JOURNAL OF ULTRASTRUCTURERESEARCH ~l, 94-105 (1975)
Desmosomal Form, Fate, and Function in Mammalian Epidermis TERENCE D. ALLEN AND CHRISTOPHER S. POTTEN Electron Microscopy and Epithelial Kinetics, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, England Received July 30, 1974; accepted October 24, 1974 Separation of epidermal desmosomal contact is achieved by two means. Proliferative cells which both migrate and divide change their cell to cell contact by an isolation and cytoplasmic separation followed by engulfment of the entire desmosome. During desquamation of cornified cells contacts are broken by an apparent enzymatic degradation mediated via the membrane coating granules (MCG). Differences in this process can be observed according to the rate of cell production. In slowly proliferating regions (ear and dorsal skin) the "stacked" cornified cells have their desmosomes replaced by a specialised peripheral band of attachment, a squamosome; in regions with a higher proliferative rate (foot) the "nonstacked" cornified cells retain many of their desmosomes. Mammalian epidermis represents a highly dynamic tissue which continuously loses d e a d , c o r n i f i e d cells from its surface, a n d r e p l a c e s t h e m b y m i t o t i c r e n e w a l in the b a s a l layer. As a c o n s e q u e n c e cells are constantly migrating both laterally and vertically. This results in continual c h a n g e s i n cell to cell a t t a c h m e n t a n d m o d i f i c a t i o n of t h e s e a t t a c h m e n t s a c c o r d i n g to t h e cell's f u n c t i o n a l role w i t h i n t h e e p i d e r m i s . T h i s c u l m i n a t e s i n h i g h l y spec i a l i s e d f o r m s of cell to cell a t t a c h m e n t ( s q u a m o s o m e s in low s t e a d y s t a t e s y s t e m s a n d m o d i f i e d d e s m o s o m e s in o t h e r systerns) w h i c h f a c i l i t a t e t h e release of t h e cell a t t h e s k i n s u r f a c e p o s s i b l y via a s e q u e n t i a l d e g e n e r a t i o n of a t t a c h m e n t . T h e r a t e s of m i g r a t i o n (cell p r o d u c t i o n ) v a r y from region to region a n d also in r e s p o n s e to e p i d e r m a l i n j u r y . T h i s p a p e r describes the morphological events which a c c o m p a n y these changes. MATERIALS AND METHODS All material was obtained from male DBA-2 mouse epidermal sites both under normal conditions and after stimulation by injury. The details of the techniques are described elsewhere (1, 26, 27, 29).
The normal and injured sites thus provided samples with a wide range of proliferative rates (1, 5, 6, 11, 26-29). For electron microscopy glutaraldehyde-osmium fixed Epon-Araldite embedded material was stained with uranyl acetate-lead citrate and viewed in an AEI EM 801 A electron microscope (I). Cell surface replicas were prepared in the following manner. The cornified cells were removed using a commercial plastic adhesive dressing ("Aeroplast" Parke, Davis Co., Detroit) and a shadowed replica prepared by evaporating platinum-carbon at an angle of 30° followed by a vertical deposition of carbon alone. The Aeroplast was then dissolved in a 50/50 mixture of ethyl acetate and acetone, releasing the replica, which was then cleaned in commercial bleach and rinsed before observation. RESULTS
B a s a l layer. T h e p r o l i f e r a t i v e b a s a l l a y e r of t h e e p i d e r m i s is c h a r a c t e r i s e d b y r o u g h l y c u b o i d a l cells p o s s e s s i n g e x t e n s i v e d e s m o s o m a l c o n t a c t s b e t w e e n cells, a n d h e m i d e s m o s o m e s to t h e b a s e m e n t m e m b r a n e (Fig. 1). D i s t r i b u t e d t h r o u g h o u t t h e b a s a l l a y e r are a p o p u l a t i o n of d e n d r i t i c n o n k e r a t i n o c y t e s ( a b o u t 12-13%) (see review 33), w h i c h are c o m p r i s e d of two cell t y p e s , L a n g e r h a n s cells (15) ( a b o u t 10%) a n d I n d e t e r m i n a t e cells (or m e l a n o c y t e s ) ( a b o u t 2-3%). T h e s e cells s h a r e t h e c h a r a c -
FIG. 1. Low power micrograph of a vertical section through mouse dorsal epidermis. The centrally postioned Langerhans cell (L) has a "clear" cytoplasm containing Langerhans cell granules (Lcg) and a membrane devoid of desmosomal attachments. It appears to be held in position by cytoplasmic extensions from the adjacent basal cells (B) which surround it above and below. Desmosomes (D) are clearly visible between these extensions, and 94 Copyright © 1975by AcademicPress, Inc. All rights of reproduction in any form reserved.
also between the basal (B) and spinous layer cell (Sp). Hemidesmosomal attachments (hd) are apparent between the basement membrane (BM) and basal cells (B). × 15 000. FIG. 2. Oblique horizontal section through mouse dorsal epidermis, showing the extreme interfolding and numerous desmosomes of cell membranes between the cells of the spinous (Sp) and granular (Gr) layers. Two dendritic extensions (De) from a Langerhans cell are also visible, and the smoother cell membranes between the basal (B) and spinous (Sp) ceils (MCG, membrane coating granules). × 37 000. 95
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ALLEN AND P O T T E N
teristic of an electron lucent cytoplasm intracellular (Figs. 3d, 4e). Once intracel("clear cells"), and a lack of any form of lular, its ultimate fate is not known. Differentiating layers. Once they have membrane attachments to the neighbouring keratinocyte or basement membrane migrated out of the proliferative zone of the (Fig. 1). They are however, securely en- epidermis, cells begin to show signs of closed by the neighbouring keratinocytes keratinisation and changes in shape, bewhich surround them with interlocking coming flattened with a more extensive cell folds of cytoplasm above and below (Fig. to cell contact via numerous desmosomes on an extensively interfolded membrane 1). During the processes of mitosis and both (Fig. 2). Cells in the spinous layer contain lateral and vertical migration, the des- cytoplasmic granules (membrane coating mosomal contacts are broken and re- granules (MCG) (12) (Fig. 2)) which appear formed. DividirLg keratinocytes are com- to be involved with desmosomal breakmonly observed with areas of relatively down in later stages of keratinisation. The desmosome-free membrane. During the flattened and well attached arrangement of course of the reaction to wounding when cells in the spinous and granular layers is migration is dramatically accelerated maintained until the final stages of kera(leading to the introduction of many inter- tinisation at the top of the granular layer. cellular spaces, cytoplasmic bridges, and During this last phase of keratinisation the vacuoles), the sequence of events leading to granular cell changes to a flat hexagonal desmosomal breakdown (intracellular lo- plate of keratin. This involves loss of the calisation and ultimate resorption) can be nucleus, all recognisable cytoplasmic orvisualised. The sequence is diagrammati- ganelles and a reduction in desmosomal contacts in "nonordered" (high steady cally represented in Fig. 4. The initial change is an "isolation" of state) tissue, or their replacement in the preexisting desmosomes, either by in- "ordered" (I, 3, 4, 17, 27) epidermis by a vagination of the plasma membrane either specialised attachment at the edges termed side, or general cytoplasmic retraction, so the squamosome (1). Cornified layer. Cornified cells that the intact desmosome is left centrally positioned in a bridge of cytoplasm be- (squames) in "ordered" epidermis (mouse tween two adjacent cells (Figs. 3a, b, 4b). dorsal or ear epidermis) are thin hexagonal From this situation, the intercellular vacu- plates of keratin arranged in precise vertioles around the cytoplasmic bridge enlarge cal columns with their cell to cell attachso that the desmosomal plaque is now at ment restricted to the edges in the small the ends of two rather thin cytoplasmic area of overlap between neighbouring colprojections (Figs. 3b, 4c). The connection umns of cells (Figs. 5a, c). In nonstacked is finally broken by the "pinching off" of epidermis, which lacks the columnar arone of these strands, leading to a com- rangement of cornified cells, such as mouse pletely unattached area of membrane on plantar epidermis, the cornified cells are one cell (Fig. 4d) and a "double desmo- thicker, shorter, and more crenellated, and some" embedded in the wall of the other. have desmosomallike attachments over This double desmosome is then engulfed their entire surface (Figs. 5b, 8a). In both by cytoplasmic sheets and thus becomes types of epidermis, there appears to be free Fla. 3a. Vertical section through mouse dorsal epidermis 12 hr after wounding showing the formation of numerous intercellular spaces (ICS) and desmosomes (D) isolated at the end of cell projections across the intercellular space. Some separation (arrows) is also apparent between the basement membrane (BM) and the hemidesmosomes (hd). × 5 000. FIG. 3b. Higher power micrograph through the region of intercellular space (ICS) showing the desmosomes isolated between the cells on cytoplasmic projections (cp), which also appear to begin to surround the isolated
d e s m o s o m e s by c y t o p l a s m i c extensions {ce). x 64 0(O. Fro. 3c. Section showing a d e s m o s o m e a t t a c h m e n t , interpreted to be in a ia',er state of breakdown. T h e c y t o p l a s m i c extension from the cell on t h e right is about to pinch off at X, and the "whole" d e s m o s o m e engulfed by the cytoplasmic e x t e n s i o n s {ee) from the cell projection on the left. x 47 000. FIG. 3d. Section t h r o u g h a d e s m o s o m e (D) interpreted to have heen completely engulfed by' the cytoplasmic e x t e n s i o n s (ce) a n d t h u s isolated from t h e previously joined adjacent cell. ,< 57 000. 97
98
ALLEN AND POTTEN
a
D CM1 \ x \ x x \ \ ~ \ x x x \ \ x x \ x \ , ~M 2 , \ \ \ \ \ \ x x x ...... :...2,\-,\\\\\\\x\,
b
C
d
space between squames, with the exception of the areas of specialised cell to cell contact. In vertical transverse sections through the edge region of adjacent columns of regularly overlapping squames in mouse dorsal skin, the squamosome appears as an electron dense region (Fig. 5c) 35 nm thick and 160 nm wide (I). Favourable vertical sections cut both parallel to and along the overlapping region of squames from adjacent columns show that the squamosome runs around the edges of the squames as a largely unbroken band (Fig. 5d). This was confirmed by cell surface replicas of the inner surface of stripped cornified cells which revealed the full three dimensional arrangement of squamosomes at the edge of the hexagonal squames (Figs. 6a, b). At higher power the squamosomal band can be subdivided into individual irregularly shaped regions arranged at right angles to the long axis (Fig. 6b). Thus, rapidly proliferating epidermis (e.g., plantar) appears to retain discrete regions of attachment (Fig. 8a) while the cornified cells in slowly proliferating regions have their attachment restricted to a peripheral band, i.e., the squamosome.
Membrane coating granule activity. e
MCG's are first seen in the spinous layer (Fig. 2). These granules become concentrated in the perinuclear region (Fig. 7b) and at the upper cell membrane in the most distal granular cell (Fig. 7a). MCG
FIG. 4. Diagrammatic representation of the sequence of events of desmosomal separation of basal cells. (a) Cell membranes i and 2 (CM1, CM2) are joined by a central desmosome D. (b, c) Intercellular spaces or vacuoles (ICV) appear between the cells and begin to isolate the desmosome (D) until it is isolated on a small cytoplasmic projection. (d, e) The cytoplasmic projection pinches off and leaves a "double desmosome" (D) embedded in cell membrane 2 (CM2) which is finally engulfed by the cytoplasmic extensions (E) from cell 2. FIG. 5a. Vertical section through the edge of two stacked columns of cornified cells (squames). The region of squame overlap (SO) is shown by the dotted lines, and is restricted. Gr, Granular cell; BC, Basal cornified ceil. × 6 000. FIa. 5b. Vertical section through a region of nonstacked epidermis (mouse foot) where the squames are not arranged in columns, and consequently overlap completely. The edges of the squames (arrowed) are thus not grouped in any way. BC, basal cornified cell; Gr, granular cell. × 5 000. Fta 5c. High power micrograph showing the squame overlap region. The squamosomes (Sq) are in transverse section, and appear as an electron dense region joining the squames together. × 26 000. FIG. 5d. Vertical section cut approximately at right angles to a similar region to that in 5c, so that the squamosomes (Sq) can be seen in longitudinal section between the squames in some regions. × 67 000.
Sa
99
Fro. 6a. Cell surface replica of stripped mouse dorsal epidermis at low power, showing the hexagonal outline of the squames (S) and characteristic ridges (arrowed) in the region of overlap at the edges. × 3 000. FIG. 6b. High power micrograph of a region of 6a close to the squame edge (se) showing the squamosome (sq) to exist as a band of material subdivided by transverse ridges. × 19 000. 100
101
102
ALLEN AND POTTEN
profiles in vertical sections clearly suggest t h a t their contents are emptied into the space between the granular and basal cornified cell (Fig. 7a inset). In horizontal sections in the intercellular zone these granules or their contents, have a " f r o t h y " a p p e a r a n c e (Fig. 8b) which surrounds the desmosomal plaques and is suggestive of an e n z y m a t i c degradation. Thus these granules a p p e a r associated with an extracellular breakdown of cell to cell contacts and also possibly the intracellular nuclear breakdown which occurs at about the same time. Both processes probably also involve a rapid and effective resorption of the breakdown products, as shown in studies with radioactively labeled compounds (8). This strongly suggests a lysosomal function for the M C G ' s which in the later stages of t e r m i n a l differentiation also appear in all cytoplasmic regions (Fig. 7b) not occupied by keratinising fibres. Cell surface replicas of cornified cells from " o r d e r e d " epidermis showed considerable surface detail highly reminiscent of freeze-etched preparations of similar material (23). Detailed examination revealed areas consistent with the idea t h a t enzymatic digestion of desmosomal plaques had occurred (Fig. 8c), where profiles interpreted (from their similarity to freeze etch profiles) as desmosomal plaques, appear to be interacted with material consistent in a p p e a r a n c e with the secretions of MCG's (cf. Figs. 8b, c). DISCUSSION Within m a m m a l i a n epidermis, separation of desmosomal contacts takes place by two distinct methods according to the position of the cell and its state of differentiation. In the cornified layers separation
appears to take place by enzymatic degradation, whereas in the basal cells, the connections appear to be isolated, pinched off in the cytoplasm on one side, and subsequently phagocytically engulfed, resulting in intracellular desmosomes and numerous cell surface projections. T h e early formation of intercellular spaces (7, 9, 10, 13, 16, 18, 19, 20) is well reported as a s t a n d a r d response to injury or disease, and also the final engulfment (2, 19, 20, 32-36) but the full sequence of events does not a p p e a r to have been previously described. T h e situation in the basal layer appears to differ sharply from that in the cornified layer, and also from the experimental separation of desmosomes by trypsinisation (24). It is interesting to note however t h a t the artifical (trypsin) enzymatic separation results in the same phagocytic engulfment, although in this case, a " h a l f d e s m o s o m e " is engulfed rather t h a n a complete one. Both observations suggest t h a t once made, the desmosome is an extremely p e r m a n e n t organelle and t h a t as a consequence of its sequential and involved formation (14, 23, 25) it must be easier to isolate, pinch off and resorb the desmosome in the basal layer, than to secrete an enzyme which will separate the actual jointing material between the two cell m e m b r a n e s . This "physical" separation would be more easily controlled t h a n an enzymatic separation, where the enzyme, once released would cause a general separation of contacts rather t h a n the preferred localised one. F u r t h e r m o r e , the i n t r a c e l l u l a r p l a q u e s would remain, and still have to be resorbed, as shown after trypsinisation (24). In the cornified layer, the main separation of desmosomal a t t a c h m e n t s occurs at the boundary of the upper granular cell
FIG. 7a. Vertical section through mouse ear epidermis in the upper region, showing the accumulation of membrane coating granule (mcg) secretion (arrowed) between the membranes of the granular cell (Gr) and basal cornified cell. The inset shows a membrane coating granule in contact with the cell membrane, apparently discharging its contents into the intercellular space adjacent to a desmosome (D). 58 000; inset × 83 000. FIG. 7b. Horizontal section (parallel to the skin surface) showing the accumulation of membrane coating granules (mcg) in the nuclear (N) region of the granular cell, and also in the cytoplasm between the fibrils (f). × 23 000.
FIG. 8a. Vertical section through the cornified layer of mouse foot, a nonstacked epidermis showing the squames (S) and the individual desmosomelike attachments between them. × 32 000. FIG 8b. Horizontal section (parallel to the skin surface) cut through the region between the basal cornified cell (BC) and the uppermost granular cell (Gr). Large regions of membrane coating granule secretion (gs) are visible, often surrounding the desmosomal plaques (d). x 33 000. 103
104
ALLEN AND POTTEN
and basal cornified cell. Here, the membrane coating granules (MCG) secrete their enzymatic contents (21, 22, 30, 31) suggested to be capable of degrading the extracellular jointing material, in a similar manner to trypsin (24). In this case however, the separation is between an almost fully differentiated and presumably dead cell (the basal cornified) and a living cell still to complete the last part of its differentiation (the uppermost granular cell). In "stacked" skin there is a complete breakdown of the preexisting desmosomal contacts and their replacement by a different type of attachment, the squamosome. Thus there appears to be no resorbtion of intracellular desmosomal material from the cornified cells, and the associated tonofilaments may well be utilised in the process of keratinisation (20). The formation of squamosomal joints presents an interesting problem. These attachments appear to be made between a dead, keratinised cell (as there is no evidence for them in the spinous and granular layers) at the same time as desmosomal contacts are being enzymatically degraded over the rest of the surface. It is however, between cells of the same column (1) that the desmosomal contacts are broken, whereas the squamosomal contacts are formed between the overlapping regions of cornified cells from adjacent columns. The squamosomes thus appear to be formed between the most recently keratinised cell in a column, and the previously keratinised cell in the adjacent column. This process may occur as follows. The secretion from the MCG's acts to break down the desmosomal attachments over the entire surface of the keratinising cells. At this time there is also a large scale reabsorption and recycling of material. Much of the MCG enzyme plus degraded attachment material
is also reabsorbed into the cells, but at the region of overlap, (between adjacent squame columns) the previously keratinised edges of the squames act as a partial barrier and cause an accumulation of degraded jointing material, which reforms to make the peripheral band of attachment, i.e., the squamosome. In suggesting this function for the MCG's, we should like to state that this may be an additional function, as well as those previously described
(12). The authors thank Mr. G. R. Bennion for technical help during the ultrastructural studies, Mr. F. Leigh for preparing the micrographs, Miss Irene Wylie, Mrs. Margaret Grimes, and Mrs. Dorothy Robinson for technical assistance with the cell kinetic studies. This work was supported by grants from the Cancer Research Campaign and the Medical Research Council. REFERENCES 1. ALLEN, T. D., AND POTTEN, C. S., J. Cell Sci. 15, 291 (1974). 2. BREATHNACH,A. S., AND WYLLIE, L. M. A., in MONTAGNA,W., and Hu, F. (Eds.), Advances in Biology of Skin, Vol. 8, The Pigmentary System, p. 97. Pergamon Press, 1967. 3. CHRISTOPHERS,E., Z. Zellforsch. 114, 441 (1971). 4. CHRISTOPHERS,E., J. Invest. Derm . 56,165 (1971). 5. CHRISTOPHERS,E., ANDLAURENCE,E. B., Virchows Arch. Zellpath. 12, 212 (1973). 6. CHRISTOPHERS,E., WOLFF, H. H., AND LAURENCE, E. B., J. Invest. Derm., in press. 7. CROFT, C. B., AND TARIN, D., J. Anat. 106, 63 (1970). 8. CUTR1GHT,D. E., AND BAUER, H,, Oral Surg. Oral Med. Oral Path. 23, 260 (1967). 9. ERIKSSON, G., Acta Dermatovener. (Stockholm) 52, 441 (1972). 10. GOTTLIEB, S. K., AND LUTZNER, M. A., J. Invest. Derm. 54, 368 (1970). 11. HAMILTON, E., AND POTTEN, C. S., Cell Tissue Kinet. 5, 505 (1972). 12. HAYWARD,A. F., ANDHACKERMANN,M., J. Ultrastruct. Res. 43, 205 (1973). 13. KRAWCZYR,W. S., in MAIBACH,H. I., and ROVEE, D. T. (Eds.), Epidermal Wound Healing; p. 123. Year Book Medical Publ., Chicago, 1972. 14. KRAWCZYK,W. S., AND WILGRAM,G. F., J. Ultra-
FIG. 8c. Cell surface replica of a region interpreted to be similar to that shown in section in Fig. 8b, i.e., a surface replica of the inner side of the basal cornified cell after stripping. Some profiles are interpreted to be intact desmosomal plaques (arrowed) whilst others are interpreted to be desmosomal plaques undergoing enzymatic degradation as a result of membrane coating granule secretion (D). × 36 000.
DESMOSOMES IN MAMMALIAN EPIDERMIS struct. Res. 45, 93 (1973). 26. 15. LANGERHANS,P., Virchows Arch. Path. Anat. 44, 27. 325 (1868). 28. 16. LESSARD,R. J., WOLFF, K., ANDWINKELMANN,R. K., J. Invest. Derm, 50, 171 (1968). 29. 17. MACKENZIE, I. C., Nature (London) 222, 881 (1969). 30. 18. MARTINEZ,I. R., in MAIBACH,H. I., and ROVEE,D. T. (Eds.), Epidermal Wound Healing, p. 323. Year Book Medical Publ., Chicago, 1972. 31. 19. MISHIMAY., AND PINKUS, H., J. Invest. Derrn. 50, 89 (1968). 20. ODLAND, G., AND ROSS, R., J. Cell Biol. 39, 135 32.
(1968). 21. OHASHI, M., SAWADA,Y., AND MAKITA, R., Acta Derm Suppl. 73, 47 (1973). 22. OLSON,R. L., NORDQUiST,R. E., ANDEVERETT,M. A., Dermatologiea. 138, 268 (1969). 23. ORWIN,D. F. G., THOMPSON,R. W., ANDFLOWER, N. E., J. Ultrastruct. Res. 45, 15 (1973). 24. OVERTON,J., J. Exp. Zool. 168, 203 (1968). 25. OVERTON,J., J. Cell Biol. 56, 636 (1973).
33. 34. 35. 36.
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POTrEN, C. S., J. Invest. Derm. 58, 180 (1972). PO~rEN, C. S., Cell Tissue Kinet. 7, 77 (1974). POTTEN, C. S., Cell Tissue Kinet, submitted
(1974). POTTEN, C. S., AND ALLEN, T. D., J. Cell Sci., in press. SO~IER, C. S., in SQUIER, C. A., and MEYER, J. (Eds.), Current Concepts of the Histology of Oral Mucosa, p. 5. C. Thomas, Illinois, 1970. SILVERMAN, S., in SQUIER, C. A. and MEYER, J. (Eds.), Current Concepts of the Histology of Oral Mucosa, p. 5. C. Thomas, Illinois, 1970. WOLFF, K., Arch. F. Klin. Exp. Derm. 229, 54 (1967). WOLFF,K., Curt. Probl. Derm. 4, 79 (1972). WOLFF, K., AND HONIGSMANN,H. J. Ultrastruct. Res. 36, 176 (1971). WOLFF,K., AND KONaAD, K., J. Ultrastruct. Res. 39,262 (1972). WOLFF, K., AND SCHREINER, E., J. Invest. Dermat. 54, 37 (1970).
Desmosomal Form, Fate, and Function in Mammalian Epidermis TERENCE D. ALLEN AND CHRISTOPHER S. POTTEN Electron Microscopy and Epithelial Kinetics, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, England Received July 30, 1974; accepted October 24, 1974 Separation of epidermal desmosomal contact is achieved by two means. Proliferative cells which both migrate and divide change their cell to cell contact by an isolation and cytoplasmic separation followed by engulfment of the entire desmosome. During desquamation of cornified cells contacts are broken by an apparent enzymatic degradation mediated via the membrane coating granules (MCG). Differences in this process can be observed according to the rate of cell production. In slowly proliferating regions (ear and dorsal skin) the "stacked" cornified cells have their desmosomes replaced by a specialised peripheral band of attachment, a squamosome; in regions with a higher proliferative rate (foot) the "nonstacked" cornified cells retain many of their desmosomes. Mammalian epidermis represents a highly dynamic tissue which continuously loses d e a d , c o r n i f i e d cells from its surface, a n d r e p l a c e s t h e m b y m i t o t i c r e n e w a l in the b a s a l layer. As a c o n s e q u e n c e cells are constantly migrating both laterally and vertically. This results in continual c h a n g e s i n cell to cell a t t a c h m e n t a n d m o d i f i c a t i o n of t h e s e a t t a c h m e n t s a c c o r d i n g to t h e cell's f u n c t i o n a l role w i t h i n t h e e p i d e r m i s . T h i s c u l m i n a t e s i n h i g h l y spec i a l i s e d f o r m s of cell to cell a t t a c h m e n t ( s q u a m o s o m e s in low s t e a d y s t a t e s y s t e m s a n d m o d i f i e d d e s m o s o m e s in o t h e r systerns) w h i c h f a c i l i t a t e t h e release of t h e cell a t t h e s k i n s u r f a c e p o s s i b l y via a s e q u e n t i a l d e g e n e r a t i o n of a t t a c h m e n t . T h e r a t e s of m i g r a t i o n (cell p r o d u c t i o n ) v a r y from region to region a n d also in r e s p o n s e to e p i d e r m a l i n j u r y . T h i s p a p e r describes the morphological events which a c c o m p a n y these changes. MATERIALS AND METHODS All material was obtained from male DBA-2 mouse epidermal sites both under normal conditions and after stimulation by injury. The details of the techniques are described elsewhere (1, 26, 27, 29).
The normal and injured sites thus provided samples with a wide range of proliferative rates (1, 5, 6, 11, 26-29). For electron microscopy glutaraldehyde-osmium fixed Epon-Araldite embedded material was stained with uranyl acetate-lead citrate and viewed in an AEI EM 801 A electron microscope (I). Cell surface replicas were prepared in the following manner. The cornified cells were removed using a commercial plastic adhesive dressing ("Aeroplast" Parke, Davis Co., Detroit) and a shadowed replica prepared by evaporating platinum-carbon at an angle of 30° followed by a vertical deposition of carbon alone. The Aeroplast was then dissolved in a 50/50 mixture of ethyl acetate and acetone, releasing the replica, which was then cleaned in commercial bleach and rinsed before observation. RESULTS
B a s a l layer. T h e p r o l i f e r a t i v e b a s a l l a y e r of t h e e p i d e r m i s is c h a r a c t e r i s e d b y r o u g h l y c u b o i d a l cells p o s s e s s i n g e x t e n s i v e d e s m o s o m a l c o n t a c t s b e t w e e n cells, a n d h e m i d e s m o s o m e s to t h e b a s e m e n t m e m b r a n e (Fig. 1). D i s t r i b u t e d t h r o u g h o u t t h e b a s a l l a y e r are a p o p u l a t i o n of d e n d r i t i c n o n k e r a t i n o c y t e s ( a b o u t 12-13%) (see review 33), w h i c h are c o m p r i s e d of two cell t y p e s , L a n g e r h a n s cells (15) ( a b o u t 10%) a n d I n d e t e r m i n a t e cells (or m e l a n o c y t e s ) ( a b o u t 2-3%). T h e s e cells s h a r e t h e c h a r a c -
FIG. 1. Low power micrograph of a vertical section through mouse dorsal epidermis. The centrally postioned Langerhans cell (L) has a "clear" cytoplasm containing Langerhans cell granules (Lcg) and a membrane devoid of desmosomal attachments. It appears to be held in position by cytoplasmic extensions from the adjacent basal cells (B) which surround it above and below. Desmosomes (D) are clearly visible between these extensions, and 94 Copyright © 1975by AcademicPress, Inc. All rights of reproduction in any form reserved.
also between the basal (B) and spinous layer cell (Sp). Hemidesmosomal attachments (hd) are apparent between the basement membrane (BM) and basal cells (B). × 15 000. FIG. 2. Oblique horizontal section through mouse dorsal epidermis, showing the extreme interfolding and numerous desmosomes of cell membranes between the cells of the spinous (Sp) and granular (Gr) layers. Two dendritic extensions (De) from a Langerhans cell are also visible, and the smoother cell membranes between the basal (B) and spinous (Sp) ceils (MCG, membrane coating granules). × 37 000. 95
96
ALLEN AND P O T T E N
teristic of an electron lucent cytoplasm intracellular (Figs. 3d, 4e). Once intracel("clear cells"), and a lack of any form of lular, its ultimate fate is not known. Differentiating layers. Once they have membrane attachments to the neighbouring keratinocyte or basement membrane migrated out of the proliferative zone of the (Fig. 1). They are however, securely en- epidermis, cells begin to show signs of closed by the neighbouring keratinocytes keratinisation and changes in shape, bewhich surround them with interlocking coming flattened with a more extensive cell folds of cytoplasm above and below (Fig. to cell contact via numerous desmosomes on an extensively interfolded membrane 1). During the processes of mitosis and both (Fig. 2). Cells in the spinous layer contain lateral and vertical migration, the des- cytoplasmic granules (membrane coating mosomal contacts are broken and re- granules (MCG) (12) (Fig. 2)) which appear formed. DividirLg keratinocytes are com- to be involved with desmosomal breakmonly observed with areas of relatively down in later stages of keratinisation. The desmosome-free membrane. During the flattened and well attached arrangement of course of the reaction to wounding when cells in the spinous and granular layers is migration is dramatically accelerated maintained until the final stages of kera(leading to the introduction of many inter- tinisation at the top of the granular layer. cellular spaces, cytoplasmic bridges, and During this last phase of keratinisation the vacuoles), the sequence of events leading to granular cell changes to a flat hexagonal desmosomal breakdown (intracellular lo- plate of keratin. This involves loss of the calisation and ultimate resorption) can be nucleus, all recognisable cytoplasmic orvisualised. The sequence is diagrammati- ganelles and a reduction in desmosomal contacts in "nonordered" (high steady cally represented in Fig. 4. The initial change is an "isolation" of state) tissue, or their replacement in the preexisting desmosomes, either by in- "ordered" (I, 3, 4, 17, 27) epidermis by a vagination of the plasma membrane either specialised attachment at the edges termed side, or general cytoplasmic retraction, so the squamosome (1). Cornified layer. Cornified cells that the intact desmosome is left centrally positioned in a bridge of cytoplasm be- (squames) in "ordered" epidermis (mouse tween two adjacent cells (Figs. 3a, b, 4b). dorsal or ear epidermis) are thin hexagonal From this situation, the intercellular vacu- plates of keratin arranged in precise vertioles around the cytoplasmic bridge enlarge cal columns with their cell to cell attachso that the desmosomal plaque is now at ment restricted to the edges in the small the ends of two rather thin cytoplasmic area of overlap between neighbouring colprojections (Figs. 3b, 4c). The connection umns of cells (Figs. 5a, c). In nonstacked is finally broken by the "pinching off" of epidermis, which lacks the columnar arone of these strands, leading to a com- rangement of cornified cells, such as mouse pletely unattached area of membrane on plantar epidermis, the cornified cells are one cell (Fig. 4d) and a "double desmo- thicker, shorter, and more crenellated, and some" embedded in the wall of the other. have desmosomallike attachments over This double desmosome is then engulfed their entire surface (Figs. 5b, 8a). In both by cytoplasmic sheets and thus becomes types of epidermis, there appears to be free Fla. 3a. Vertical section through mouse dorsal epidermis 12 hr after wounding showing the formation of numerous intercellular spaces (ICS) and desmosomes (D) isolated at the end of cell projections across the intercellular space. Some separation (arrows) is also apparent between the basement membrane (BM) and the hemidesmosomes (hd). × 5 000. FIG. 3b. Higher power micrograph through the region of intercellular space (ICS) showing the desmosomes isolated between the cells on cytoplasmic projections (cp), which also appear to begin to surround the isolated
d e s m o s o m e s by c y t o p l a s m i c extensions {ce). x 64 0(O. Fro. 3c. Section showing a d e s m o s o m e a t t a c h m e n t , interpreted to be in a ia',er state of breakdown. T h e c y t o p l a s m i c extension from the cell on t h e right is about to pinch off at X, and the "whole" d e s m o s o m e engulfed by the cytoplasmic e x t e n s i o n s {ee) from the cell projection on the left. x 47 000. FIG. 3d. Section t h r o u g h a d e s m o s o m e (D) interpreted to have heen completely engulfed by' the cytoplasmic e x t e n s i o n s (ce) a n d t h u s isolated from t h e previously joined adjacent cell. ,< 57 000. 97
98
ALLEN AND POTTEN
a
D CM1 \ x \ x x \ \ ~ \ x x x \ \ x x \ x \ , ~M 2 , \ \ \ \ \ \ x x x ...... :...2,\-,\\\\\\\x\,
b
C
d
space between squames, with the exception of the areas of specialised cell to cell contact. In vertical transverse sections through the edge region of adjacent columns of regularly overlapping squames in mouse dorsal skin, the squamosome appears as an electron dense region (Fig. 5c) 35 nm thick and 160 nm wide (I). Favourable vertical sections cut both parallel to and along the overlapping region of squames from adjacent columns show that the squamosome runs around the edges of the squames as a largely unbroken band (Fig. 5d). This was confirmed by cell surface replicas of the inner surface of stripped cornified cells which revealed the full three dimensional arrangement of squamosomes at the edge of the hexagonal squames (Figs. 6a, b). At higher power the squamosomal band can be subdivided into individual irregularly shaped regions arranged at right angles to the long axis (Fig. 6b). Thus, rapidly proliferating epidermis (e.g., plantar) appears to retain discrete regions of attachment (Fig. 8a) while the cornified cells in slowly proliferating regions have their attachment restricted to a peripheral band, i.e., the squamosome.
Membrane coating granule activity. e
MCG's are first seen in the spinous layer (Fig. 2). These granules become concentrated in the perinuclear region (Fig. 7b) and at the upper cell membrane in the most distal granular cell (Fig. 7a). MCG
FIG. 4. Diagrammatic representation of the sequence of events of desmosomal separation of basal cells. (a) Cell membranes i and 2 (CM1, CM2) are joined by a central desmosome D. (b, c) Intercellular spaces or vacuoles (ICV) appear between the cells and begin to isolate the desmosome (D) until it is isolated on a small cytoplasmic projection. (d, e) The cytoplasmic projection pinches off and leaves a "double desmosome" (D) embedded in cell membrane 2 (CM2) which is finally engulfed by the cytoplasmic extensions (E) from cell 2. FIG. 5a. Vertical section through the edge of two stacked columns of cornified cells (squames). The region of squame overlap (SO) is shown by the dotted lines, and is restricted. Gr, Granular cell; BC, Basal cornified ceil. × 6 000. FIa. 5b. Vertical section through a region of nonstacked epidermis (mouse foot) where the squames are not arranged in columns, and consequently overlap completely. The edges of the squames (arrowed) are thus not grouped in any way. BC, basal cornified cell; Gr, granular cell. × 5 000. Fta 5c. High power micrograph showing the squame overlap region. The squamosomes (Sq) are in transverse section, and appear as an electron dense region joining the squames together. × 26 000. FIG. 5d. Vertical section cut approximately at right angles to a similar region to that in 5c, so that the squamosomes (Sq) can be seen in longitudinal section between the squames in some regions. × 67 000.
Sa
99
Fro. 6a. Cell surface replica of stripped mouse dorsal epidermis at low power, showing the hexagonal outline of the squames (S) and characteristic ridges (arrowed) in the region of overlap at the edges. × 3 000. FIG. 6b. High power micrograph of a region of 6a close to the squame edge (se) showing the squamosome (sq) to exist as a band of material subdivided by transverse ridges. × 19 000. 100
101
102
ALLEN AND POTTEN
profiles in vertical sections clearly suggest t h a t their contents are emptied into the space between the granular and basal cornified cell (Fig. 7a inset). In horizontal sections in the intercellular zone these granules or their contents, have a " f r o t h y " a p p e a r a n c e (Fig. 8b) which surrounds the desmosomal plaques and is suggestive of an e n z y m a t i c degradation. Thus these granules a p p e a r associated with an extracellular breakdown of cell to cell contacts and also possibly the intracellular nuclear breakdown which occurs at about the same time. Both processes probably also involve a rapid and effective resorption of the breakdown products, as shown in studies with radioactively labeled compounds (8). This strongly suggests a lysosomal function for the M C G ' s which in the later stages of t e r m i n a l differentiation also appear in all cytoplasmic regions (Fig. 7b) not occupied by keratinising fibres. Cell surface replicas of cornified cells from " o r d e r e d " epidermis showed considerable surface detail highly reminiscent of freeze-etched preparations of similar material (23). Detailed examination revealed areas consistent with the idea t h a t enzymatic digestion of desmosomal plaques had occurred (Fig. 8c), where profiles interpreted (from their similarity to freeze etch profiles) as desmosomal plaques, appear to be interacted with material consistent in a p p e a r a n c e with the secretions of MCG's (cf. Figs. 8b, c). DISCUSSION Within m a m m a l i a n epidermis, separation of desmosomal contacts takes place by two distinct methods according to the position of the cell and its state of differentiation. In the cornified layers separation
appears to take place by enzymatic degradation, whereas in the basal cells, the connections appear to be isolated, pinched off in the cytoplasm on one side, and subsequently phagocytically engulfed, resulting in intracellular desmosomes and numerous cell surface projections. T h e early formation of intercellular spaces (7, 9, 10, 13, 16, 18, 19, 20) is well reported as a s t a n d a r d response to injury or disease, and also the final engulfment (2, 19, 20, 32-36) but the full sequence of events does not a p p e a r to have been previously described. T h e situation in the basal layer appears to differ sharply from that in the cornified layer, and also from the experimental separation of desmosomes by trypsinisation (24). It is interesting to note however t h a t the artifical (trypsin) enzymatic separation results in the same phagocytic engulfment, although in this case, a " h a l f d e s m o s o m e " is engulfed rather t h a n a complete one. Both observations suggest t h a t once made, the desmosome is an extremely p e r m a n e n t organelle and t h a t as a consequence of its sequential and involved formation (14, 23, 25) it must be easier to isolate, pinch off and resorb the desmosome in the basal layer, than to secrete an enzyme which will separate the actual jointing material between the two cell m e m b r a n e s . This "physical" separation would be more easily controlled t h a n an enzymatic separation, where the enzyme, once released would cause a general separation of contacts rather t h a n the preferred localised one. F u r t h e r m o r e , the i n t r a c e l l u l a r p l a q u e s would remain, and still have to be resorbed, as shown after trypsinisation (24). In the cornified layer, the main separation of desmosomal a t t a c h m e n t s occurs at the boundary of the upper granular cell
FIG. 7a. Vertical section through mouse ear epidermis in the upper region, showing the accumulation of membrane coating granule (mcg) secretion (arrowed) between the membranes of the granular cell (Gr) and basal cornified cell. The inset shows a membrane coating granule in contact with the cell membrane, apparently discharging its contents into the intercellular space adjacent to a desmosome (D). 58 000; inset × 83 000. FIG. 7b. Horizontal section (parallel to the skin surface) showing the accumulation of membrane coating granules (mcg) in the nuclear (N) region of the granular cell, and also in the cytoplasm between the fibrils (f). × 23 000.
FIG. 8a. Vertical section through the cornified layer of mouse foot, a nonstacked epidermis showing the squames (S) and the individual desmosomelike attachments between them. × 32 000. FIG 8b. Horizontal section (parallel to the skin surface) cut through the region between the basal cornified cell (BC) and the uppermost granular cell (Gr). Large regions of membrane coating granule secretion (gs) are visible, often surrounding the desmosomal plaques (d). x 33 000. 103
104
ALLEN AND POTTEN
and basal cornified cell. Here, the membrane coating granules (MCG) secrete their enzymatic contents (21, 22, 30, 31) suggested to be capable of degrading the extracellular jointing material, in a similar manner to trypsin (24). In this case however, the separation is between an almost fully differentiated and presumably dead cell (the basal cornified) and a living cell still to complete the last part of its differentiation (the uppermost granular cell). In "stacked" skin there is a complete breakdown of the preexisting desmosomal contacts and their replacement by a different type of attachment, the squamosome. Thus there appears to be no resorbtion of intracellular desmosomal material from the cornified cells, and the associated tonofilaments may well be utilised in the process of keratinisation (20). The formation of squamosomal joints presents an interesting problem. These attachments appear to be made between a dead, keratinised cell (as there is no evidence for them in the spinous and granular layers) at the same time as desmosomal contacts are being enzymatically degraded over the rest of the surface. It is however, between cells of the same column (1) that the desmosomal contacts are broken, whereas the squamosomal contacts are formed between the overlapping regions of cornified cells from adjacent columns. The squamosomes thus appear to be formed between the most recently keratinised cell in a column, and the previously keratinised cell in the adjacent column. This process may occur as follows. The secretion from the MCG's acts to break down the desmosomal attachments over the entire surface of the keratinising cells. At this time there is also a large scale reabsorption and recycling of material. Much of the MCG enzyme plus degraded attachment material
is also reabsorbed into the cells, but at the region of overlap, (between adjacent squame columns) the previously keratinised edges of the squames act as a partial barrier and cause an accumulation of degraded jointing material, which reforms to make the peripheral band of attachment, i.e., the squamosome. In suggesting this function for the MCG's, we should like to state that this may be an additional function, as well as those previously described
(12). The authors thank Mr. G. R. Bennion for technical help during the ultrastructural studies, Mr. F. Leigh for preparing the micrographs, Miss Irene Wylie, Mrs. Margaret Grimes, and Mrs. Dorothy Robinson for technical assistance with the cell kinetic studies. This work was supported by grants from the Cancer Research Campaign and the Medical Research Council. REFERENCES 1. ALLEN, T. D., AND POTTEN, C. S., J. Cell Sci. 15, 291 (1974). 2. BREATHNACH,A. S., AND WYLLIE, L. M. A., in MONTAGNA,W., and Hu, F. (Eds.), Advances in Biology of Skin, Vol. 8, The Pigmentary System, p. 97. Pergamon Press, 1967. 3. CHRISTOPHERS,E., Z. Zellforsch. 114, 441 (1971). 4. CHRISTOPHERS,E., J. Invest. Derm . 56,165 (1971). 5. CHRISTOPHERS,E., ANDLAURENCE,E. B., Virchows Arch. Zellpath. 12, 212 (1973). 6. CHRISTOPHERS,E., WOLFF, H. H., AND LAURENCE, E. B., J. Invest. Derm., in press. 7. CROFT, C. B., AND TARIN, D., J. Anat. 106, 63 (1970). 8. CUTR1GHT,D. E., AND BAUER, H,, Oral Surg. Oral Med. Oral Path. 23, 260 (1967). 9. ERIKSSON, G., Acta Dermatovener. (Stockholm) 52, 441 (1972). 10. GOTTLIEB, S. K., AND LUTZNER, M. A., J. Invest. Derm. 54, 368 (1970). 11. HAMILTON, E., AND POTTEN, C. S., Cell Tissue Kinet. 5, 505 (1972). 12. HAYWARD,A. F., ANDHACKERMANN,M., J. Ultrastruct. Res. 43, 205 (1973). 13. KRAWCZYR,W. S., in MAIBACH,H. I., and ROVEE, D. T. (Eds.), Epidermal Wound Healing; p. 123. Year Book Medical Publ., Chicago, 1972. 14. KRAWCZYK,W. S., AND WILGRAM,G. F., J. Ultra-
FIG. 8c. Cell surface replica of a region interpreted to be similar to that shown in section in Fig. 8b, i.e., a surface replica of the inner side of the basal cornified cell after stripping. Some profiles are interpreted to be intact desmosomal plaques (arrowed) whilst others are interpreted to be desmosomal plaques undergoing enzymatic degradation as a result of membrane coating granule secretion (D). × 36 000.
DESMOSOMES IN MAMMALIAN EPIDERMIS struct. Res. 45, 93 (1973). 26. 15. LANGERHANS,P., Virchows Arch. Path. Anat. 44, 27. 325 (1868). 28. 16. LESSARD,R. J., WOLFF, K., ANDWINKELMANN,R. K., J. Invest. Derm, 50, 171 (1968). 29. 17. MACKENZIE, I. C., Nature (London) 222, 881 (1969). 30. 18. MARTINEZ,I. R., in MAIBACH,H. I., and ROVEE,D. T. (Eds.), Epidermal Wound Healing, p. 323. Year Book Medical Publ., Chicago, 1972. 31. 19. MISHIMAY., AND PINKUS, H., J. Invest. Derrn. 50, 89 (1968). 20. ODLAND, G., AND ROSS, R., J. Cell Biol. 39, 135 32.
(1968). 21. OHASHI, M., SAWADA,Y., AND MAKITA, R., Acta Derm Suppl. 73, 47 (1973). 22. OLSON,R. L., NORDQUiST,R. E., ANDEVERETT,M. A., Dermatologiea. 138, 268 (1969). 23. ORWIN,D. F. G., THOMPSON,R. W., ANDFLOWER, N. E., J. Ultrastruct. Res. 45, 15 (1973). 24. OVERTON,J., J. Exp. Zool. 168, 203 (1968). 25. OVERTON,J., J. Cell Biol. 56, 636 (1973).
33. 34. 35. 36.
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POTrEN, C. S., J. Invest. Derm. 58, 180 (1972). PO~rEN, C. S., Cell Tissue Kinet. 7, 77 (1974). POTTEN, C. S., Cell Tissue Kinet, submitted
(1974). POTTEN, C. S., AND ALLEN, T. D., J. Cell Sci., in press. SO~IER, C. S., in SQUIER, C. A., and MEYER, J. (Eds.), Current Concepts of the Histology of Oral Mucosa, p. 5. C. Thomas, Illinois, 1970. SILVERMAN, S., in SQUIER, C. A. and MEYER, J. (Eds.), Current Concepts of the Histology of Oral Mucosa, p. 5. C. Thomas, Illinois, 1970. WOLFF, K., Arch. F. Klin. Exp. Derm. 229, 54 (1967). WOLFF,K., Curt. Probl. Derm. 4, 79 (1972). WOLFF, K., AND HONIGSMANN,H. J. Ultrastruct. Res. 36, 176 (1971). WOLFF,K., AND KONaAD, K., J. Ultrastruct. Res. 39,262 (1972). WOLFF, K., AND SCHREINER, E., J. Invest. Dermat. 54, 37 (1970).
