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A study of ultrathin frozen sections of granular cells in newborn rat epidermis
JOURNAL OF ULTRASTRUCTURERESEARCH 70, 8-14
(1980)
A Study of Ultrathin Frozen Sections of Granular Cells in Newborn Rat Epidermis SHIGEO KAKIMI, KIMIE FUKUYAMA, AND WILLIAM L. EPSTEIN
Department of Dermatology, School of Medicine, University of California, San Francisco, California 94143 Received June 26, 1979 The ultrastructure of granular cells of newborn rat epidermis was investigated in sections prepared by cryomicrotomy and stained negatively with phosphotungstic acid. Tonofilaments, keratohyalin granules, mitochondria, membrane coating granules, and membranes of cells and nuclei were seen in negative contrast. Filaments in keratohyalin granules were randomly oriented and formed a meshwork, while tonofflaments appeared in a group with a parallel arrangement. Their diameter was significantly smaller than filaments seen in cornified cells. Two distinct layers were demonstrable in membranes of nuclei and mitochondria, but not in the plasma membrane or membrane coating granules. These results indicate that use of cryomicrotomy offers advantages for the elucidation of the ultrastructure appearance of epidermis which are not obtainable by conventional techniques. Keratinocytes of mammalian epidermis contain a large numbers of filaments. The ultrastructure of the filaments as observed in tissues fixed in osmium tetroxide, with or without prefixation, dehydrated and embedded in plastic, has been extensively documented (1, 11). T h e i n f o r m a t i o n g e n erated by those Studies has provided the basis for understanding of the appearance of epidermal cells in both normal and pathological conditions. R e c e n t l y , S j S s t r a n d a n d B e r n h a r d (13) a n d S j S s t r 5 m a n d S q u i r e (14) r e v i e w e d t h e remarkable progress being made in the use of cryoultramicrotomy. The process eliminates damage to macromolecules which m a y b e i n t r o d u c e d d u r i n g c o n v e n t i o n a l tissue preparation for electron microscopy a n d h a s b e e n p a r t i c u l a r l y u s e f u l in t h e o b servation of internal details of myofibrils and mitochondria membrane. In this report we describe electron microscopic findings in g r a n u l a r c e l l s o f n e w b o r n r a t e p i d e r m i s prepared by cryoultramicrotomy and negative staining. MATERIALS AND METHODS Shaved biopsy specimens, 3 x 5 x 2 mm, from 2day-old rats (Sprague-Dawley strain) were fixed in 0022-5320/80/010008-07502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min. They were cut into smaller pieces with pyramid shapes with a base of 1 x 1 mm and fixed for an additional 30 min then washed for 24 hr in three changes of 0.05 M phosphate buffer containing 5% sucrose, pH 7.4. Sucrose was infused at 4°C into the tissues by a modification of the technique described by Tokuyasu (17). They were immersed in~ 10% sucrose (w/v) solution buffered with 0.05 M phosphate buffer, pH 7.4, for 2 hr and in 30 and 50% sucrose solutions with the same buffer for 2 hr each. The tissues were then mounted one by one on a flat-tip type special holder (LKB, Rockville, Md.). Efforts were made to obtain both epidermis and dermis at the cutting edge. Freon 22 (Pameco Aire, Oakland, Calif.) was chilled to -160°C in liquid nitrogen and tissue in the holder was rapidly frozen in the Freon 22. Sectioning was done with a 14 800 CryoKit attached Ultratom III (LKB, Rockville, Md.). Glass knives prepared by a LKB knife maker with a scoring angle of 50 ° were used. Temperature was controlled at -100°C for the knife side and -120°C for the specimen side before cutting but set at -60°C for the knife side and -80°C for the specimen side during cutting. The clearance angle was 8 ° and usually cut at a speed of 10 mm/sec. Sections which showed a cellophane-like thickness were collected by the tip of an eyelash probe and picked up with a wire loop of 2 mm in diameter coated with a saturated sucrose solution. The sections were transferred to a stainless-steel grid coated with 0.3% Formvar in 1,2dichloroethane and carbon evaporation. As soon as the sections were thawed, the grid was floated on the surface of a water deposit, the section side down, to wash out the sucrose. Excess water was removed from
CRYOULTRA
MICROTOMY
the grid and the sections were negatively stained with 1.5% phosphotungstic acid (PTA) for 5 sec. All samples were examined with a Siemens Elmiskop IA electron microscope at 80 kV. Photographs were taken at magnification of 20 000 or 40 000 and enlarged to a magnification of 35 000-70 000 by printing. The magnification was corrected by use of a grating replica (Ernest F, Fullam, Inc., Schenectady, N.Y.). RESULTS
Different cell layers of the epidermis and the dermis were easily recognizable at low magnification. However, the images observed in negative stained skin sections differed considerably from those prepared by conventional techniques. Figure 1 represents a typical view of granular cells. The most noticeable feature was that PTA did not stain keratohyalin granules and they appeared electron lucent in contrast to their electron density as seen in positive stained specimens. Each cell and nucleus was distinctly outlined by its unstained membrane. Intercellular spaces appeared to be stained as a continuous line. A large number of small particles scattered in the cytoplasm was also observed. These unstained components in granular cells were found to be mostly small granules with a lamellated structure (Odland bodies (10), keratinosomes (18), membrane coating granules (8)) and mitochondria. At higher magnification they exhibited essentially the same basic structure as reported by conventional electron microscopy, but the distribution of electron density was different due to the negative staining. The membrane-coating granules showed a regular repeating pattern; dark line-to-dark line periodicity was about 8.8 nm (Fig. 2a). In mitochondria the outer and inner membranes were seen in negative contrast (Fig. 2b). Numerous infoldings of the membrane also were well preserved. Some desmosomes were also studied under high magnification. Figure 2c shows the typical appearance of a cell junction found in the lower granular cells. The trilaminar plasma membrane appeared as a white line and identification of each leaf-
OF GRANULAR
CELLS
9
let was not possible. These findings contrast with the appearance of the nuclear membrane where both inner and outer leaflets showed independent negative contrast (Fig. 1). At the desmosome region the marginal plaques and intercellular contact layer, densely stained in conventional preparation (6, 12), were observed unstained. Distribution and ultrastructure of tonofibrils were examined in granular cells. Although it was not quantified, more tonofilaments seemed to be present in the frozen sections than in Epon-embedded preparations stained in different ways. Filaments were often grouped in parallel arrangement and showed direct contact with keratohyalin granules (Fig. 3). Under higher magnification keratohyalin granules were found to consist of a large number of filaments (Fig. 4a). The filaments were randomly oriented and formed a meshwork, but at the edges of the granules it was possible to observe continuation of these filaments with tonofilaments. Since the filaments of granular cells are considered to become keratin filaments in cornified cells, we compared the ultrastructure of filaments in keratohyalin granules and tonofflaments with those filaments seen in cornified cells. Figures 4b and c illustrate a negative stained cornified cell demonstrating a fibril pattern almost identical to the keratin pattern observed in plastic embedded sections (2, 3). Most of the filaments were thicker than tonofilaments and were individually dispersed in an electron-opaque matrix. Table i summarizes the diameters of filaments in keratohyalin granules and tonofilaments in granular cells along with those of cornified cells. The average diameters of keratohyalin granular filaments and tonofilaments did not show any statistical difference, but the difference between that of cornified cells was significantly greater than that of tonofilaments (P > 0.00001). DISCUSSION
The epidermis of newborn rats was cut
10
KAKIMI, FUKUYAMA, AND EPSTEIN
CRYOULTRA MICROTOMY OF GRANULAR CELLS
11
Fro. 1. Granular cells of newborn rat epidermis, fixed in glutaraldehyde, frozen after infusion of sucrose, and cut by cryomicrotomy. 1.5% phosphotungstic acid was used to stain the section negatively. Outline of cells and nucleus is seen clearly and keratohyalin granules (k) show a negative contrast. × 14 500. Fro. 2. (a) A small granule seen in granular cells with a regular periodicity of approximately 8.8 +_ 0.1 nm (n = 9). The outer membrane appears as a white line. (b) Mitochondria are recognized by their typical membranous appearance. (c) Granular cell boarders are delineated by the negative image of their plasma membranes. Desmosome junctions are identified by insertion of filaments and symmetrical arrangement of the marginal plaques. (a) and (b), × 280 000; (c), × 62 000. under low temperature without dehydration and plastic embedding. Although there were some technical difficulties associated w i t h t h e p r o c e s s i n v o l v e d in c r y o m i c r o t o m y , i n v e s t i g a t i o n of g r a n u l a r a n d c o r n i f i e d cells w a s p o s s i b l e a f t e r n e g a t i v e s t a i n i n g with PTA. Filaments, keratohyalin granules, n u c l e a r m e m b r a n e , p l a s m a m e m brane, mitochondria, and lamellated bodies
w e r e s e e n in n e g a t i v e c o n t r a s t . N i c o l s o n (9) s t u d i e d c h l o r o p l a s t s e m b e d d e d in b o v i n e serum albumin and reported that negative s t a i n i n g o c c u r s in h y d r o p h i l i c r e g i o n s b u t n o t in h y d r o p h o b i c a r e a s . T h e p r e s e n t findings, t h u s , c o i n c i d e w i t h t h e p h y s i o c h e m i c a l characterization reported by Swanbeck and T h y r e s s o n (15), w h o s u g g e s t e d t h a t k e r a tohyalin granules, filaments, and mem-
12
KAKIMI, FUKUYAMA, AND EPSTEIN
FIo. 3. Negatively stained keratohyalin granules (k) and groups of tonofflaments (t). They are connected at edges of the granules, x 96 000. FIG. 4. (a) Higher magnification of tonofibrils and filaments in keratohyalin granules; ( ) indicates 50 nm. (b) and (c) Filaments in cornified cells appear thicker and more loosely embedded in the matrix. ( ) indicates 50 nm. (a), × 350 000; (b), x 154 000; (c), × 350 000. b r a n e s of t h e n u c l e u s a n d m i t o c h o n d r i a a r e h y d r o p h o b i c c o m p o n e n t s o f g r a n u l a r cells. The internal structure of keratohyalin g r a n u l e s is o b s c u r e d b y t h e e l e c t r o n - d e n s e s t a i n in s k i n p r o c e s s e d b y c o n v e n t i o n a l t e c h n i q u e s . S e v e r a l a t t e m p t s to r e m o v e t h e electron-dense material from the granules h a v e b e e n r e p o r t e d (5, 16). H o w e v e r , d e m -
o n s t r a t i o n of f i l a m e n t s in k e r a t o h y a l i n g r a n u l e s h a s n o t b e e n successful. C r y o m i c r o t o m y of s k i n a n d u s e o f n e g a t i v e s t a i n i n g a l l o w e d us to i n v e s t i g a t e t h e s e f i l a m e n t s . They appeared more irregular than tonofilaments and formed a loosely arranged meshwork. Connection of the filaments and t o n o f i l a m e n t s w a s s e e n a t t h e e d g e of k e r -
CRYOULTRA MICROTOMY OF GRANULAR CELLS
13
14
KAKIMI, FUKUYAMA, AND EPSTEIN TABLE I DIAMETERS (nm) OF TONOFILAMENTS
ANn FILAMENTS IN KERATOHYALIN GRANULES ANn CORNIFIED CELLS a
Filament Tonofflaments
Keratohyalin granules
Cornified cells
3.6 _+0.1
3.4 _+ 0.1
7.3 +_0.3
Numbers represent averages of 15 randomly selected filaments. atohyalin granules suggesting that tonofilaments undergo morphological changes most probably by association with so-called keratohyalin material observed by conventional techniques as osmiophilic subs t a n c e s . O n t h e o t h e r h a n d , e v e n b y conv e n t i o n a l p r o c e s s i n g , f i l a m e n t s in c o r n i f i e d cells a p p e a r u n s t a i n e d (4); t h u s , t h e n e g a tive staining technique which we used demonstrated essentially the same ultrastructure with a keratin pattern. Brody measu r e d t h e d i a m e t e r o f t o n o f i l a m e n t s in m a n (3) a n d g u i n e a p i g s (2) a n d n o t e d a n inc r e a s e in t h e size o f f i l a m e n t s in c o r n i f i e d cells. A s i m i l a r i n c r e a s e w a s c o n f i r m e d in the present study. M e m b r a n e c o a t i n g g r a n u l e s a n d t h e i r lam e l l a e g a v e a n e g a t i v e c o n t r a s t r e s p o n s e in t h e c y t o p l a s m of g r a n u l a r cells. H o w e v e r , i t w a s n o t p o s s i b l e t o i d e n t i f y t h e m in i n t e r c e l l u l a r s p a c e s a f t e r t h e y w e r e e x t r u d e d (8). Previous studies indicated that these granules are surrounded by trilaminar memb r a n e s s i m i l a r t o t h o s e s e e n in t h e p l a s m a m e m b r a n e (7). N e g a t i v e s t a i n i n g f a i l e d to reveal the inner and outer leaves of the m e m b r a n e a n d t h e y a p p e a r e d as a w h i t e line e n c i r c l i n g t h e g r a n u l e s . T h e s a m e s t a i n i n g p a t t e r n w a s s e e n in t h e p l a s m a m e m b r a n e o f g r a n u l a r cells; t h e t w o l e a v e s were not separately identified. In contrast,
two layers of membrane were clearly seen in n u c l e a r m e m b r a n e s a n d m i t o c h o n d r i a . T h e r e s u l t s m a y b e i n t e r p r e t e d as a d e m o n s t r a t i o n of d i f f e r e n c e s in c h e m i c a l a n d physical properties of the two types of m e m b r a n e e x i s t i n g in e p i d e r m a l cells.
This study was supported by National Institutes of Health Grant AM-12433, a grant from the Skin Disease Research Foundation of San Francisco, and Biomedical Research Grant RR05355 (Dr. Fukuyama). REFERENCES 1. BREATHNACH, A. S. (1975) J. Invest. Dermatol.
65, 2. 2. BRODY, I. (1959} 3". Ultrastruct. Res. 2, 482. 3. BROD¥, I. (1960) J. Ultrastruct. Res. 4, 264.
4. BROD¥, I. (1964) The Epidermis, p. 251, Academic Press, New York. 5. FUKUYAMA,K., BUXMAN, M. M., AND EPSTEIN, W. L. (1968) J. Invest. Dermatol. 51, 355. 6. KOMURA,J., AND OFUJI, S. (1967) J. Invest. Dermatol. 48, 304. 7. MARTINEZ,I. R., JR., ANDPETERS,A. (1971) Amer. J. Anat. 103, 93. 8. MATOLTSY,A. G., AND PARAKKAL,P. F. (1965) J. Cell Biol. 24, 297. 9. NICOLSON, G. L. {1971) J. Cell Biol. 50, 258. 10. ODLAND,G. F. (1960) J. Invest. Dermatol. 34, 11. 11. ODLAND, G. F., AND REED, T. H. (1967) Ultrastructure of Normal and Abnormal Skin, p. 54, Lea & Febiger, Philadelphia. 12. RAKNERUD,N. (1975) J. Ultrastruct. Res. 52, 32. 13. SJOSTRAND,f. S., AND BERNHARD, W. (1976) J. Ultrastruct. Res. 56, 233. 14. SJOSTROM, M., AND SQUIRE, J. M. (1977) J. Microsc. 111, 239. 15. SWANBECK,G., AND THYRESSON, N. (1965) Acta Derm. Venereol. 45, 21. 16. TEZUKA, T., AND FREEDBERG, I. M. (1972) Biochim. Biophys. Acta 261, 402. 17. TOKUYASU,K. T. (1973) J. Cell Biol. 57, 551. 18. WILGRAM,G., CAULFIELD,J. S., AND MADGIC, E. B. (1964) The Epidermis, p. 275, Academic Press, New York.
(1980)
A Study of Ultrathin Frozen Sections of Granular Cells in Newborn Rat Epidermis SHIGEO KAKIMI, KIMIE FUKUYAMA, AND WILLIAM L. EPSTEIN
Department of Dermatology, School of Medicine, University of California, San Francisco, California 94143 Received June 26, 1979 The ultrastructure of granular cells of newborn rat epidermis was investigated in sections prepared by cryomicrotomy and stained negatively with phosphotungstic acid. Tonofilaments, keratohyalin granules, mitochondria, membrane coating granules, and membranes of cells and nuclei were seen in negative contrast. Filaments in keratohyalin granules were randomly oriented and formed a meshwork, while tonofflaments appeared in a group with a parallel arrangement. Their diameter was significantly smaller than filaments seen in cornified cells. Two distinct layers were demonstrable in membranes of nuclei and mitochondria, but not in the plasma membrane or membrane coating granules. These results indicate that use of cryomicrotomy offers advantages for the elucidation of the ultrastructure appearance of epidermis which are not obtainable by conventional techniques. Keratinocytes of mammalian epidermis contain a large numbers of filaments. The ultrastructure of the filaments as observed in tissues fixed in osmium tetroxide, with or without prefixation, dehydrated and embedded in plastic, has been extensively documented (1, 11). T h e i n f o r m a t i o n g e n erated by those Studies has provided the basis for understanding of the appearance of epidermal cells in both normal and pathological conditions. R e c e n t l y , S j S s t r a n d a n d B e r n h a r d (13) a n d S j S s t r 5 m a n d S q u i r e (14) r e v i e w e d t h e remarkable progress being made in the use of cryoultramicrotomy. The process eliminates damage to macromolecules which m a y b e i n t r o d u c e d d u r i n g c o n v e n t i o n a l tissue preparation for electron microscopy a n d h a s b e e n p a r t i c u l a r l y u s e f u l in t h e o b servation of internal details of myofibrils and mitochondria membrane. In this report we describe electron microscopic findings in g r a n u l a r c e l l s o f n e w b o r n r a t e p i d e r m i s prepared by cryoultramicrotomy and negative staining. MATERIALS AND METHODS Shaved biopsy specimens, 3 x 5 x 2 mm, from 2day-old rats (Sprague-Dawley strain) were fixed in 0022-5320/80/010008-07502.00/0 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min. They were cut into smaller pieces with pyramid shapes with a base of 1 x 1 mm and fixed for an additional 30 min then washed for 24 hr in three changes of 0.05 M phosphate buffer containing 5% sucrose, pH 7.4. Sucrose was infused at 4°C into the tissues by a modification of the technique described by Tokuyasu (17). They were immersed in~ 10% sucrose (w/v) solution buffered with 0.05 M phosphate buffer, pH 7.4, for 2 hr and in 30 and 50% sucrose solutions with the same buffer for 2 hr each. The tissues were then mounted one by one on a flat-tip type special holder (LKB, Rockville, Md.). Efforts were made to obtain both epidermis and dermis at the cutting edge. Freon 22 (Pameco Aire, Oakland, Calif.) was chilled to -160°C in liquid nitrogen and tissue in the holder was rapidly frozen in the Freon 22. Sectioning was done with a 14 800 CryoKit attached Ultratom III (LKB, Rockville, Md.). Glass knives prepared by a LKB knife maker with a scoring angle of 50 ° were used. Temperature was controlled at -100°C for the knife side and -120°C for the specimen side before cutting but set at -60°C for the knife side and -80°C for the specimen side during cutting. The clearance angle was 8 ° and usually cut at a speed of 10 mm/sec. Sections which showed a cellophane-like thickness were collected by the tip of an eyelash probe and picked up with a wire loop of 2 mm in diameter coated with a saturated sucrose solution. The sections were transferred to a stainless-steel grid coated with 0.3% Formvar in 1,2dichloroethane and carbon evaporation. As soon as the sections were thawed, the grid was floated on the surface of a water deposit, the section side down, to wash out the sucrose. Excess water was removed from
CRYOULTRA
MICROTOMY
the grid and the sections were negatively stained with 1.5% phosphotungstic acid (PTA) for 5 sec. All samples were examined with a Siemens Elmiskop IA electron microscope at 80 kV. Photographs were taken at magnification of 20 000 or 40 000 and enlarged to a magnification of 35 000-70 000 by printing. The magnification was corrected by use of a grating replica (Ernest F, Fullam, Inc., Schenectady, N.Y.). RESULTS
Different cell layers of the epidermis and the dermis were easily recognizable at low magnification. However, the images observed in negative stained skin sections differed considerably from those prepared by conventional techniques. Figure 1 represents a typical view of granular cells. The most noticeable feature was that PTA did not stain keratohyalin granules and they appeared electron lucent in contrast to their electron density as seen in positive stained specimens. Each cell and nucleus was distinctly outlined by its unstained membrane. Intercellular spaces appeared to be stained as a continuous line. A large number of small particles scattered in the cytoplasm was also observed. These unstained components in granular cells were found to be mostly small granules with a lamellated structure (Odland bodies (10), keratinosomes (18), membrane coating granules (8)) and mitochondria. At higher magnification they exhibited essentially the same basic structure as reported by conventional electron microscopy, but the distribution of electron density was different due to the negative staining. The membrane-coating granules showed a regular repeating pattern; dark line-to-dark line periodicity was about 8.8 nm (Fig. 2a). In mitochondria the outer and inner membranes were seen in negative contrast (Fig. 2b). Numerous infoldings of the membrane also were well preserved. Some desmosomes were also studied under high magnification. Figure 2c shows the typical appearance of a cell junction found in the lower granular cells. The trilaminar plasma membrane appeared as a white line and identification of each leaf-
OF GRANULAR
CELLS
9
let was not possible. These findings contrast with the appearance of the nuclear membrane where both inner and outer leaflets showed independent negative contrast (Fig. 1). At the desmosome region the marginal plaques and intercellular contact layer, densely stained in conventional preparation (6, 12), were observed unstained. Distribution and ultrastructure of tonofibrils were examined in granular cells. Although it was not quantified, more tonofilaments seemed to be present in the frozen sections than in Epon-embedded preparations stained in different ways. Filaments were often grouped in parallel arrangement and showed direct contact with keratohyalin granules (Fig. 3). Under higher magnification keratohyalin granules were found to consist of a large number of filaments (Fig. 4a). The filaments were randomly oriented and formed a meshwork, but at the edges of the granules it was possible to observe continuation of these filaments with tonofilaments. Since the filaments of granular cells are considered to become keratin filaments in cornified cells, we compared the ultrastructure of filaments in keratohyalin granules and tonofflaments with those filaments seen in cornified cells. Figures 4b and c illustrate a negative stained cornified cell demonstrating a fibril pattern almost identical to the keratin pattern observed in plastic embedded sections (2, 3). Most of the filaments were thicker than tonofilaments and were individually dispersed in an electron-opaque matrix. Table i summarizes the diameters of filaments in keratohyalin granules and tonofilaments in granular cells along with those of cornified cells. The average diameters of keratohyalin granular filaments and tonofilaments did not show any statistical difference, but the difference between that of cornified cells was significantly greater than that of tonofilaments (P > 0.00001). DISCUSSION
The epidermis of newborn rats was cut
10
KAKIMI, FUKUYAMA, AND EPSTEIN
CRYOULTRA MICROTOMY OF GRANULAR CELLS
11
Fro. 1. Granular cells of newborn rat epidermis, fixed in glutaraldehyde, frozen after infusion of sucrose, and cut by cryomicrotomy. 1.5% phosphotungstic acid was used to stain the section negatively. Outline of cells and nucleus is seen clearly and keratohyalin granules (k) show a negative contrast. × 14 500. Fro. 2. (a) A small granule seen in granular cells with a regular periodicity of approximately 8.8 +_ 0.1 nm (n = 9). The outer membrane appears as a white line. (b) Mitochondria are recognized by their typical membranous appearance. (c) Granular cell boarders are delineated by the negative image of their plasma membranes. Desmosome junctions are identified by insertion of filaments and symmetrical arrangement of the marginal plaques. (a) and (b), × 280 000; (c), × 62 000. under low temperature without dehydration and plastic embedding. Although there were some technical difficulties associated w i t h t h e p r o c e s s i n v o l v e d in c r y o m i c r o t o m y , i n v e s t i g a t i o n of g r a n u l a r a n d c o r n i f i e d cells w a s p o s s i b l e a f t e r n e g a t i v e s t a i n i n g with PTA. Filaments, keratohyalin granules, n u c l e a r m e m b r a n e , p l a s m a m e m brane, mitochondria, and lamellated bodies
w e r e s e e n in n e g a t i v e c o n t r a s t . N i c o l s o n (9) s t u d i e d c h l o r o p l a s t s e m b e d d e d in b o v i n e serum albumin and reported that negative s t a i n i n g o c c u r s in h y d r o p h i l i c r e g i o n s b u t n o t in h y d r o p h o b i c a r e a s . T h e p r e s e n t findings, t h u s , c o i n c i d e w i t h t h e p h y s i o c h e m i c a l characterization reported by Swanbeck and T h y r e s s o n (15), w h o s u g g e s t e d t h a t k e r a tohyalin granules, filaments, and mem-
12
KAKIMI, FUKUYAMA, AND EPSTEIN
FIo. 3. Negatively stained keratohyalin granules (k) and groups of tonofflaments (t). They are connected at edges of the granules, x 96 000. FIG. 4. (a) Higher magnification of tonofibrils and filaments in keratohyalin granules; ( ) indicates 50 nm. (b) and (c) Filaments in cornified cells appear thicker and more loosely embedded in the matrix. ( ) indicates 50 nm. (a), × 350 000; (b), x 154 000; (c), × 350 000. b r a n e s of t h e n u c l e u s a n d m i t o c h o n d r i a a r e h y d r o p h o b i c c o m p o n e n t s o f g r a n u l a r cells. The internal structure of keratohyalin g r a n u l e s is o b s c u r e d b y t h e e l e c t r o n - d e n s e s t a i n in s k i n p r o c e s s e d b y c o n v e n t i o n a l t e c h n i q u e s . S e v e r a l a t t e m p t s to r e m o v e t h e electron-dense material from the granules h a v e b e e n r e p o r t e d (5, 16). H o w e v e r , d e m -
o n s t r a t i o n of f i l a m e n t s in k e r a t o h y a l i n g r a n u l e s h a s n o t b e e n successful. C r y o m i c r o t o m y of s k i n a n d u s e o f n e g a t i v e s t a i n i n g a l l o w e d us to i n v e s t i g a t e t h e s e f i l a m e n t s . They appeared more irregular than tonofilaments and formed a loosely arranged meshwork. Connection of the filaments and t o n o f i l a m e n t s w a s s e e n a t t h e e d g e of k e r -
CRYOULTRA MICROTOMY OF GRANULAR CELLS
13
14
KAKIMI, FUKUYAMA, AND EPSTEIN TABLE I DIAMETERS (nm) OF TONOFILAMENTS
ANn FILAMENTS IN KERATOHYALIN GRANULES ANn CORNIFIED CELLS a
Filament Tonofflaments
Keratohyalin granules
Cornified cells
3.6 _+0.1
3.4 _+ 0.1
7.3 +_0.3
Numbers represent averages of 15 randomly selected filaments. atohyalin granules suggesting that tonofilaments undergo morphological changes most probably by association with so-called keratohyalin material observed by conventional techniques as osmiophilic subs t a n c e s . O n t h e o t h e r h a n d , e v e n b y conv e n t i o n a l p r o c e s s i n g , f i l a m e n t s in c o r n i f i e d cells a p p e a r u n s t a i n e d (4); t h u s , t h e n e g a tive staining technique which we used demonstrated essentially the same ultrastructure with a keratin pattern. Brody measu r e d t h e d i a m e t e r o f t o n o f i l a m e n t s in m a n (3) a n d g u i n e a p i g s (2) a n d n o t e d a n inc r e a s e in t h e size o f f i l a m e n t s in c o r n i f i e d cells. A s i m i l a r i n c r e a s e w a s c o n f i r m e d in the present study. M e m b r a n e c o a t i n g g r a n u l e s a n d t h e i r lam e l l a e g a v e a n e g a t i v e c o n t r a s t r e s p o n s e in t h e c y t o p l a s m of g r a n u l a r cells. H o w e v e r , i t w a s n o t p o s s i b l e t o i d e n t i f y t h e m in i n t e r c e l l u l a r s p a c e s a f t e r t h e y w e r e e x t r u d e d (8). Previous studies indicated that these granules are surrounded by trilaminar memb r a n e s s i m i l a r t o t h o s e s e e n in t h e p l a s m a m e m b r a n e (7). N e g a t i v e s t a i n i n g f a i l e d to reveal the inner and outer leaves of the m e m b r a n e a n d t h e y a p p e a r e d as a w h i t e line e n c i r c l i n g t h e g r a n u l e s . T h e s a m e s t a i n i n g p a t t e r n w a s s e e n in t h e p l a s m a m e m b r a n e o f g r a n u l a r cells; t h e t w o l e a v e s were not separately identified. In contrast,
two layers of membrane were clearly seen in n u c l e a r m e m b r a n e s a n d m i t o c h o n d r i a . T h e r e s u l t s m a y b e i n t e r p r e t e d as a d e m o n s t r a t i o n of d i f f e r e n c e s in c h e m i c a l a n d physical properties of the two types of m e m b r a n e e x i s t i n g in e p i d e r m a l cells.
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