Cell Tiss. Res. 185, 527-534 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Freeze Fracture Studies on the Annelid Septate Junction Ulrich Welsch Anatomisches Institut der Universitfit Kiel, Germany Wolfgang Buchheim Institut ffir Physik der Bundesanstalt ffir Milchforschung, Kiel, Germany
Summary. Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80-120 A, the distance between two particles (centre to centre) is 150-250 A. Additionally striking rows of large particles (long diameter 150-200 A). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100-150 A, centre to centre distance approx. 250 A) whereas the particles of the extracellular face (EF, mean diameter 200-250 A) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species. Key words: Septate j unction - Annelids (Lumbricus terrestris and Tubifex spec.) - Epidermis - Freeze-fracture.
In an effort to bring order into a wealth of individual observations Staehelin (1974) suggested a preliminary classification of septate junctions into three major groups: a) Hydra type, b) pleated sheat type, c) continuous type. Trying to apply this classification to his observations on polychaete septate junctions Baskin (1976) found that the epidermal septate junctions of polychaetes do not fit, in all respects, into one of Staehelin's three types, but instead exhibited a number of characters of their own. Since Baskin (1976) did not use freeze-fracture preparations for his analysis we found it worthwhile to apply this technique to annelid material in order to find more structural details about the septate junctions in these animals.
Sendoffprint requeststo: Prof. U. Welsch,AnatomischesInstitut der Universidit,Olshausenstr. 40-60, D-2300 Kiel, Federal Republic of Germany
528
U. Welsch and W. Buchheim
Materials and Methods Small pieces(about 1 x 1 mm) of the body wall of Lumbricusterrestriswere transferred for 10mins into a 30 % glycerol/saline mixture for cryoprotective pretreatment. Tubifex spec. (diameter approx. 0.3 mm) was prepared without cryoprotection by using slices of approx. 0.5 mm thickness. Cryofixation was performed under usual conditions, using gold sample holders (Balzers) and Freon 22 (-160~C) as coolant. Freeze-etching preparation was carried out in a Balzers 360 M unit at an object temperature of - 105~C and an etching period of 1 min. For replication electron gun evaporation of platinum/carbon (approx. 17 A. thickness, shadowing angle 45~ and carbon (approx. 100 A) was used. Cleaning of the replicas was achieved by sodium hypochlorite solution. Results A. Lumbricus The septate junction occupies the apical quarter o f the epidermal cells. The protoplasmic face (PF, B r a n t o n et al., 1975) o f the plasma m e m b r a n e o f the epidermal cells is characterized in the area o f the septate junction by variously arranged rows o f particles(Fig. 1). The general direction ofthese rows runs roughly parallel to the epithelial surface; occasionally the majority o f rows can be directed vertically to the epithelial surface. In the upper two thirds or three quarters o f the junctional area these rows form a rather loose system and take an undulating course. Here they are separated by distances o f between about 500 and 2000A. In the lower third or quarter the rows are rather straight (Fig. 2a) and usually run closely together separated by a space o f 150 to 200 A. In spite o f their often irregular course one can follow individual rows over long distances. N o instance o f a fusion o f two rows into one was found. Rarely a row can form a loop and run back. The a p p a r e n t diameter o f the individual particles is about 80-120A, the distance between two particles (centre to centre) generally measures 150-250A. In addition to the above mentioned rows a different type o f particle-row (Fig. 2b) is regularly to be observed in the P F o f the lower part o f the junction and often isolated from the above mentioned system. This second type o f particle is relatively large and o f elongated shape (long diameter 150-200A, distance from adjacent particles generally 200-250A). The long axis o f these particles is perpendicularly directed to the direction o f the row (Fig. 2b). They occasionally were observed to terminate (or start) at a certain point o f the membrane. A t this terminal or beginning section o f the row, the particles are distinctly smaller and o f more globular shape (Fig. 2b). So far no definite statements can be given on the appearance o f the extracellular face (EF, B r a n t o n et al., 1975) since that face was not clearly exposed in our preparations. B. Tubifex A l t h o u g h the material o f Tubifex was not pretreated with an antifreeze large areas o f fractured plasma membranes could be observed. The structural integrity o f the m e m b r a n e s was not seriously affected by a limited degree o f ice crystal formation in the extracellular and intracellular fluids. Both fracture faces (EF and PF) could be inspected frequently (Fig. 3).
Fig. 1. Area of the septate junction in the epidermis of Lumbricus. Cu cuticle, Mv microvilli, double arrows: area with particle rows running closely together (basal part of the junction), asterisks: area with loosely arranged particle rows (apical part of the junction), x 72,000
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U. Welsch and W. Buchheim
Fig. 2a and b. Epidermal cell of Lumbricus. a Particle rows in the protoplasmic face (PF) of a fractured plasma membrane, ap apical, ba basal part of the junction, b Row with large particles, a, b x 80,000
Annelid Septate Junction
531
Fig. 3. General view of a septate junction in the epidermis of Tubifex. EF, P F extracellular and protoplasmic face, My regions of microviUi, x 20,000
Fig. 4. a Epidermis of Tubifex (detail from Fig. 3). The selected area shows the arrangement of membrane particles of the septate junction on both protoplasmic (PF) and extracellular (EF) faces of the plasma membrane; is: intercellular space, b Ladder-shaped structure in the protoplasmic face (PF) of a fractured plasma membrane (probably intestinal epithelium or chloragogue tissue) of Tub~,x. 1, 2: cytoplasm of neighbouring cells, a, b x 80,000
Annelid SeptateJunction
533
In the epidermis the protoplasmic face (PF) is of similar appearance as in
Lumbrieus. It contains rows of distinct individual particles (mean diameter 100-150N, centre to centre distance 200-300A) (Fig. 4a). The extracellular face (EF) contains densely arranged strands of particles (Figs. 3 and 4a). These particles are of distinctly different appearance if compared with those of the protoplasmic face. Their diameter generally measures 250 A, they are relatively flat and so closely attached to each other (Fig. 4a) that they form continuous strands. These strands mostly take an undulating course. Frequently up to 10 and more strands are closely attached to each other. In such areas usually a two-dimensional periodicity of the membrane particles and correspondingly arranged small depressions between the single strands exist. On the whole the junctional area of the Tubifex epidermis is characterized by considerably denser arranged rows of particles than in Lumbricus. In the membranes (PF) of the intestinal epithelium (or possibly chloragogue tissue) peculiar ladder shaped structures have been detected which cannot be assigned to any of the known junctional types (Fig. 4 b). The individual elements of this structure measured approx. 50~ in thickness and 300-500A in length and consisted of closely arranged 50A subunits. The elongated elements were mostly grouped in parallel with a distance of 150-200/k.
Discussion
The present investigation of the annelid septate junctions has revealed a much less regular construction than one could expect after the appearance in sectioned material in which this type of intercellular junction is of rather uniform and regular structure (Baskin, 1976; Coggeshall, 1966; Storch and Welsch, 1970). In the freezefractured preparations the general arrangement of the membrane particles is surprisingly variable and not even the same in the two annelid species investigated in the present paper. It is difficult to explain this discrepancy between sectioned and fractured material. In the terrestrial species (Lumbricus) the membrane particles form rows often - especially in the apical region - widely separated from each other by distances up to 2000 N. They take and undulating course. In the basal part of the junction the particle rows are running more closely together. In the apical part of the Lumbricus septate junction the arrangement of particles resembles that of the curved ridges of the septate junctions of the continuous type (Gilula, 1974; Staehelin, 1974). It is possibly this more loosely constructed part of the junction into which ruthenium red penetrates (Baskin, 1976). The individual particles in the protoplasmic membrane face, which are sometimes believed to represent the structural connection between septa and membrane (literature see Staehelin, 1974), are roughly of the same dimensions as in molluscs and arthropods (Gilula et al., 1970; Gilula, 1974; Staehelin, 1974; own observations). A new feature are the more or less isolated rows of large particles in the Lumbricus epidermis and the ladder shaped structures in plasma membranes of Tubifex, the significance of which remains unknown. Of special interest are the findings in the Tubifex epidermis. In this animal the protoplasmic and extracellular face bear membrane particles. These also are arranged in rows; however, they stand much closer together and the individual
534
U. Welsch and W. Buchheim
strands which they form are usually also in much closer contact than in Lumbricus. Thus, in the fresh water species Tubifex the general compact construction of the junction to some degree resembles that of the septate junctions of the pleated sheat type in molluscs and arthropods (Gilula et al., 1970; Staehelin, 1974). In the EF the peculiarly flattened particles of several neighbouring strands can lie so closely together that they form a regular two-dimensional pattern, which resembles the images of the enface views of the lanthanum infiltrated polychaete junctions (Baskin, 1976). The particles of the PF seem to fit into depressions which can be recognized between neighbouring strands of the EF. Our results to some degree support the speculation of Baskin (1976) that the surface of the membrane in the septate junction is characterized by regularly arranged particles, separated by narrow spaces. In any case, in Tubifex (and probably also in Lumbricus) the plasma membrane contains particles, which structurally differ from each other, those of the extracellular face even being larger than those of the protoplasmic face. Although the present investigation has shown that there are fundamental similarities in structure between annelid, molluscan and arthropod septate junctions it also supports the view of Baskin (1976) who found that the annelid septate junction has many characteristics of its own.
References Baskin, D.G.: The fine structure of polychaete septate junctions. Cell Tiss. Res. 174, 55 67 (1976) Branton, D., Bullivant, S., Gilula, N.B., Karnovsky, M.J., Moor, H., M(ihlethaler, K., Northcote, D.H., Packer, L., Satir, B., Satir, P., Speth, V., Staehelin, L.A., Steere, R.L., Weinstein, R.S.: Freezeetching nomenclature. Science 190, 54-56 (1975) Coggeshall, R.E.: A fine structural analysis of the epidermis of the earthworm, Lumbricus terrestris L. J. Cell Biol. 28, 95-104 (1966) Gilula, N.B.: Junctions between cells. In: Cell communication (R. Cox, ed.), pp. 1 29. New York: Wiley 1974 Gilula, N.B., Branton, D., Satir, P.: The septate junction: A structural basis for intercellular coupling. Proc. nat. Acad. Sci. (Wash.) 67, 213 220 (1970) Staehelin, L.A.: Structure and function of intercellular junctions. Int. Rev. Cytol. 39, 191-283 (1974) Storch, V., Welsch, U.: ~ber die Feinstruktur der Polychaetenepidermis (Annelida). Z. Morph. Tiere 66, 310-322 (1970)
Accepted June 6, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Freeze Fracture Studies on the Annelid Septate Junction Ulrich Welsch Anatomisches Institut der Universitfit Kiel, Germany Wolfgang Buchheim Institut ffir Physik der Bundesanstalt ffir Milchforschung, Kiel, Germany
Summary. Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80-120 A, the distance between two particles (centre to centre) is 150-250 A. Additionally striking rows of large particles (long diameter 150-200 A). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100-150 A, centre to centre distance approx. 250 A) whereas the particles of the extracellular face (EF, mean diameter 200-250 A) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species. Key words: Septate j unction - Annelids (Lumbricus terrestris and Tubifex spec.) - Epidermis - Freeze-fracture.
In an effort to bring order into a wealth of individual observations Staehelin (1974) suggested a preliminary classification of septate junctions into three major groups: a) Hydra type, b) pleated sheat type, c) continuous type. Trying to apply this classification to his observations on polychaete septate junctions Baskin (1976) found that the epidermal septate junctions of polychaetes do not fit, in all respects, into one of Staehelin's three types, but instead exhibited a number of characters of their own. Since Baskin (1976) did not use freeze-fracture preparations for his analysis we found it worthwhile to apply this technique to annelid material in order to find more structural details about the septate junctions in these animals.
Sendoffprint requeststo: Prof. U. Welsch,AnatomischesInstitut der Universidit,Olshausenstr. 40-60, D-2300 Kiel, Federal Republic of Germany
528
U. Welsch and W. Buchheim
Materials and Methods Small pieces(about 1 x 1 mm) of the body wall of Lumbricusterrestriswere transferred for 10mins into a 30 % glycerol/saline mixture for cryoprotective pretreatment. Tubifex spec. (diameter approx. 0.3 mm) was prepared without cryoprotection by using slices of approx. 0.5 mm thickness. Cryofixation was performed under usual conditions, using gold sample holders (Balzers) and Freon 22 (-160~C) as coolant. Freeze-etching preparation was carried out in a Balzers 360 M unit at an object temperature of - 105~C and an etching period of 1 min. For replication electron gun evaporation of platinum/carbon (approx. 17 A. thickness, shadowing angle 45~ and carbon (approx. 100 A) was used. Cleaning of the replicas was achieved by sodium hypochlorite solution. Results A. Lumbricus The septate junction occupies the apical quarter o f the epidermal cells. The protoplasmic face (PF, B r a n t o n et al., 1975) o f the plasma m e m b r a n e o f the epidermal cells is characterized in the area o f the septate junction by variously arranged rows o f particles(Fig. 1). The general direction ofthese rows runs roughly parallel to the epithelial surface; occasionally the majority o f rows can be directed vertically to the epithelial surface. In the upper two thirds or three quarters o f the junctional area these rows form a rather loose system and take an undulating course. Here they are separated by distances o f between about 500 and 2000A. In the lower third or quarter the rows are rather straight (Fig. 2a) and usually run closely together separated by a space o f 150 to 200 A. In spite o f their often irregular course one can follow individual rows over long distances. N o instance o f a fusion o f two rows into one was found. Rarely a row can form a loop and run back. The a p p a r e n t diameter o f the individual particles is about 80-120A, the distance between two particles (centre to centre) generally measures 150-250A. In addition to the above mentioned rows a different type o f particle-row (Fig. 2b) is regularly to be observed in the P F o f the lower part o f the junction and often isolated from the above mentioned system. This second type o f particle is relatively large and o f elongated shape (long diameter 150-200A, distance from adjacent particles generally 200-250A). The long axis o f these particles is perpendicularly directed to the direction o f the row (Fig. 2b). They occasionally were observed to terminate (or start) at a certain point o f the membrane. A t this terminal or beginning section o f the row, the particles are distinctly smaller and o f more globular shape (Fig. 2b). So far no definite statements can be given on the appearance o f the extracellular face (EF, B r a n t o n et al., 1975) since that face was not clearly exposed in our preparations. B. Tubifex A l t h o u g h the material o f Tubifex was not pretreated with an antifreeze large areas o f fractured plasma membranes could be observed. The structural integrity o f the m e m b r a n e s was not seriously affected by a limited degree o f ice crystal formation in the extracellular and intracellular fluids. Both fracture faces (EF and PF) could be inspected frequently (Fig. 3).
Fig. 1. Area of the septate junction in the epidermis of Lumbricus. Cu cuticle, Mv microvilli, double arrows: area with particle rows running closely together (basal part of the junction), asterisks: area with loosely arranged particle rows (apical part of the junction), x 72,000
530
U. Welsch and W. Buchheim
Fig. 2a and b. Epidermal cell of Lumbricus. a Particle rows in the protoplasmic face (PF) of a fractured plasma membrane, ap apical, ba basal part of the junction, b Row with large particles, a, b x 80,000
Annelid Septate Junction
531
Fig. 3. General view of a septate junction in the epidermis of Tubifex. EF, P F extracellular and protoplasmic face, My regions of microviUi, x 20,000
Fig. 4. a Epidermis of Tubifex (detail from Fig. 3). The selected area shows the arrangement of membrane particles of the septate junction on both protoplasmic (PF) and extracellular (EF) faces of the plasma membrane; is: intercellular space, b Ladder-shaped structure in the protoplasmic face (PF) of a fractured plasma membrane (probably intestinal epithelium or chloragogue tissue) of Tub~,x. 1, 2: cytoplasm of neighbouring cells, a, b x 80,000
Annelid SeptateJunction
533
In the epidermis the protoplasmic face (PF) is of similar appearance as in
Lumbrieus. It contains rows of distinct individual particles (mean diameter 100-150N, centre to centre distance 200-300A) (Fig. 4a). The extracellular face (EF) contains densely arranged strands of particles (Figs. 3 and 4a). These particles are of distinctly different appearance if compared with those of the protoplasmic face. Their diameter generally measures 250 A, they are relatively flat and so closely attached to each other (Fig. 4a) that they form continuous strands. These strands mostly take an undulating course. Frequently up to 10 and more strands are closely attached to each other. In such areas usually a two-dimensional periodicity of the membrane particles and correspondingly arranged small depressions between the single strands exist. On the whole the junctional area of the Tubifex epidermis is characterized by considerably denser arranged rows of particles than in Lumbricus. In the membranes (PF) of the intestinal epithelium (or possibly chloragogue tissue) peculiar ladder shaped structures have been detected which cannot be assigned to any of the known junctional types (Fig. 4 b). The individual elements of this structure measured approx. 50~ in thickness and 300-500A in length and consisted of closely arranged 50A subunits. The elongated elements were mostly grouped in parallel with a distance of 150-200/k.
Discussion
The present investigation of the annelid septate junctions has revealed a much less regular construction than one could expect after the appearance in sectioned material in which this type of intercellular junction is of rather uniform and regular structure (Baskin, 1976; Coggeshall, 1966; Storch and Welsch, 1970). In the freezefractured preparations the general arrangement of the membrane particles is surprisingly variable and not even the same in the two annelid species investigated in the present paper. It is difficult to explain this discrepancy between sectioned and fractured material. In the terrestrial species (Lumbricus) the membrane particles form rows often - especially in the apical region - widely separated from each other by distances up to 2000 N. They take and undulating course. In the basal part of the junction the particle rows are running more closely together. In the apical part of the Lumbricus septate junction the arrangement of particles resembles that of the curved ridges of the septate junctions of the continuous type (Gilula, 1974; Staehelin, 1974). It is possibly this more loosely constructed part of the junction into which ruthenium red penetrates (Baskin, 1976). The individual particles in the protoplasmic membrane face, which are sometimes believed to represent the structural connection between septa and membrane (literature see Staehelin, 1974), are roughly of the same dimensions as in molluscs and arthropods (Gilula et al., 1970; Gilula, 1974; Staehelin, 1974; own observations). A new feature are the more or less isolated rows of large particles in the Lumbricus epidermis and the ladder shaped structures in plasma membranes of Tubifex, the significance of which remains unknown. Of special interest are the findings in the Tubifex epidermis. In this animal the protoplasmic and extracellular face bear membrane particles. These also are arranged in rows; however, they stand much closer together and the individual
534
U. Welsch and W. Buchheim
strands which they form are usually also in much closer contact than in Lumbricus. Thus, in the fresh water species Tubifex the general compact construction of the junction to some degree resembles that of the septate junctions of the pleated sheat type in molluscs and arthropods (Gilula et al., 1970; Staehelin, 1974). In the EF the peculiarly flattened particles of several neighbouring strands can lie so closely together that they form a regular two-dimensional pattern, which resembles the images of the enface views of the lanthanum infiltrated polychaete junctions (Baskin, 1976). The particles of the PF seem to fit into depressions which can be recognized between neighbouring strands of the EF. Our results to some degree support the speculation of Baskin (1976) that the surface of the membrane in the septate junction is characterized by regularly arranged particles, separated by narrow spaces. In any case, in Tubifex (and probably also in Lumbricus) the plasma membrane contains particles, which structurally differ from each other, those of the extracellular face even being larger than those of the protoplasmic face. Although the present investigation has shown that there are fundamental similarities in structure between annelid, molluscan and arthropod septate junctions it also supports the view of Baskin (1976) who found that the annelid septate junction has many characteristics of its own.
References Baskin, D.G.: The fine structure of polychaete septate junctions. Cell Tiss. Res. 174, 55 67 (1976) Branton, D., Bullivant, S., Gilula, N.B., Karnovsky, M.J., Moor, H., M(ihlethaler, K., Northcote, D.H., Packer, L., Satir, B., Satir, P., Speth, V., Staehelin, L.A., Steere, R.L., Weinstein, R.S.: Freezeetching nomenclature. Science 190, 54-56 (1975) Coggeshall, R.E.: A fine structural analysis of the epidermis of the earthworm, Lumbricus terrestris L. J. Cell Biol. 28, 95-104 (1966) Gilula, N.B.: Junctions between cells. In: Cell communication (R. Cox, ed.), pp. 1 29. New York: Wiley 1974 Gilula, N.B., Branton, D., Satir, P.: The septate junction: A structural basis for intercellular coupling. Proc. nat. Acad. Sci. (Wash.) 67, 213 220 (1970) Staehelin, L.A.: Structure and function of intercellular junctions. Int. Rev. Cytol. 39, 191-283 (1974) Storch, V., Welsch, U.: ~ber die Feinstruktur der Polychaetenepidermis (Annelida). Z. Morph. Tiere 66, 310-322 (1970)
Accepted June 6, 1977
