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Inclusion bodies in fibroblast-like cells in the mucosa of the guinea pig ureter
© 1971 by Academic Press, Inc.
J. U:LTRASTRrOCTURERZS~ARCH34, 175--180 (1971)
175
Inclusion Bodies in Fibroblast-like Cells in the Mucosa of the Guinea Pig
Ureter
1
YASUO UEHARA AND a . BURNSTOCK
Department of Zoology, University of Melbourne, Parkville 3052, Victoria, Australia Received May 19, 1970, and in revised form July 6, 1970
Although there have been a number of ultrastructural studies of the mammalian ureter (1, 4, 7, 9), the presence of inclusion bodies has been noted only once in specialized cells of the rat ureter (4). During the course of a detailed study of the guinea pig ureter, highly ordered inclusion bodies were found in fibroblast-like cells, which are coupled by tight junctions to form a continuous cytoplasmic layer just beneath the transitional epithelium. These inclusion bodies were examined in guinea pig ureters which were fixed initially with phosphate buffered OsO4 (pH 7.4) for 1 hour, immersed in 4 % unbuffered glutaraldehyde for 1 hour, followed by postosmification. The tissue was then block-stained with uranyl acetate solution (T. Kanaseki, Y. Uehara, and M. Imaizumi, in preparation) and, after brief dehydration, embedded in Araldite, prior to sectioning and examination under an Hitachi l l B electron microscope. The cells containing inclusion bodies possess a well developed rough surfaced endoplasmic reticulum and lack investing basal lamina (Fig. 1). The inclusion bodies are usually roughly rectangular in shape and are surrounded by a single limiting membrane with no attached ribosomal particles. They show considerable variation in their internal structure between two extremes. Some inclusion bodies contain electron dense crystalloids which are either homogeneous in appearance (Figs. 1 and 7) or exhibit a highly ordered pattern of alternating light and dense, parallel lines about 20 • wide (Figs. 2, 5, and 6). The appearance of the repeating periodicity varies; this may indicate differences in crystal structure or merely represent different appearances depending on the sectioned angle to the crystal lattice. Other inclusion bodies consist of hollow, cylindrical structures with an outer diameter ranging from 200 to 450 ~ and a wall thickness of 30 to 80 ~. The electron dense wall of the cylinders appears to have a globular subunit structure, but no regular arrangement has been demonstrated (Fig. 4). These cylinders are oriented either randomly (Fig. 3) or are closely packed in 1 This work was supported by a grant from the Australian Research Grants Committee.
176
UEHARA AND BURNSTOCK
parallel to each other (Figs. 6 and 7). Sometimes inclusion bodies contain both cylinders and electron dense crystalloid material (Figs. 5 and 6); the cylinders are sometimes oriented nearly perpendicularly to the long axis of the crystalloid and are closely associated with it (Fig. 5). Transitional forms where bundles of cylinders lie in parallel to the electron dense crystalloid with a repeating periodicity, are also seen (Fig. 6). Coexistence of electron dense crystalloids and cylinders in a single inclusion body and the presence of transitional forms suggest a maturation process of highly ordered crystalloids from cylindrical structures, and may provide a useful system for examining protein assembly mechanisms. Highly ordered crystalloid forms of inclusion bodies have been described in other cell types, such as the liver cells of the salamander (2), the epithelial cells of the intestine (10, 12), the kidney proximal tubules (5, 8), and the thyroid follicular cells (13, 14). These are somewhat similar in appearance to the crystalloid inclusions reported here, but their structural organization and the width of repeating periods are different. Inclusion bodies which contain cylindrical structures have also been described in receptor cells of the special cutaneous organ of the electric fish (11) and in microbodies of rat liver cells (6). However, the cylindrical structures in these cell types are different in outer diameter and wall thickness from those in the inclusion bodies in the ureter described here, and do not show any sign of coexistence with electron dense crystalloids. Hicks (4) reported a peculiar type of cell ("bundle cell"), which contained a bundle of fine, tubular crystallites, in the transitional epithelial layer of rat ureter. Although these crystallites, demonstrated in low resolution electron micrographs, appear to have similar profiles to the cylindrical structures in the inclusion bodies in guinea pig fibroblast-like cells, the location and structural features of rat "bundle cells" is different. The inclusion bodies sometimes contain lipid droplets (Fig. 2), which commonly occur in lysosomal structures. Moreover, membrane-bound structures were found (inset of Fig. 7) which are similar in appearance either to lysosomes or to the cylindertype inclusion bodies. Thus it cannot be excluded that the inclusion bodies in this material, which are bound by a single membrane and have considerable morphological heterogeneity are related to lysosomes (3). The crystalloid inclusions in thyroid folFro. 1. Inclusion body consisting of an electron dense matrix which shows no distinctly ordered pattern. There is a fairly constant space of less than I00 ~ in width between the limiting membrane (double arrow) and electron dense matrix. Rectangular, less electron dense areas (r) are usually seen in the matrix; E, rough-surfaced endoplasmic reticulum containing fine granular or filamentous material; c, collagenous fibrils, x 83 000. Fro. 2. High power electron micrograph of an inclusion body, which contains an electron dense crystalloid exhibiting a 20 ~ lamellar periodicity. Arrow shows an electron transparent space between the limiting membrane (L) and crystalloid matrix, r, rectangular less electron dense area. x 220 000.
t
INCLUSION BODIES IN URETER MUCOSA
179
FIG. 7. An electron micrograph showing four inclusion bodies with different internal structures. The inclusion body indicated b y / 1 is without lamellar periodicity. The inclusion bodies 12 and Is consist of closely packed parallel cylinders cut longitudinally. The inclusion body Ia contains transversely sectioned, closely packed cylinders with indistinct wall structure. The fused walls of adjacent cylinders form a dark line which varies in width; they are oriented approximately parallel to each other. The inclusion body 12 contains a lipid droplet (arrow). x 82 000. Inset: An electron micrograph of a structure bounded by a limiting membrane and consisting of an intermediate electron dense matrix with the suggestion of cross sectioned cylindrical structures (arrow). The structure shows a close resemblance to some lysosomes, x 60 000.
FIG. 3. Inclusion body containing randomly oriented cylindrical structures. Note the variation in the size of cylinders. The length of the cylinders sometimes measures up to 0.5/z in a single section. m, mitochondria, x 120 000. F I 6 . 4 . High power electron micrograph of a cross section of the cylindrical structures (Cy) within an inclusion body, showing some indication of globular subunit structure in the wall. Arrow indicates a connection between adjacent cylinders. Intermediate electron dense substance (e) is seen outside or inside the cylinders. L, limiting membrane of inclusion body. Note that the cylinders vary in wall thickness, x 300 000. FIG. 5. An inclusion body containing both cylindrical structures (Cy) and electron dense crystalloids (C1 and C2) suspended in a large electron transparent space (s) surrounded by a limiting membrane (L). The crystalloid matrix C1 indicates a lamellar periodicity of 20 A. Connection between the adjacent cylinders is indicated by arrow. Double arrow shows the close association between the cylinders and electron dense crystalloid, e, electron dense substance scattered around the cylinder and crystalloid matrix, x 150 000. Fro. 6. Inclusion body containing a bundle of closely packed cylinders which are juxtaposed to a crystalloid matrix exhibiting clear 20 A lamellar periodicity. The profile suggests that the cylinders are the precursor structure of a highly organized crystalloid matrix. × 200 000.
180
UEHARA AND BURNSTOCK
licular cells have been suggested to be derived from lysosomal granules (14). Moreover, some crystalloid inclusion bodies have been shown to have acid phosphatase activity, suggesting that lysosomes may consist almost entirely of material which is sufficiently pure to attain a crystallized state (8). Further studies, including the use of histochemical methods, are necessary to clarify the nature and function of these inclusion bodies. REFERENCES 1. BERGMAN,R. A., Bull. Johns Hopkins Hosp. 102, 195 (1958). 2. FAWCETT,D. W., in MENGE,A. C. (Ed.), Lipid Transport. Thomas, Springfield, Illinois, 1964.
3. FAWCETT,D. W., in The Cell, Its Organelles and Inclusions, p. 189. Saunders, Philadelphia, Pennsylvania, 1966. 4. HICKS, R. M., J. Cell Biol. 26, 25 (1965). 5. HIMMELrtOC~,S. R. and KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 9, 893 (1961). 6. HRUBAN,Z. and SWIFT, H., Science 146, 1316 (1964). 7. LEESON,C. R. and LEESON,T. S., Acta Anat. 62, 60 (1965). 8. MAUNSBACH,A. B., J. Ultrastruct. Res. 14, 167, 189 (1966). 9. NOTLEY, R. G., J. Anat. 10S, 393 (1969). 10. SILVA,D. G., J. Ultrastruct. Res. 18, 127 (1967). 11. WACHTEL,A. W. and SZAMIER,R. B., J. Ultrastruct. Res. 27, 361 (1969). 12. WESLEY,R. K. A. and JENSEN,T. E., J. Ultrastruct. Res. 27, 306 (1969). 13. YOSHIMURA,F. and IRIE, M., Z. Zellforsch. Mikrosk. Anat. 55, 204 (1961). 14. YOUSON,J. and VAN HEYNIGEN,H., Amer. J. Anat. 122, 377 (1968).
J. U:LTRASTRrOCTURERZS~ARCH34, 175--180 (1971)
175
Inclusion Bodies in Fibroblast-like Cells in the Mucosa of the Guinea Pig
Ureter
1
YASUO UEHARA AND a . BURNSTOCK
Department of Zoology, University of Melbourne, Parkville 3052, Victoria, Australia Received May 19, 1970, and in revised form July 6, 1970
Although there have been a number of ultrastructural studies of the mammalian ureter (1, 4, 7, 9), the presence of inclusion bodies has been noted only once in specialized cells of the rat ureter (4). During the course of a detailed study of the guinea pig ureter, highly ordered inclusion bodies were found in fibroblast-like cells, which are coupled by tight junctions to form a continuous cytoplasmic layer just beneath the transitional epithelium. These inclusion bodies were examined in guinea pig ureters which were fixed initially with phosphate buffered OsO4 (pH 7.4) for 1 hour, immersed in 4 % unbuffered glutaraldehyde for 1 hour, followed by postosmification. The tissue was then block-stained with uranyl acetate solution (T. Kanaseki, Y. Uehara, and M. Imaizumi, in preparation) and, after brief dehydration, embedded in Araldite, prior to sectioning and examination under an Hitachi l l B electron microscope. The cells containing inclusion bodies possess a well developed rough surfaced endoplasmic reticulum and lack investing basal lamina (Fig. 1). The inclusion bodies are usually roughly rectangular in shape and are surrounded by a single limiting membrane with no attached ribosomal particles. They show considerable variation in their internal structure between two extremes. Some inclusion bodies contain electron dense crystalloids which are either homogeneous in appearance (Figs. 1 and 7) or exhibit a highly ordered pattern of alternating light and dense, parallel lines about 20 • wide (Figs. 2, 5, and 6). The appearance of the repeating periodicity varies; this may indicate differences in crystal structure or merely represent different appearances depending on the sectioned angle to the crystal lattice. Other inclusion bodies consist of hollow, cylindrical structures with an outer diameter ranging from 200 to 450 ~ and a wall thickness of 30 to 80 ~. The electron dense wall of the cylinders appears to have a globular subunit structure, but no regular arrangement has been demonstrated (Fig. 4). These cylinders are oriented either randomly (Fig. 3) or are closely packed in 1 This work was supported by a grant from the Australian Research Grants Committee.
176
UEHARA AND BURNSTOCK
parallel to each other (Figs. 6 and 7). Sometimes inclusion bodies contain both cylinders and electron dense crystalloid material (Figs. 5 and 6); the cylinders are sometimes oriented nearly perpendicularly to the long axis of the crystalloid and are closely associated with it (Fig. 5). Transitional forms where bundles of cylinders lie in parallel to the electron dense crystalloid with a repeating periodicity, are also seen (Fig. 6). Coexistence of electron dense crystalloids and cylinders in a single inclusion body and the presence of transitional forms suggest a maturation process of highly ordered crystalloids from cylindrical structures, and may provide a useful system for examining protein assembly mechanisms. Highly ordered crystalloid forms of inclusion bodies have been described in other cell types, such as the liver cells of the salamander (2), the epithelial cells of the intestine (10, 12), the kidney proximal tubules (5, 8), and the thyroid follicular cells (13, 14). These are somewhat similar in appearance to the crystalloid inclusions reported here, but their structural organization and the width of repeating periods are different. Inclusion bodies which contain cylindrical structures have also been described in receptor cells of the special cutaneous organ of the electric fish (11) and in microbodies of rat liver cells (6). However, the cylindrical structures in these cell types are different in outer diameter and wall thickness from those in the inclusion bodies in the ureter described here, and do not show any sign of coexistence with electron dense crystalloids. Hicks (4) reported a peculiar type of cell ("bundle cell"), which contained a bundle of fine, tubular crystallites, in the transitional epithelial layer of rat ureter. Although these crystallites, demonstrated in low resolution electron micrographs, appear to have similar profiles to the cylindrical structures in the inclusion bodies in guinea pig fibroblast-like cells, the location and structural features of rat "bundle cells" is different. The inclusion bodies sometimes contain lipid droplets (Fig. 2), which commonly occur in lysosomal structures. Moreover, membrane-bound structures were found (inset of Fig. 7) which are similar in appearance either to lysosomes or to the cylindertype inclusion bodies. Thus it cannot be excluded that the inclusion bodies in this material, which are bound by a single membrane and have considerable morphological heterogeneity are related to lysosomes (3). The crystalloid inclusions in thyroid folFro. 1. Inclusion body consisting of an electron dense matrix which shows no distinctly ordered pattern. There is a fairly constant space of less than I00 ~ in width between the limiting membrane (double arrow) and electron dense matrix. Rectangular, less electron dense areas (r) are usually seen in the matrix; E, rough-surfaced endoplasmic reticulum containing fine granular or filamentous material; c, collagenous fibrils, x 83 000. Fro. 2. High power electron micrograph of an inclusion body, which contains an electron dense crystalloid exhibiting a 20 ~ lamellar periodicity. Arrow shows an electron transparent space between the limiting membrane (L) and crystalloid matrix, r, rectangular less electron dense area. x 220 000.
t
INCLUSION BODIES IN URETER MUCOSA
179
FIG. 7. An electron micrograph showing four inclusion bodies with different internal structures. The inclusion body indicated b y / 1 is without lamellar periodicity. The inclusion bodies 12 and Is consist of closely packed parallel cylinders cut longitudinally. The inclusion body Ia contains transversely sectioned, closely packed cylinders with indistinct wall structure. The fused walls of adjacent cylinders form a dark line which varies in width; they are oriented approximately parallel to each other. The inclusion body 12 contains a lipid droplet (arrow). x 82 000. Inset: An electron micrograph of a structure bounded by a limiting membrane and consisting of an intermediate electron dense matrix with the suggestion of cross sectioned cylindrical structures (arrow). The structure shows a close resemblance to some lysosomes, x 60 000.
FIG. 3. Inclusion body containing randomly oriented cylindrical structures. Note the variation in the size of cylinders. The length of the cylinders sometimes measures up to 0.5/z in a single section. m, mitochondria, x 120 000. F I 6 . 4 . High power electron micrograph of a cross section of the cylindrical structures (Cy) within an inclusion body, showing some indication of globular subunit structure in the wall. Arrow indicates a connection between adjacent cylinders. Intermediate electron dense substance (e) is seen outside or inside the cylinders. L, limiting membrane of inclusion body. Note that the cylinders vary in wall thickness, x 300 000. FIG. 5. An inclusion body containing both cylindrical structures (Cy) and electron dense crystalloids (C1 and C2) suspended in a large electron transparent space (s) surrounded by a limiting membrane (L). The crystalloid matrix C1 indicates a lamellar periodicity of 20 A. Connection between the adjacent cylinders is indicated by arrow. Double arrow shows the close association between the cylinders and electron dense crystalloid, e, electron dense substance scattered around the cylinder and crystalloid matrix, x 150 000. Fro. 6. Inclusion body containing a bundle of closely packed cylinders which are juxtaposed to a crystalloid matrix exhibiting clear 20 A lamellar periodicity. The profile suggests that the cylinders are the precursor structure of a highly organized crystalloid matrix. × 200 000.
180
UEHARA AND BURNSTOCK
licular cells have been suggested to be derived from lysosomal granules (14). Moreover, some crystalloid inclusion bodies have been shown to have acid phosphatase activity, suggesting that lysosomes may consist almost entirely of material which is sufficiently pure to attain a crystallized state (8). Further studies, including the use of histochemical methods, are necessary to clarify the nature and function of these inclusion bodies. REFERENCES 1. BERGMAN,R. A., Bull. Johns Hopkins Hosp. 102, 195 (1958). 2. FAWCETT,D. W., in MENGE,A. C. (Ed.), Lipid Transport. Thomas, Springfield, Illinois, 1964.
3. FAWCETT,D. W., in The Cell, Its Organelles and Inclusions, p. 189. Saunders, Philadelphia, Pennsylvania, 1966. 4. HICKS, R. M., J. Cell Biol. 26, 25 (1965). 5. HIMMELrtOC~,S. R. and KARNOVSKY,M. J., J. Biophys. Biochem. Cytol. 9, 893 (1961). 6. HRUBAN,Z. and SWIFT, H., Science 146, 1316 (1964). 7. LEESON,C. R. and LEESON,T. S., Acta Anat. 62, 60 (1965). 8. MAUNSBACH,A. B., J. Ultrastruct. Res. 14, 167, 189 (1966). 9. NOTLEY, R. G., J. Anat. 10S, 393 (1969). 10. SILVA,D. G., J. Ultrastruct. Res. 18, 127 (1967). 11. WACHTEL,A. W. and SZAMIER,R. B., J. Ultrastruct. Res. 27, 361 (1969). 12. WESLEY,R. K. A. and JENSEN,T. E., J. Ultrastruct. Res. 27, 306 (1969). 13. YOSHIMURA,F. and IRIE, M., Z. Zellforsch. Mikrosk. Anat. 55, 204 (1961). 14. YOUSON,J. and VAN HEYNIGEN,H., Amer. J. Anat. 122, 377 (1968).