which moreover treated (1 hr) or not with glycerol before freezing in for a given type exhibit some morphological melting nitrogen. Sections and freeze-fracture replicas were examined or structural differences. Both intercellular in a Hitachi HU-l I E electron microscope. and intramembrane structures have been studied comparatively using sections of RESULTS AND INTERPRETATION lanthanum-impregnated tissues and freezeIn monolayered epithelia such as those fracture replicas. A tridimensional interpreexamined, any three contiguous cells are tation of the observed data has been perapically linked in pairs through three bicelformed. luiar septate junctions and also together The results of this work are reported in through a tricellular junction (Fig. 1). This two papers dealing with the smooth and the tricellular junction follows a more or less pleated septate junctions, respectively. sinuous line which we call the "juncture However, because of the common characline." This is the axis of the "juncture teristics recognized in all cases for regions space" lying between the three cell corners. of tricellular junctions the observations are Near the juncture space the bicellular discussed together in the second paper (I1). Preliminary results have been pub- junctions undergo morphological changes. lished previously in abstract form (10) and Thus it appears necessary to distinguish and to successively describe the principal in a review article by two of us (13). and the marginal (i.e., the area near the MATERIALS AND TECHNIQUES juncture space) regions of these bicellular Materials junctions before examining the tricellular Some of the observations were carried out on the junctions and the specialized devices enmidgut posterior caeca of Orchestia cavimana (Cruscountered at their level.
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C FIG. 1. (a) An interpretative diagram of the cross-sectioned parts of three cells involved in three bicellular junctions (B) and a tricellular junction (T). Both inter- and intramembrane junctional components are shown. JS, junctional strand, the structural unit of bicellular junctions, composed of a septum (S) stretched between two intramembrane particle rows (R). LS, limiting strand, forming the limit of a bicellular junction where it abuts against a tricellular junction. P, partition, the structural unit of tricellular junctions, composed of a diaphragm (D) located in the juncture space and linked to three doublets of juncture particles (J). The diaphragm includes a central vesicular part and a thinner periphery connected with the limiting strands and the corner membranes. (b) and (c) Limiting septa (LS) and diaphragms (D) as they appear in longitudinal sections along the planes X X ' (b) and YY' (c) shown on diagram (a). See detailed description in the text and compare with Figs. 4 and 5.
1. Posterior Caecum of Orchestia (Crustacea) During the intermolt (state C), the epithelial cells are columnar, about 3-8/xm in
width and 50 /xm in height. Their lateral faces are smooth and linked apically by a zonular smooth septate junction (6) and more basally by numerous macular gap junctions (5).
FIGS. 2-5. Caecum of Orchestia. Unstained sections of lanthanum-impregnated specimens, showing intermembrane junctional components in negative contrast. FIG. 2. Section of the apical region of the epithelium, below the microvilli (MV), showing three cells (C l, C2, C3), the bicellular junction (B) between C1 and C2, and the tricellular junction (T) between C1, C2, and C3. In the principal region of the bicellular junction (on the left), the septa (S) run parallel to the apical surface. In the marginal region (i.e., near the tricellular junction), they curve toward the base, loop (*), and then run parallel to the juncture line as they come back toward the apex. The juncture space includes a series of regularly superposed diaphragms (D), x 60 000. FIGS. 3-5. Tricellular junctions (T) and adjoining parts of tangentially sectioned bicellular junctions. In the marginal regions of bicellular junctions several septa run parallel to the juncture line because of their previous looping course (*). Interseptal spaces are commonly large enough to include pillars (P). Segments of septa follow each other in various ways (arrows l, 2, 3; compare with Fig. 12b) to form the limiting septa (LS). Tricellular junctions are characterized by the occurrence of diaphragms (D). These are regularly superposed in the juncture space and look like bridges which include a vesicular part and thinner lateral arms. In Fig. 4, at center and bottom, the vesicular part is linked on the left to a cell corner membrane (M) by a very short arm and on the right to a limiting septum (LS) by a longer arm (compare with Fig. lb). On Fig. 4 at the top and on Fig. 5, the sectioning incidence is somewhat different and the vesicular part bears two symmetrical arms, each inserted on a limiting septum (compare with Fig. lc). Fig. 3, × 225 000; Figs. 4, 5, x 300 000.
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FIG. 6. A three-dimensional diagram of the intermembrane structures; that is, the septa (S) of the three bicellular junctions (B1, B2, B3) and the diaphragms (D) of the tricellular junction (T) which link the membranes (M1, M2, M3) of three cells. The diaphragms are arranged in a regular file in the tricellular juncture space and each one is normally connected to the three discontinuous limiting septa (LS) and to the three cell corner membranes surrounding this space.
1.1 Principal regions of bicellular junctions. As previously reported (6), the septa look like smooth ribbons, well delineated by lanthanum (Figs. 2 to 5), and the freezecleaved membranes of fixed specimens show particle ridges on the P faces or com-
plementary furrows on the E faces (Figs. 7 to 10). The junctional strands (septa and particle ridges or furrows), although slightly wavy, are on the whole parallel to the apical surface (Figs. 2, 7, 8, 12a). Arrays of close and equidistant septa are frequent and in these cases each interseptal space (5-6 nm) includes a row of pillars. However, in some places septa are more widely spaced and the interseptal spaces include numerous and randomly distributed pillars. The depth of the junction ranges from 0.12 to 1 /xm and the number of strands from 3 to 30. It must be stressed though that these large variations occur between different junctions and not along a given one, at least as far as the principal regions are considered.
1.2 Marginal regions of bicellular junctions. The differences between bicellular junctions as regards their depth and strand number are progressively reduced as they approach a tricellular junction because supplementary strands can appear above those coming from the principal region (Figs. 8, 12a). Characteristic morphological changes are observed in the marginal regions of all bicellular junctions. Their depth suddenly increases and usually reaches 1.6-2/xm, although a depth of only 1.2/zm is reached when two tricellular junctions are close to each other. In these marginal regions, 0.3 to 0.6 ~m in width, the junctional strands become more spaced and their course is modified to different degrees depending on their position. The basal strands curve toward the cell base, then with a hairpin loop curve back toward the apical surface to
Fi~s. 7 AND 8. Caecum of Orchestia. Freeze-fracture replicas of fixed specimens, each including two bicellular junctions and one tricellular (T) junction. The bicellular junctions are characterized by particle ridges (R) on the P faces (P) and complementary furrows (F) on the E faces (E). In the principal regions of the junctions, ridges and furrows are close to each other and parallel to the apical surface covered by microvilli (MV). In the marginal regions (M), the junction depth greatly increases and the ridges or furrows become more widely spaced. Those of the basal half exhibit a conspicuous hairpin pattern (*) and then run in a basoapical direction, following each other to form a limiting strand (LS) on each side of the tricellular junctions (T). These are seen in both figures on a P face and appear as a crest bearing a series of juncture particles. G, E-Type gap junctions; TS, twisted apical supplementary strands, x 50 000.
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which they are then perpendicular (Figs. 2, 7, 8, 12a). Most of the strands run parallel to the tricellular juncture line before ending and their terminal segments overlap and follow each other in progression (Figs. 5, 10, and 12b, arrow 1) to form a discontinuous "limiting strand" including a "limiting septum." In some places the overlapping of the terminal segments is reduced or absent (Figs. 4 and 12b, arrow 2) and moreover some strands recurve instead of ending, leaving a larger gap in the limiting strand between the two curves moving in opposite directions (Figs. 3 and 12b, arrow 3). Nevertheless, a limiting strand always occurs where a bicellular junction abuts against a tricellular junction (Figs. 1, 3-10, and 12). The median strands behave somewhat like the basal ones except that they either do not curve toward the base, or only form a short loop before following a basoapical course (Fig. 12a). The apical strands are not involved in making the limiting strands; they turn back without any previous bending toward the base so that the limbs of the loops are parallel to the apical surface (Fig. 12a). The most apical strands sometimes form concentric loops or spirals (Figs. 7, 8, 12a). Two facts should be emphasized. First, the information given by either sections of lanthanum-impregnated tissues or freezefracture replicas is quite similar even though the replicas furnish more extensive picture areas. This is new indirect evidence of the presumed association between septa and intramembrane particle rows. Second,
bicellular junctions structurally appear to be well separated and autonomous, the strands never passing over a juncture line from one bicellular junction to another, and the limiting strands, although discontinuous, are really the borders of the bicellular junctions. 1.3. Tricellular junctions. Whatever the previous course and orientation of the plasma membranes, the dihedral angles of the three apposed cells always appear to be equal (120°) around the juncture space (Fig. 11). The limiting strands of the three convergent bicellular junctions, only two of which can be simultaneously seen in tangential sections (Figs. 4, 5) and on freezefracture replicas (Figs. 7-10, 12), are parallel and separated by 25-30 nm. Together with the corner membranes of the three cells they circumscribe the juncture space where a series of superposed diaphragms is found (Fig. 6). The center-to-center distance between the diaphragms ranges from 13 to 18 nm (average 16.5 rim). In longitudinal sections of tricellular junctions these diaphragms are well delineated by lanthanum. They appear as transverse bridges, each with a thicker central part and thinner lateral arms. The central parts have the shape of a flattened ring (9to 10-nm maximum thickness) surrounding an ovoid space filled by lanthanum (Figs. 3-5). According to the angle of incidence of the section the detailed aspects are somewhat different. In some places, the septa of only one bicellular junction are observed (Figs. lb, 3, 4) and on the other side
FIGS. 9 AND 10. Caecum of Orchestia. Freeze-fracture replicas of fixed specimens. On the E faces (E), a tricellular junction (T) can be recognized as a groove bearing some juncture particles and the imprints (arrowheads on Fig. 10) of others. Typically two juncture particles lie side by side forming a doublet (D) or are fused into a rodlet (R). On each side of the groove the basal furrows of the bicellular junctions display their characteristic looping pattern (*). It can be seen in Fig. 10 that the terminal parts of the furrows overlap and follow each other to form the border (limiting strand, LS) of the bicellular junctions. Their ends (arrows marked 1) are located just beside a juncture particle. Fig. 9, ×100000; Fig. 10, x 175 000. FIG. 11. Caecum of Orchestia. Conventional section showing three bicellular junctions (B) abutting onto a transversely cut tricellular junction (T). At this point the three plasma membranes always form equal angles. Septa of the bicellular junctions and diaphragms of the tricellular junction are not visible as is common in conventional sections. × 350 000.
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FIG. 12. Diagrams summarizing information obtained from freeze-fracture replicas of Orchestia caecum. (a) A tricellular junction (T) is located in between two bicellular junctions, the principal regions of which include either (on the left) rather numerous strands, or (on the right) only a few. In both cases their marginal regions (MR) display an increased depth and the characteristic looping pattern of the strands (see more details in the text). A, Apical strands; B, basal strands; M, median strands; SS, supplementary strands; TS, twisted supplementary strands. (b) A tricellular junction (T) is characterized by a row of regularly spaced juncture particle doublets (D). These lie on a membrane strip bordered on both sides by a limiting strand (LS), which is actually a succession of strand segments which follow each other in various ways (arrows 1, 2, 3) before ending or curving. The end points are located beside a juncture particle doublet (arrows 1, 2).
of the juncture space a faint electron-translucent line eventually appears (Fig. 4), which probably represents the corner membrane of a cell. In these cases the bridges appear asymmetric. The ring-shaped part is more distant from the septa side than from the other side and it is linked to the observed limiting septum by a long, clearly seen arm, whereas on the opposite side it is connected to the plasma membrane directly or through a very short arm. In other places the septa of two bicellular junctions are visible and the bridges appear symmetrically organized (Figs. lc, 5). The ringshaped part (15-16 nm in width) lies in the middle of the juncture space and is joined to each limiting septum by lateral arms of equal length. In some sections the two types of images can be seen in succession (Fig. 4) because of a slight tilting of the whole junction within the depth of the section. These data indicate that the bridges are true diaphragms which compartmentalize the juncture space (Fig. 6). Each one has
an inflated central part with a biconvex lens shape and a thinner periphery which is linked to the limiting septa of the surrounding three bicellular junctions and to the three cell corner membranes. The peripheral part has been tentatively pictured as a continuous and homogeneous platform (Figs. la, 6). However, this cannot be definitely ascertained and it is possible that it is heterogeneous or made of independent arms. On freeze-fracture replicas the limiting strands (particle rows on the P faces or complementary furrows on the E faces) of the bicellular junctions are separated by a membrane strip seen as a crest on the P faces (Figs. 7, 8) and as a groove on the E faces (Figs. 9, 10). At this level the membrane includes a row of juncture particle pairs (called "doublets") which mostly adhere to the P faces but in some places are observed on the E faces. The two particles of a doublet are close together and are sometimes joined to form a small rod (called "rodlet") (Fig. 10). The average di-
TRICELLULAR JUNCTIONS, I
ameter of the juncture particles (7.5 nm) is larger than that of the strand particles (5 nm). The successive doublets are regularly ordered in a ladder-like way, with an average center-to-center spacing of 16.5 nm, that is to say, the same value as that reported for the diaphragm spacing. This fact favors the idea that the particle doublets are the intramembrane counterparts of the diaphragms. Further evidence of this is provided by the fact that along a limiting strand the ends of the composing segments are located at the level of a diaphragm on the one hand and of a doublet on the other (see especially Fig. 4, arrow 2, and Fig. 10, arrow 1, and compare with Fig. 12b, arrows 2 and 1). Consequently the structural units of tricellular junctions, called "partitions" (13), appear to include an extracellular diaphragm and three doublets of intramembrane juncture particles.
2. Midgut of Blaberus (Insecta) In last instar nymphs, as well as in adults, mature epithelial cells are at least 100/xm in height and about 15/xm in diameter. Lateral membranes are smooth except in the subapical region where more or less deep foldings are always observed. As in the midgut of other insect species (13), the cells are linked by a well-developed zonular smooth septate junction which begins immediately below the microvilli since it is not preceded by a belt desmosome. Some small-sized macular desmosomes are included in the apical part of this smooth septate junction. Rather numerous gap junctions are either intercalated in the septate junction, mostly in its basal half, or located just below the septate junction.
2.1. Principal regions of bicellular junctions. The septa of bicellular junctions (Figs. 13-15) resemble those observed ifi Orchestia except that they are somewhat thicker. Freeze-cleaved junctional membranes give different pictures depending on the previous treatment of the specimens. This has also been observed in the midgut or Malpighian tubules of other arthropod
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species (2, 4, 8). When a fixation step is used (Figs. 17, 18) the two fracture faces are more or less similar. On both faces, each strand is seen as a succession of furrows and particle files, the particles often being closely apposed in ridges. Furrows and ridges (or particle files) are of quite variable length. The partitioning of the membrane particles between the two cleavage halves changes when the specimens have not been fixed, regardless of whether or not they were treated with glycerol. In these cases (Figs. 16, 19-22), almost all the particles adhere to the E faces so that these bear continuous ridges which are easily resolved as apposed particles whereas the P faces are characterized by complementary furrows. This means that the picture is then the reverse of that reported for the smooth septate junctions of Orchestia. It should be added that the partitioning of the gap junction particles is not modified by the fixation. In all instances these particles are observed on the E faces and the complementary pits on the P faces (type E gap junctions). The junctional strands are much more numerous and are also more sinuous and variably distributed than in Orchestia. The most apical strands are very close to each other and are typically organized in stacks (Figs. 13, 14, 16) where many of the interseptal spaces are very narrow and thus do not include pillars. In many places the stacked strands lie parallel to the apical surface (Figs. 13, 14, 16) although they can diverge and curve. Below the apical strip showing this organization, the junctional strands follow a very tortuous path and are unevenly spaced (Figs. 15-22). In some places several strands run in parallel and very close to each other, the interseptal spaces being very narrow and often without pillars. More frequently, however, the strands are variably spaced due to the curves and loops that they describe and numerous randomly distributed pillars are present in the interseptal spaces unless such a space is occupied by a gap junction.
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The average depth of the bicellular smooth septate junctions in Blaberus is 15 /zm. The number of the strands is more than a hundred although this is hard to state precisely due to the large junction depth, the tortuous path of the strands, and the paucity of extended cleavage areas obtained
for the apical region, probably because of the membrane foldings.
2.2. Marginal regions of bicellular junctions. Around any tricellular juncture space, each bicellular junction is clearly bordered by a limiting strand which, as in Orchestia, is formed by a succession of
FIGS. 13-15. Midgut of Blaberus. Unstained sections of lanthanum-impregnated tissues which show intermembrane junctional components in negative contrast. Each figure includes a tricellular junction (T) and adjacent parts of two bicellular junctions. FIG. 13. Near the apex most septa of the bicellular junctions are very close to each other and organized in stacks although some can diverge and curve (arrow). Pillars (P) are present in the interseptal spaces only where the spaces are large enough. In the principal region (a) of a bicellular junction the septa are mostly parallel to the apical surface covered by microvilli (MV), while in the marginal regions (b) they are parallel to the tricellular junction (T). Here the juncture space includes a series of diaphragms (D). x 90 000. FIC. 14. At the top of the figure a stack of septa (a), with pillars (P) in some interseptal spaces, is located just below the microvilli (not included) and the septa are parallel to the apical surface. On the right the septa of another stack (b) curve to become parallel to a tricellular junction (T). The juncture space is bordered on each side by a limiting septum (LS) which, as can be seen on the right-hand side, is composed of a succession of segments following each other. The diaphragms (D) located in the juncture space display a central annular part and lateral arms which are crossed by a thin plate (PL). Successive plates may form a ribbon (R) parallel to, but thinner than, the limiting septa. Most spaces between diaphragms are of equal size although one is slightly larger (arrow 1) and two others are twice as large (arrows 2 and 3). x 150 000. FIG. 15. Deeper in the junctional area, the septa are more frequently curved or looped and unevenly arranged, often with large interseptal spaces including numerous pillars (P). Some septa follow each other (arrowheads) to form a limiting septum (LS) on each side of the tricellular junction (T). At the center, each diaphragm shows two symmetrical arms crossed by a thin plate (PL) and inserted onto a limiting septum. On the right, the annular part of the diaphragms is linked on one side to a cell corner membrane (M) either directly or by a short arm, and on the other side to a limiting septum (LS) by a larger arm. x 200 000. FIG. 16. Midgut of Blaberus. Freeze-fracture replica of a glycerinated unfixed specimen. Apical regions of three cells (C 1, C2, C3) covered by microvilli (MV) and two bicellular junctions (B1, B2), linking, respectively, CI to C2 and C2 to C3, are shown. The bicellular junctions are characterized by particle rows on the E faces (E) and complementary furrows on the P faces (P). Just below the microvilli furrows (F) are very close to each other and mostly parallel to the epithelium surface while deeper they are variably spaced and curved. On the left toward the top cell C2 terminates and the two bicellular junctions abut onto each side of a specialized membrane strip which thus can be identified as part of a tricellular junction (T). Here, on an E face, the specialized strip is groove shaped and bears juncture particles in a ladder-like arrangement. Moreover, some strands of the bicellular junctions run in parallel to the rims of the groove. (Blisters in junctions were caused by a slightly hypertonic glycerol solution.) x 55 000. FIGS. 17-22. Midgut of Blaberus. Freeze-fracture replicas of fixed (Figs. 17, 18) and unfixed (Figs. 19-22) specimens, each showing a tricellular junction (T) either on an E face (Figs. 17, 19, 21) or a P face (Figs. 18, 20, 22) and the adjoining parts of two bicellular junctions. The occurrence of a tricellular junction (T) is indicated by an elongated membrane strip more or less groove shaped on E faces (E, Figs. 17, 19, 21) and crest shaped on P faces (P, Figs. 18, 20, 22). In both fixed and unfixed tissues, the E-face grooves bear juncture particles (J) forming doublets (D) or fused in rodlets (R) while complementary imprints (i) (paired pits or short furrows) as well as a few particles (J, Fig. 22) are observed on the P-face crests. Pits are more obvious on replicas of unfixed specimens (Figs. 20, 22). The imprints are regularly spaced (Figs. 20, 22) but plastic deformation produces a less regular pattern of the particle doublets (Fig. 21). Interruptions in the series of juncture particles sometimes occur, especially in the basal junctional area (*, Fig. 17). Bicellular junctions of fixed tissues display particle rows interrupted by furrows on both E (E, Fig. 17) and P (P, Fig. 18) faces. On replicas of unfixed tissues particle rows are observed on E faces (E, Figs. 19, 21) and complementary furrows on P faces (P, Figs. 20, 22). Particle rows and furrows conspicuously meander except on each side of tricellular junctions where following segments form a limiting strand (LS, chiefly visible on Fig. 22, left side), usually paralleled by one or more other strands. Figs. 17, 18; x 65000; Figs. 19, 20; x 60000; Figs. 21, 22; x 200000.
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strand segments (Figs. 14, 15, and 17-22). These are of unequal length and commonly overlap and follow each other (Figs. 14, 15, 22) so that some other strands usually run in parallel with the limiting strand. This orientation of the strands characterizes the marginal regions of the bicellular junctions. However, these regions are less distinct from the principal regions than in Orchestia, first, because they do not show an increased depth, at least not a significant one, and second, because the change in orientation of the strands is less remarkable due to their tortuous path. More exactly, as regards this last point, the situation is variable. Near the apical surface the strands are close to each other and frequently organized in stacks similar to those of the principal regions. All the strands of a stack often curve together so as to become parallel to the juncture line (Fig. 14). Deeper in the junctional area the strands are more variably spaced and their path more tortuous. As shown on Fig. 15, either one single strand or an array of several parallel strands can curve or loop before running alongside a tricellular junction. 2.3. Tricellularjunctions. The two limiting strands which can be simultaneously o b s e r v e d in t a n g e n t i a l s e c t i o n s or on freeze-fracturereplicas are parallel and distant by 35 nm. The juncture space is compartmentalized by a long series of diaphragms sometimes observed in conventional sections but usually seen to better advantage after lanthanum impregnation. In most places these diaphragms are regularly ordered with a center-to-center spacing of 17.5-20 nm (average 19 nm). However, in some places two diaphragms may be further apart (Fig. 14, arrow 1), and it may even appear as if one diaphragm is missing altogether (Fig. 14, arrows 2 and 3). In longitudinal sections of tricellular junctions the diaphragms look like bridges which resemble those of Orchestia since they include a vesicular part and thin lateral arms. Depending on the sectioning inci-
dence angle, the vesicular part appears to bear either two long symmetrical arms linked to limiting septa (Figs. 14, 15), or only one such arm and a very short one which is connected to a cell corner membrane (Fig. 15, on the right side). However, each long arm has the peculiarity of being crossed by a thin plate lying in parallel with the adjacent limiting septum (Figs. 14, 15). The ends of these plates are very near those of the adjacent diaphragms so that the successive plates often appear as a thin continuous ribbon (Fig. 14). Each cell corner membrane has a row of j u n c t u r e particle d o u b l e t s w h i c h after freeze-cleavage adhere mostly to the E face in the case of both fixed (Fig. 17) and unfixed (Figs. 16, 19, 21) tissues. Hence, in the latter case the doublets are observed on the same fracture face as the particle rows, and the P faces bear the complementary depressions of both the doublets and the particle rows (furrows) (Figs. 20, 22). The juncture particles are about 11 nm in diameter (as against 9 nm for the particles of the rows). They are very close to each other and may appear fused to form a rodlet (Fig. 21), the complementary imprint of which is a short furrow (Figs. 20, 22). A cell corner membrane involved in a tricellular junction therefore appears as a specialized membrane strip, either groove shaped and bearing juncture particles on the E faces, or crest shaped and adorned with parallel short furrows on the P faces. On both faces a ladder-like aspect is obvious. However, the particles show plastic deformation (Fig. 21) so that they commonly appear less regularly ordered and spaced than the complementary pits. The centerto-center spacing of the doublet particles or their imprints ranges from 18 to 21 nm (19.5 nm on average), a value very close to that reported for the diaphragm spacing (19 nm). Occasionally, as in the case of some diaphragms, some doublets are more widely spaced with random interruptions in the doublet rows. This occurs more frequently in the basal junctional region (Fig. 17). All
TRICELLULAR JUNCTIONS, I
these data agree with the presumed association between diaphragms and particle doublets (partitions).
3. Final Remarks
2.
The posterior caeca of Orchestia and the midgut of Blaberus were chosen for this work because previous observations had shown some morphological and structural differences as regards their bicellular septate junctions. Despite this fact, in both tissues the tricellular junctions exhibit the same fundamental characteristic features with only minor variations. It should be added that sections of lanthanum-impregnated tissues and/or freeze-fracture replicas of midgut from other insect species (the termite Kalotermes flavicollis, the cockroaches Periplaneta americana and Blattella germanica) gave results quite similar to those obtained for Blaberus. The general bearing of these observations will be clearer after comparison with tissues containing pleated septate junctions (see (11)).
3.
REFERENCES
4. 5. 6. 7.
8. 9.
I0. 11. 12. 13. 14.
1. BRANTON, D., BULLIVANT, S., GILULA, N. B., 15. KARNOVSKY, M. J., MOOR, H., MUHLETHAL-
151
ER, K., NORTHCOTE,D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L., AND WEINSTEIN, R. S. (1975) Science 190, 54-56. DALLAL R. (1976) J. Submicrosc. Cytol. 8, 163174. ESCAtG, J., AND NICHOLAS,G. (1976) C. R. Hebd. S e a n c e s Acad. Sci. Ser. D 283, 1245-1248. FLOWER, N. E., AND FILSHIE, B. K. (1975) J. Cell Sci. 17, 221-239. GP,AF, F. (1978) C. R. Hebd. Seances Acad. Sci. Ser. D 287, 41--44. GRAF, F. (1978) Biol. Cell. 33, 55-62. GREEN, C. R. (1978) Proceedings of the 9th International Congress on Electron Microscopy, Vol. II, pp. 338-339, Toronto. LANE, N. J., AND HARRISON, B. J. (1978) J. Ultrastruct. Res. 64, 85-97. NOIROT, CH., AND NOIROT-T~MOTH~E,C. (1967) C. R. Hebd. Seances Acad. Sci. Ser. D 264, 2796-2798. NOIROT-TIMOTHI~E,C., GRAF, F., AND NOIROT, Ca. (1978) Biol. Cell. 33, 24a. NOIROT-TIMOTHEE,C., GRAF, F., AND NOIROT, Cry. (1982) J. Ultrastruct. Res. 78, 152-165. NOIROT-TIMOTHI~E,C., AND NOIROT, CH. (1973) J. Microsc. 17, 169-184. NOIROT-TIMOTHI~E,C., AND NOIROT, CH. (1980) Int. Rev. Cytol. 63, 97-140. REVEL, J. P., AND KARNOVSKY, M. J. (1967) J. Cell Biol. 33, C7-C12. STAEHELIN, L, A. (1974) Int. Rev. Cytol. 39, 191283.
(1982)
The Specialization of Septate Junctions in Regions of Tricellular Junctions I. Smooth Septate Junctions (=Continuous Junctions) F . G R A F , * C . N O I R O T - T I M O T H I ~ E , t AND C H . NOIROT~
*Laboratoire de Biologie animale et gdndrale, and ?Laboratoire de Zoologie, Universit( de Dijon, Boulevard Gabriel, 21100 Dijon, France Received June 1l, 1980, and in revised form August 19, 1981 Some regions from the midgut of a crustacean and an insect were comparatively studied, both in sections of lanthanum-impregnated tissues and on freeze-fracture replicas. The epithelial cells a r e apically joined by zonular smooth septate junctions which are actually composed of alternating parts built and shared in common by either two cells (bicellular junctions) or three cells (tricellular junctions). The bicellular junctions are composed of strands which include a smooth extracellular septum stretched between two intramembrane particle rows. The strands run nearly or preferentially parallel to the apical surface, except near the cell corners where they become parallel to the axes of the tricellular junctions. This change in strand orientation characterizes the marginal regions of the bicellular junctions, which moreover are bordered by a limiting strand. At tricellular junctions each cell corner membrane displays a series of regularly spaced doublets of juncture particles, somewhat larger than the strand particles. The doublets are apparently the intramembrahe components of junctional units (partitions), the chief part of which is an extracellular diaphragm showing a central vesicular part and a thinner periphery. The diaphragms compartmentalize the extracellular space and are linked to the limiting strands of the bicellular junctions abutting onto a tricellular junction.
In arthropods, as in most other inverte- ber and pattern showing a good correlation brates, epithelial cells are joined by an api- with the extracellular septa. Thus it is incal junctional complex which commonly in- ferred that each septum is associated with cludes a zonular septate junction. There is two particle rows, one in each plasma memno general agreement about the functional brane, the whole constituting the structural significance of the septate junctions though unit of the junctions and called the juncvarious data suggest that besides their role tional strand (13). in cellular cohesion they probably restrict Several types of septate junctions are the paracellular movement of fluids and recognized, two of which commonly occur thus are functionally similar to the tight in arthropods (7, 13, 15). In pleated septate junctions of vertebrate epithelia (review in junctions (4, 13), the septa show regularly (13)). spaced (20-22 nm) fine pleats and the cenThe structural organization of the septate ter-to-center spacing of the particles along junctions has mainly been studied in re- a row ranges from 10 to 20 nm. In all degions where two cells are linked. Such bi- scribed cases most particles preferentially cellular junctions are first characterized on adhere to the protoplasmic cleavage face sections by ribbon-like septa which span (P face) (I) on freeze-fracture replicas. In the intercellular space and join the two ad- smooth septate or continuous junctions (4, jacent plasma membranes. Moreover, the 9, 13, 15), the septa are parallel-sided (not freeze-fracture technique allows recogni- pleated) and the particles of a row are very tion of particle rows which are in alignment close to each other, so that they frequently in both membranes, and which have a num- form ridges. Depending on the species, tis136 0022-5320/82/020136-16502.00/0 Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
TRICELLULAR JUNCTIONS, I
137
sue, or preparative procedure, most parti- tacea, Amphipoda). Others were made on the midgut cles adhere either to the protoplasmic (P) and the anterior caeca of the midgut from several species of cockroaches, usually adults or last instar or to the exoplasmic (E) fracture face, or nymphs of Blaberus craniifer (Insecta, Dictyoptera). show no preferential binding. Moreover, small intermembrane pillars without intra- Techniques membrane counterparts are commonly ob1. Conventional sections. These were prepared as served in the interseptal spaces as soon as described elsewhere (6, 12). 2. Lanthanum impregnation. A lanthanum solution these are large enough to accommodate at was prepared according to the procedure of Revel and least one row of pillars. Karnovsky (14) and added to the fixation and washing A zonular junction also includes regions mixtures (1% final lanthanum concentration). Tissues where three cells are joined. Some features were fixed (18 hr) in a solution of 2% formaldehyde of these regions of tricellular junctions, (freshly prepared from paraformaldehyde) and 3% glustudied in sections, have been previously taraldehyde in 0.08 or 0.1 M cacodylate buffer. They were washed in the same buffer, postfixed (1 hr) in reported in the case of pleated septate junc- buffered 1% OSO4, then dehydrated in an ethanol setions (12). In these regions several septa of ries and embedded in an Araldite-Epon mixture. Secthe convergent bicellular junctions run in tions were examined without further contrast enparallel alongside the junction line between hancement. 3. Freeze-fracture replicas. Most specimens were the three cells and the most central septa briefly (about 0.5 hr) fixed in cacodylate-buffered 2% are joined by a series of regularly spaced glutaraldehyde and infiltrated with a buffered 40% sobridges, with each bridge showing a ring- lution of glycerol for at least 12 hr. Some of these shaped central part and thin lateral arms. specimens were frozen in Freon 21 and stored in liquid In order to obtain new and more precise nitrogen, then fractured and replicated in a BAF 301 information on the regions of tricellular (Balzers) apparatus. Other specimens were frozen in melting nitrogen before processing in a Cryofract 250 junctions, tissues have been selected where (Reichert) apparatus (3). For the Blaberus midgut the septate junctions are of either the pleat- some tissue specimens were not fixed and were either ed or the smooth type and which moreover treated (1 hr) or not with glycerol before freezing in for a given type exhibit some morphological melting nitrogen. Sections and freeze-fracture replicas were examined or structural differences. Both intercellular in a Hitachi HU-l I E electron microscope. and intramembrane structures have been studied comparatively using sections of RESULTS AND INTERPRETATION lanthanum-impregnated tissues and freezeIn monolayered epithelia such as those fracture replicas. A tridimensional interpreexamined, any three contiguous cells are tation of the observed data has been perapically linked in pairs through three bicelformed. luiar septate junctions and also together The results of this work are reported in through a tricellular junction (Fig. 1). This two papers dealing with the smooth and the tricellular junction follows a more or less pleated septate junctions, respectively. sinuous line which we call the "juncture However, because of the common characline." This is the axis of the "juncture teristics recognized in all cases for regions space" lying between the three cell corners. of tricellular junctions the observations are Near the juncture space the bicellular discussed together in the second paper (I1). Preliminary results have been pub- junctions undergo morphological changes. lished previously in abstract form (10) and Thus it appears necessary to distinguish and to successively describe the principal in a review article by two of us (13). and the marginal (i.e., the area near the MATERIALS AND TECHNIQUES juncture space) regions of these bicellular Materials junctions before examining the tricellular Some of the observations were carried out on the junctions and the specialized devices enmidgut posterior caeca of Orchestia cavimana (Cruscountered at their level.
138
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C FIG. 1. (a) An interpretative diagram of the cross-sectioned parts of three cells involved in three bicellular junctions (B) and a tricellular junction (T). Both inter- and intramembrane junctional components are shown. JS, junctional strand, the structural unit of bicellular junctions, composed of a septum (S) stretched between two intramembrane particle rows (R). LS, limiting strand, forming the limit of a bicellular junction where it abuts against a tricellular junction. P, partition, the structural unit of tricellular junctions, composed of a diaphragm (D) located in the juncture space and linked to three doublets of juncture particles (J). The diaphragm includes a central vesicular part and a thinner periphery connected with the limiting strands and the corner membranes. (b) and (c) Limiting septa (LS) and diaphragms (D) as they appear in longitudinal sections along the planes X X ' (b) and YY' (c) shown on diagram (a). See detailed description in the text and compare with Figs. 4 and 5.
1. Posterior Caecum of Orchestia (Crustacea) During the intermolt (state C), the epithelial cells are columnar, about 3-8/xm in
width and 50 /xm in height. Their lateral faces are smooth and linked apically by a zonular smooth septate junction (6) and more basally by numerous macular gap junctions (5).
FIGS. 2-5. Caecum of Orchestia. Unstained sections of lanthanum-impregnated specimens, showing intermembrane junctional components in negative contrast. FIG. 2. Section of the apical region of the epithelium, below the microvilli (MV), showing three cells (C l, C2, C3), the bicellular junction (B) between C1 and C2, and the tricellular junction (T) between C1, C2, and C3. In the principal region of the bicellular junction (on the left), the septa (S) run parallel to the apical surface. In the marginal region (i.e., near the tricellular junction), they curve toward the base, loop (*), and then run parallel to the juncture line as they come back toward the apex. The juncture space includes a series of regularly superposed diaphragms (D), x 60 000. FIGS. 3-5. Tricellular junctions (T) and adjoining parts of tangentially sectioned bicellular junctions. In the marginal regions of bicellular junctions several septa run parallel to the juncture line because of their previous looping course (*). Interseptal spaces are commonly large enough to include pillars (P). Segments of septa follow each other in various ways (arrows l, 2, 3; compare with Fig. 12b) to form the limiting septa (LS). Tricellular junctions are characterized by the occurrence of diaphragms (D). These are regularly superposed in the juncture space and look like bridges which include a vesicular part and thinner lateral arms. In Fig. 4, at center and bottom, the vesicular part is linked on the left to a cell corner membrane (M) by a very short arm and on the right to a limiting septum (LS) by a longer arm (compare with Fig. lb). On Fig. 4 at the top and on Fig. 5, the sectioning incidence is somewhat different and the vesicular part bears two symmetrical arms, each inserted on a limiting septum (compare with Fig. lc). Fig. 3, × 225 000; Figs. 4, 5, x 300 000.
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GRAF, NOIROT-TIMOTHI~E, AND NOIROT
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FIG. 6. A three-dimensional diagram of the intermembrane structures; that is, the septa (S) of the three bicellular junctions (B1, B2, B3) and the diaphragms (D) of the tricellular junction (T) which link the membranes (M1, M2, M3) of three cells. The diaphragms are arranged in a regular file in the tricellular juncture space and each one is normally connected to the three discontinuous limiting septa (LS) and to the three cell corner membranes surrounding this space.
1.1 Principal regions of bicellular junctions. As previously reported (6), the septa look like smooth ribbons, well delineated by lanthanum (Figs. 2 to 5), and the freezecleaved membranes of fixed specimens show particle ridges on the P faces or com-
plementary furrows on the E faces (Figs. 7 to 10). The junctional strands (septa and particle ridges or furrows), although slightly wavy, are on the whole parallel to the apical surface (Figs. 2, 7, 8, 12a). Arrays of close and equidistant septa are frequent and in these cases each interseptal space (5-6 nm) includes a row of pillars. However, in some places septa are more widely spaced and the interseptal spaces include numerous and randomly distributed pillars. The depth of the junction ranges from 0.12 to 1 /xm and the number of strands from 3 to 30. It must be stressed though that these large variations occur between different junctions and not along a given one, at least as far as the principal regions are considered.
1.2 Marginal regions of bicellular junctions. The differences between bicellular junctions as regards their depth and strand number are progressively reduced as they approach a tricellular junction because supplementary strands can appear above those coming from the principal region (Figs. 8, 12a). Characteristic morphological changes are observed in the marginal regions of all bicellular junctions. Their depth suddenly increases and usually reaches 1.6-2/xm, although a depth of only 1.2/zm is reached when two tricellular junctions are close to each other. In these marginal regions, 0.3 to 0.6 ~m in width, the junctional strands become more spaced and their course is modified to different degrees depending on their position. The basal strands curve toward the cell base, then with a hairpin loop curve back toward the apical surface to
Fi~s. 7 AND 8. Caecum of Orchestia. Freeze-fracture replicas of fixed specimens, each including two bicellular junctions and one tricellular (T) junction. The bicellular junctions are characterized by particle ridges (R) on the P faces (P) and complementary furrows (F) on the E faces (E). In the principal regions of the junctions, ridges and furrows are close to each other and parallel to the apical surface covered by microvilli (MV). In the marginal regions (M), the junction depth greatly increases and the ridges or furrows become more widely spaced. Those of the basal half exhibit a conspicuous hairpin pattern (*) and then run in a basoapical direction, following each other to form a limiting strand (LS) on each side of the tricellular junctions (T). These are seen in both figures on a P face and appear as a crest bearing a series of juncture particles. G, E-Type gap junctions; TS, twisted apical supplementary strands, x 50 000.
TRICELLULAR JUNCTIONS, I
141
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GRAF, NOIROT-TIMOTHI~E, AND NOIROT
which they are then perpendicular (Figs. 2, 7, 8, 12a). Most of the strands run parallel to the tricellular juncture line before ending and their terminal segments overlap and follow each other in progression (Figs. 5, 10, and 12b, arrow 1) to form a discontinuous "limiting strand" including a "limiting septum." In some places the overlapping of the terminal segments is reduced or absent (Figs. 4 and 12b, arrow 2) and moreover some strands recurve instead of ending, leaving a larger gap in the limiting strand between the two curves moving in opposite directions (Figs. 3 and 12b, arrow 3). Nevertheless, a limiting strand always occurs where a bicellular junction abuts against a tricellular junction (Figs. 1, 3-10, and 12). The median strands behave somewhat like the basal ones except that they either do not curve toward the base, or only form a short loop before following a basoapical course (Fig. 12a). The apical strands are not involved in making the limiting strands; they turn back without any previous bending toward the base so that the limbs of the loops are parallel to the apical surface (Fig. 12a). The most apical strands sometimes form concentric loops or spirals (Figs. 7, 8, 12a). Two facts should be emphasized. First, the information given by either sections of lanthanum-impregnated tissues or freezefracture replicas is quite similar even though the replicas furnish more extensive picture areas. This is new indirect evidence of the presumed association between septa and intramembrane particle rows. Second,
bicellular junctions structurally appear to be well separated and autonomous, the strands never passing over a juncture line from one bicellular junction to another, and the limiting strands, although discontinuous, are really the borders of the bicellular junctions. 1.3. Tricellular junctions. Whatever the previous course and orientation of the plasma membranes, the dihedral angles of the three apposed cells always appear to be equal (120°) around the juncture space (Fig. 11). The limiting strands of the three convergent bicellular junctions, only two of which can be simultaneously seen in tangential sections (Figs. 4, 5) and on freezefracture replicas (Figs. 7-10, 12), are parallel and separated by 25-30 nm. Together with the corner membranes of the three cells they circumscribe the juncture space where a series of superposed diaphragms is found (Fig. 6). The center-to-center distance between the diaphragms ranges from 13 to 18 nm (average 16.5 rim). In longitudinal sections of tricellular junctions these diaphragms are well delineated by lanthanum. They appear as transverse bridges, each with a thicker central part and thinner lateral arms. The central parts have the shape of a flattened ring (9to 10-nm maximum thickness) surrounding an ovoid space filled by lanthanum (Figs. 3-5). According to the angle of incidence of the section the detailed aspects are somewhat different. In some places, the septa of only one bicellular junction are observed (Figs. lb, 3, 4) and on the other side
FIGS. 9 AND 10. Caecum of Orchestia. Freeze-fracture replicas of fixed specimens. On the E faces (E), a tricellular junction (T) can be recognized as a groove bearing some juncture particles and the imprints (arrowheads on Fig. 10) of others. Typically two juncture particles lie side by side forming a doublet (D) or are fused into a rodlet (R). On each side of the groove the basal furrows of the bicellular junctions display their characteristic looping pattern (*). It can be seen in Fig. 10 that the terminal parts of the furrows overlap and follow each other to form the border (limiting strand, LS) of the bicellular junctions. Their ends (arrows marked 1) are located just beside a juncture particle. Fig. 9, ×100000; Fig. 10, x 175 000. FIG. 11. Caecum of Orchestia. Conventional section showing three bicellular junctions (B) abutting onto a transversely cut tricellular junction (T). At this point the three plasma membranes always form equal angles. Septa of the bicellular junctions and diaphragms of the tricellular junction are not visible as is common in conventional sections. × 350 000.
T R I C E L L U L A R JUNCTIONS, I
143
144
GRAF, NOIROT-TIMOTHI~E, AND NOIROT LS A.
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MR
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FIG. 12. Diagrams summarizing information obtained from freeze-fracture replicas of Orchestia caecum. (a) A tricellular junction (T) is located in between two bicellular junctions, the principal regions of which include either (on the left) rather numerous strands, or (on the right) only a few. In both cases their marginal regions (MR) display an increased depth and the characteristic looping pattern of the strands (see more details in the text). A, Apical strands; B, basal strands; M, median strands; SS, supplementary strands; TS, twisted supplementary strands. (b) A tricellular junction (T) is characterized by a row of regularly spaced juncture particle doublets (D). These lie on a membrane strip bordered on both sides by a limiting strand (LS), which is actually a succession of strand segments which follow each other in various ways (arrows 1, 2, 3) before ending or curving. The end points are located beside a juncture particle doublet (arrows 1, 2).
of the juncture space a faint electron-translucent line eventually appears (Fig. 4), which probably represents the corner membrane of a cell. In these cases the bridges appear asymmetric. The ring-shaped part is more distant from the septa side than from the other side and it is linked to the observed limiting septum by a long, clearly seen arm, whereas on the opposite side it is connected to the plasma membrane directly or through a very short arm. In other places the septa of two bicellular junctions are visible and the bridges appear symmetrically organized (Figs. lc, 5). The ringshaped part (15-16 nm in width) lies in the middle of the juncture space and is joined to each limiting septum by lateral arms of equal length. In some sections the two types of images can be seen in succession (Fig. 4) because of a slight tilting of the whole junction within the depth of the section. These data indicate that the bridges are true diaphragms which compartmentalize the juncture space (Fig. 6). Each one has
an inflated central part with a biconvex lens shape and a thinner periphery which is linked to the limiting septa of the surrounding three bicellular junctions and to the three cell corner membranes. The peripheral part has been tentatively pictured as a continuous and homogeneous platform (Figs. la, 6). However, this cannot be definitely ascertained and it is possible that it is heterogeneous or made of independent arms. On freeze-fracture replicas the limiting strands (particle rows on the P faces or complementary furrows on the E faces) of the bicellular junctions are separated by a membrane strip seen as a crest on the P faces (Figs. 7, 8) and as a groove on the E faces (Figs. 9, 10). At this level the membrane includes a row of juncture particle pairs (called "doublets") which mostly adhere to the P faces but in some places are observed on the E faces. The two particles of a doublet are close together and are sometimes joined to form a small rod (called "rodlet") (Fig. 10). The average di-
TRICELLULAR JUNCTIONS, I
ameter of the juncture particles (7.5 nm) is larger than that of the strand particles (5 nm). The successive doublets are regularly ordered in a ladder-like way, with an average center-to-center spacing of 16.5 nm, that is to say, the same value as that reported for the diaphragm spacing. This fact favors the idea that the particle doublets are the intramembrane counterparts of the diaphragms. Further evidence of this is provided by the fact that along a limiting strand the ends of the composing segments are located at the level of a diaphragm on the one hand and of a doublet on the other (see especially Fig. 4, arrow 2, and Fig. 10, arrow 1, and compare with Fig. 12b, arrows 2 and 1). Consequently the structural units of tricellular junctions, called "partitions" (13), appear to include an extracellular diaphragm and three doublets of intramembrane juncture particles.
2. Midgut of Blaberus (Insecta) In last instar nymphs, as well as in adults, mature epithelial cells are at least 100/xm in height and about 15/xm in diameter. Lateral membranes are smooth except in the subapical region where more or less deep foldings are always observed. As in the midgut of other insect species (13), the cells are linked by a well-developed zonular smooth septate junction which begins immediately below the microvilli since it is not preceded by a belt desmosome. Some small-sized macular desmosomes are included in the apical part of this smooth septate junction. Rather numerous gap junctions are either intercalated in the septate junction, mostly in its basal half, or located just below the septate junction.
2.1. Principal regions of bicellular junctions. The septa of bicellular junctions (Figs. 13-15) resemble those observed ifi Orchestia except that they are somewhat thicker. Freeze-cleaved junctional membranes give different pictures depending on the previous treatment of the specimens. This has also been observed in the midgut or Malpighian tubules of other arthropod
145
species (2, 4, 8). When a fixation step is used (Figs. 17, 18) the two fracture faces are more or less similar. On both faces, each strand is seen as a succession of furrows and particle files, the particles often being closely apposed in ridges. Furrows and ridges (or particle files) are of quite variable length. The partitioning of the membrane particles between the two cleavage halves changes when the specimens have not been fixed, regardless of whether or not they were treated with glycerol. In these cases (Figs. 16, 19-22), almost all the particles adhere to the E faces so that these bear continuous ridges which are easily resolved as apposed particles whereas the P faces are characterized by complementary furrows. This means that the picture is then the reverse of that reported for the smooth septate junctions of Orchestia. It should be added that the partitioning of the gap junction particles is not modified by the fixation. In all instances these particles are observed on the E faces and the complementary pits on the P faces (type E gap junctions). The junctional strands are much more numerous and are also more sinuous and variably distributed than in Orchestia. The most apical strands are very close to each other and are typically organized in stacks (Figs. 13, 14, 16) where many of the interseptal spaces are very narrow and thus do not include pillars. In many places the stacked strands lie parallel to the apical surface (Figs. 13, 14, 16) although they can diverge and curve. Below the apical strip showing this organization, the junctional strands follow a very tortuous path and are unevenly spaced (Figs. 15-22). In some places several strands run in parallel and very close to each other, the interseptal spaces being very narrow and often without pillars. More frequently, however, the strands are variably spaced due to the curves and loops that they describe and numerous randomly distributed pillars are present in the interseptal spaces unless such a space is occupied by a gap junction.
146
GRAF, NOIROT-TIMOTHI~E, AND NOIROT
The average depth of the bicellular smooth septate junctions in Blaberus is 15 /zm. The number of the strands is more than a hundred although this is hard to state precisely due to the large junction depth, the tortuous path of the strands, and the paucity of extended cleavage areas obtained
for the apical region, probably because of the membrane foldings.
2.2. Marginal regions of bicellular junctions. Around any tricellular juncture space, each bicellular junction is clearly bordered by a limiting strand which, as in Orchestia, is formed by a succession of
FIGS. 13-15. Midgut of Blaberus. Unstained sections of lanthanum-impregnated tissues which show intermembrane junctional components in negative contrast. Each figure includes a tricellular junction (T) and adjacent parts of two bicellular junctions. FIG. 13. Near the apex most septa of the bicellular junctions are very close to each other and organized in stacks although some can diverge and curve (arrow). Pillars (P) are present in the interseptal spaces only where the spaces are large enough. In the principal region (a) of a bicellular junction the septa are mostly parallel to the apical surface covered by microvilli (MV), while in the marginal regions (b) they are parallel to the tricellular junction (T). Here the juncture space includes a series of diaphragms (D). x 90 000. FIC. 14. At the top of the figure a stack of septa (a), with pillars (P) in some interseptal spaces, is located just below the microvilli (not included) and the septa are parallel to the apical surface. On the right the septa of another stack (b) curve to become parallel to a tricellular junction (T). The juncture space is bordered on each side by a limiting septum (LS) which, as can be seen on the right-hand side, is composed of a succession of segments following each other. The diaphragms (D) located in the juncture space display a central annular part and lateral arms which are crossed by a thin plate (PL). Successive plates may form a ribbon (R) parallel to, but thinner than, the limiting septa. Most spaces between diaphragms are of equal size although one is slightly larger (arrow 1) and two others are twice as large (arrows 2 and 3). x 150 000. FIG. 15. Deeper in the junctional area, the septa are more frequently curved or looped and unevenly arranged, often with large interseptal spaces including numerous pillars (P). Some septa follow each other (arrowheads) to form a limiting septum (LS) on each side of the tricellular junction (T). At the center, each diaphragm shows two symmetrical arms crossed by a thin plate (PL) and inserted onto a limiting septum. On the right, the annular part of the diaphragms is linked on one side to a cell corner membrane (M) either directly or by a short arm, and on the other side to a limiting septum (LS) by a larger arm. x 200 000. FIG. 16. Midgut of Blaberus. Freeze-fracture replica of a glycerinated unfixed specimen. Apical regions of three cells (C 1, C2, C3) covered by microvilli (MV) and two bicellular junctions (B1, B2), linking, respectively, CI to C2 and C2 to C3, are shown. The bicellular junctions are characterized by particle rows on the E faces (E) and complementary furrows on the P faces (P). Just below the microvilli furrows (F) are very close to each other and mostly parallel to the epithelium surface while deeper they are variably spaced and curved. On the left toward the top cell C2 terminates and the two bicellular junctions abut onto each side of a specialized membrane strip which thus can be identified as part of a tricellular junction (T). Here, on an E face, the specialized strip is groove shaped and bears juncture particles in a ladder-like arrangement. Moreover, some strands of the bicellular junctions run in parallel to the rims of the groove. (Blisters in junctions were caused by a slightly hypertonic glycerol solution.) x 55 000. FIGS. 17-22. Midgut of Blaberus. Freeze-fracture replicas of fixed (Figs. 17, 18) and unfixed (Figs. 19-22) specimens, each showing a tricellular junction (T) either on an E face (Figs. 17, 19, 21) or a P face (Figs. 18, 20, 22) and the adjoining parts of two bicellular junctions. The occurrence of a tricellular junction (T) is indicated by an elongated membrane strip more or less groove shaped on E faces (E, Figs. 17, 19, 21) and crest shaped on P faces (P, Figs. 18, 20, 22). In both fixed and unfixed tissues, the E-face grooves bear juncture particles (J) forming doublets (D) or fused in rodlets (R) while complementary imprints (i) (paired pits or short furrows) as well as a few particles (J, Fig. 22) are observed on the P-face crests. Pits are more obvious on replicas of unfixed specimens (Figs. 20, 22). The imprints are regularly spaced (Figs. 20, 22) but plastic deformation produces a less regular pattern of the particle doublets (Fig. 21). Interruptions in the series of juncture particles sometimes occur, especially in the basal junctional area (*, Fig. 17). Bicellular junctions of fixed tissues display particle rows interrupted by furrows on both E (E, Fig. 17) and P (P, Fig. 18) faces. On replicas of unfixed tissues particle rows are observed on E faces (E, Figs. 19, 21) and complementary furrows on P faces (P, Figs. 20, 22). Particle rows and furrows conspicuously meander except on each side of tricellular junctions where following segments form a limiting strand (LS, chiefly visible on Fig. 22, left side), usually paralleled by one or more other strands. Figs. 17, 18; x 65000; Figs. 19, 20; x 60000; Figs. 21, 22; x 200000.
TRICELLULAR JUNCTIONS, I
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strand segments (Figs. 14, 15, and 17-22). These are of unequal length and commonly overlap and follow each other (Figs. 14, 15, 22) so that some other strands usually run in parallel with the limiting strand. This orientation of the strands characterizes the marginal regions of the bicellular junctions. However, these regions are less distinct from the principal regions than in Orchestia, first, because they do not show an increased depth, at least not a significant one, and second, because the change in orientation of the strands is less remarkable due to their tortuous path. More exactly, as regards this last point, the situation is variable. Near the apical surface the strands are close to each other and frequently organized in stacks similar to those of the principal regions. All the strands of a stack often curve together so as to become parallel to the juncture line (Fig. 14). Deeper in the junctional area the strands are more variably spaced and their path more tortuous. As shown on Fig. 15, either one single strand or an array of several parallel strands can curve or loop before running alongside a tricellular junction. 2.3. Tricellularjunctions. The two limiting strands which can be simultaneously o b s e r v e d in t a n g e n t i a l s e c t i o n s or on freeze-fracturereplicas are parallel and distant by 35 nm. The juncture space is compartmentalized by a long series of diaphragms sometimes observed in conventional sections but usually seen to better advantage after lanthanum impregnation. In most places these diaphragms are regularly ordered with a center-to-center spacing of 17.5-20 nm (average 19 nm). However, in some places two diaphragms may be further apart (Fig. 14, arrow 1), and it may even appear as if one diaphragm is missing altogether (Fig. 14, arrows 2 and 3). In longitudinal sections of tricellular junctions the diaphragms look like bridges which resemble those of Orchestia since they include a vesicular part and thin lateral arms. Depending on the sectioning inci-
dence angle, the vesicular part appears to bear either two long symmetrical arms linked to limiting septa (Figs. 14, 15), or only one such arm and a very short one which is connected to a cell corner membrane (Fig. 15, on the right side). However, each long arm has the peculiarity of being crossed by a thin plate lying in parallel with the adjacent limiting septum (Figs. 14, 15). The ends of these plates are very near those of the adjacent diaphragms so that the successive plates often appear as a thin continuous ribbon (Fig. 14). Each cell corner membrane has a row of j u n c t u r e particle d o u b l e t s w h i c h after freeze-cleavage adhere mostly to the E face in the case of both fixed (Fig. 17) and unfixed (Figs. 16, 19, 21) tissues. Hence, in the latter case the doublets are observed on the same fracture face as the particle rows, and the P faces bear the complementary depressions of both the doublets and the particle rows (furrows) (Figs. 20, 22). The juncture particles are about 11 nm in diameter (as against 9 nm for the particles of the rows). They are very close to each other and may appear fused to form a rodlet (Fig. 21), the complementary imprint of which is a short furrow (Figs. 20, 22). A cell corner membrane involved in a tricellular junction therefore appears as a specialized membrane strip, either groove shaped and bearing juncture particles on the E faces, or crest shaped and adorned with parallel short furrows on the P faces. On both faces a ladder-like aspect is obvious. However, the particles show plastic deformation (Fig. 21) so that they commonly appear less regularly ordered and spaced than the complementary pits. The centerto-center spacing of the doublet particles or their imprints ranges from 18 to 21 nm (19.5 nm on average), a value very close to that reported for the diaphragm spacing (19 nm). Occasionally, as in the case of some diaphragms, some doublets are more widely spaced with random interruptions in the doublet rows. This occurs more frequently in the basal junctional region (Fig. 17). All
TRICELLULAR JUNCTIONS, I
these data agree with the presumed association between diaphragms and particle doublets (partitions).
3. Final Remarks
2.
The posterior caeca of Orchestia and the midgut of Blaberus were chosen for this work because previous observations had shown some morphological and structural differences as regards their bicellular septate junctions. Despite this fact, in both tissues the tricellular junctions exhibit the same fundamental characteristic features with only minor variations. It should be added that sections of lanthanum-impregnated tissues and/or freeze-fracture replicas of midgut from other insect species (the termite Kalotermes flavicollis, the cockroaches Periplaneta americana and Blattella germanica) gave results quite similar to those obtained for Blaberus. The general bearing of these observations will be clearer after comparison with tissues containing pleated septate junctions (see (11)).
3.
REFERENCES
4. 5. 6. 7.
8. 9.
I0. 11. 12. 13. 14.
1. BRANTON, D., BULLIVANT, S., GILULA, N. B., 15. KARNOVSKY, M. J., MOOR, H., MUHLETHAL-
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ER, K., NORTHCOTE,D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L., AND WEINSTEIN, R. S. (1975) Science 190, 54-56. DALLAL R. (1976) J. Submicrosc. Cytol. 8, 163174. ESCAtG, J., AND NICHOLAS,G. (1976) C. R. Hebd. S e a n c e s Acad. Sci. Ser. D 283, 1245-1248. FLOWER, N. E., AND FILSHIE, B. K. (1975) J. Cell Sci. 17, 221-239. GP,AF, F. (1978) C. R. Hebd. Seances Acad. Sci. Ser. D 287, 41--44. GRAF, F. (1978) Biol. Cell. 33, 55-62. GREEN, C. R. (1978) Proceedings of the 9th International Congress on Electron Microscopy, Vol. II, pp. 338-339, Toronto. LANE, N. J., AND HARRISON, B. J. (1978) J. Ultrastruct. Res. 64, 85-97. NOIROT, CH., AND NOIROT-T~MOTH~E,C. (1967) C. R. Hebd. Seances Acad. Sci. Ser. D 264, 2796-2798. NOIROT-TIMOTHI~E,C., GRAF, F., AND NOIROT, Ca. (1978) Biol. Cell. 33, 24a. NOIROT-TIMOTHEE,C., GRAF, F., AND NOIROT, Cry. (1982) J. Ultrastruct. Res. 78, 152-165. NOIROT-TIMOTHI~E,C., AND NOIROT, CH. (1973) J. Microsc. 17, 169-184. NOIROT-TIMOTHI~E,C., AND NOIROT, CH. (1980) Int. Rev. Cytol. 63, 97-140. REVEL, J. P., AND KARNOVSKY, M. J. (1967) J. Cell Biol. 33, C7-C12. STAEHELIN, L, A. (1974) Int. Rev. Cytol. 39, 191283.