Desmosome Differentiation During Epidermal Cornification: New Observations Obtained from Intermediate Voltage Electron Microscopy
t Carolyn A. Larabell, * Kimie Fukuyama, t and William L. Epstein:j: "Intermediate Voltage Electron Microscope Facility, Lawrence Berkeley Laboratory, University of Berkeley, California; and t Oepartment of Dermatology, University of California, San Francisco, California, U.S.A.
Human epidermis was examined under 400-kV intermediate voltage electron microscopy using epon sections cut 5 - 8 times thicker than usual ultrathin sections. Asymmetry of desmosomal structures occurred as cells became cornified. Electron-dense proteins deposited at the inner leaflet of the plasma membrane of corneocytes do not extend into the desmosomal area, which connects to granular cells. Stereo micrographs revealed the existence of two different cellular
During the late stages of differentiation, morphology of the epidermal cells is altered and terminally differentiated cells appear to be 1) lacking a nucleus, 2) filled with keratin fibers embedded in an amorphous matrix, and 3) outlined by a thickened plasma
membrane due to depositing of electron-dense components assembled just beneath the inner leaflet. The membrane change, which was first reported in an early ultrastructural description [1], stimulated biochemical [2] and morphologic (3) studies to elucidate the composition of this thickened membrane, and led to more recent definitive investigations (e.g., [4-11 D. On the other hand, morphologic analysis has been neglected owing to the comprehensive description of initial publications and the unavailability of newer tools . Intermediate voltage electron microscopy (IVEM) allows examination of 0.25-0.5-)1m-thick tissue sections, which permits observation of the total mass of some intracellular organelle complexes in a single section. In this report we present three-dimensional views of the organization of desmosomes in the first layer of human cornified cells after plasma membrane thickening has occurred.
MATERIALS AND METHODS
Normal skin biopsies (3 mm in diameter) obtained from healthy volunteers were minced and fixed in 3% glutaraldehyde and 2% OS04, both buffered with 0.025 M phosphate buffer, pH 7.4. The specimens were treated in 2% uranyl acetate dissolved in 80% EtOH for 1 h prior to dehydration in EtOH. They were embedded in an epon mixture. Sections 0.25 - 0.5 )1m thick were placed on copper grids and stained with uranyl acetate and lead citrate. They were examined at 400 kV in the lEOL 4000 EX lVEM. Stereo micro-
Manuscript received August II, 1992; accepted for publication December 23, 1992.
Reprint requests to: Dr. Kimie Fukuyama, Department of Dermatology, University of California San Francisco, San Francisco, CA 94143-0536.
Abbreviations: CTEM, conventional transmission electron microscopy; IVEM, intermediate voltage electron microscopy.
elements at the cell surface confirming that the plasma membrane first thickened in areas without desmosomes. Examination of the three-dimensional nature of desmosomes and keratin-filament aggregation without serial sectioning and/or selecting a strict angle of the tissues will allow us to extend our ultrastructural knowledge. ] Invest Dermatol 101:103-104, 1993
graphs were obtained by tilting the specimen ± 6 0 using a eucentric goniometer.
RESULTS
The general view of the epidermis seen by IVEM at low power, such as magnification X 2000 - 3000, is identical to that reported with conventional transmission electron microscopy (CTEM). Morphology of keratin filaments, keratohyalin granules, desmosomes, and nuclei were readily observed. In the IVEM, small structures, such as polysomes and lamellar granules, demonstrated much less contrast than those seen in CTEM. However, thick sections facilitated visualization of entire structures, such as desmosomes with superior internal details (Fig 1). The different layers and general symmetry of desmosomal organization in spinous and granular cells were more readily detected in the thicker sections than usually observed by CTEM. Furthermore, the lack of symmetry in desmosomes connecting granular cells and cornified cells became quite obvious. Whereas tonofilaments and the plaque on the granular cell side retained the fine structure comparable to more proximal cells, the intercellular layers had become indistinct and the specific desmosomal substructure had totally disappeared from the cornified cell side. The abrupt termination of the thickened plasma membrane at the edge of the conjunctional area also was noted in relatively thinner sections (0.25 -0.3)1m thick) as shown in Fig 1. Electron dense zone measuring 16.3 ± 2.4 nm (mean ± SO, n = 23) covered the inner surface of the non-desmosomal area of cornified cells.
Stereo micrographs allow demonstration of the thickened plasma membrane and the desmosomal regions of cornified cells facing granular cells within the same plane of a single section (Fig 2). What becomes readily apparent in relatively thicker sections (approximately 0.5 )1m), without use of serial sections, is the presence of two different margins of cornified cells. One is the electrondense zone; another is the electron lucent cytoplasm of the desmosome area without membrane changes. The conjunctional sites devoid of thickening of the plasma membrane seemed to be retracted inward toward the granular cells. The observation indicated that plasma-membrane thickening does not occur randomly, and a free surface inner leaflet of the plasma membrane may be required for
0022-202Xj93jS06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc.
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104 LARABELL BT AL
Figure 1. Desmosomes of granular cells and a lowermost cornified cell. Although desmosomes in granular cells show a well recognized ultrastructure, those in the corneocyte have a homogenous appearance without attachment plaques and inserting tonofilaments. Electron-dense proteins deposited in the cornified cell plasma membrane do not extend into the desmosomal areas (arrowheads). Bundles of keratin filaments surrounded by electron-dense substance is seen in cornified cells but also in granular cells (arrows). Bar, 0.3 /lm.
depositing newly crosslinked proteins. Disappearance of the attachment plaques in more differentiated corneocytes coincided with protein deposition at the plasma membrane in those sites.
DISCUSSION
In this study, sections of human skin cut about 5 to 7 times thicker than those examined with CTEM were viewed under IVEM. The main advantage of this approach is the ability to visualize the ultrastructure of thick sections (due to the increased accelerating voltage of the IVEM), which contain several levels of cellular constituents. The additional information obtained allowed us to analyze the ultrastructure of keratinocytes at different depths. In addition, the IVEM is equipped with a goniometer that permits acquisition of stereo pairs of images. The three-dimensional nature of desmosomes, particularly in relation to thickening of the plasma membrane at the junction between granular and cornified cells, was demonstrated. Thickening of the entire plasma membrane does not occur in cornified cells as long as the intracellular desmosome structure exists. A biochemical basis for lack of protein deposition at the desmosome area remains speculative, though it may be related to activation of membrane-bound transglutaminase. Whereas the majority ofliterature concerning ultrastructure of keratinocytes has ignored such subtleties, the failure of plasma membrane thickening in areas with retained desmosomes has been reported by CTEM [12] . This discrete membrane appearance was demonstrable when the thin sections were cut exactl y perpendicular to the desmosomal area and photomicrographs somewhat overexposed [12]. In the case of CTEM, such observation may be considered as "selective" and difficult to separate from random or irregular findings. With IVEM most desmosomes in thick sections reveal the two different types of the cell borders in their three-dimensional architecture, and the observation becomes consistent and reliable without exceptional skills in ultrathin sectioning.
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Figure 2. A pair of stereo micrographs demonstrating rwo different characteristics of the plasma membrane of cornified cells facing towards granular cells. At one level the thickened plasma membrane is seen as a continuous line whereas in another plane desmosomes cOlmect to granular cells without thickening of the membrane. Bar, 0.2 /lm.
This work was supported by NIH gra"ts RR 05097 to tlte West Coast Facility for I"termediate Voltage Electroll Microscope alld AR 12433 (KF) .
REFERENCES
1. Brody H: The keratinization of epidermal cells of normal guinea pig skin as revealed by electron microscopy. J Ultrastr Res 2:482-511, 1959
2. Matoltsy AG , Matoltsy MN: The membrane protein of horny cells. J Invest Dermatol 46:127 -129, 1966
3. Farbman AI: Plasma membrane changes during keratinization. Anat Rec 156:269-282, 1966
4. Watt FA, Green H: Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell BioI 90:738- 742,1981
5. Zettergren JG, Peterson LL, Wucpper .!CD: Keratolinin: the so luble substrate of epidermal transglutaminase from human and bovine tissue. Proc Natl Acad Sci USA 81:238-242,1984
6. Buxman MM, Wuepper !CD: Cellular localization of epidermal transglutaminase: a histochemical and immunochemical study. J Histochem Cytochem 26:340-348, 1978
7. Thacher SM, Rice RH: Keratinocyte-specific transglutarninase of cultured human epidermal cells: relation to cross- linked envelope formation and terminal differentiation. Cell 40:685-695,1985
8. Fukuyama K, Ohtani 0 , Hibino T, Epstein WL: Cellular localization of thiol-proteinase inhibitor. Cell Tissue Res 223:313-323,1982
9. Fukuyama K, Ito Y, Yabe K, Epstein WL: Immunological detection of a cysteine protease in the skin and other tissues. Cell Tissue Res 240:417-423, 1985
10. Fukuyama K, Epstein WL: A comparative autoradiographic study of keratohyalin granules containing cysteine and histidine. J Ultrastr Res 51:314-325,1975
11 . Mehrel T, Hohl 0, Rothnagel JA, Longley MA, Bundman D, Cheng C, Lichti U, Bisher ME, Steven AC, Steinert PM, Yuspa SH, Roop DR: Identification of a major keratinocyte cell envelope protein, Loricrin. Cell 61:1103-1112, 1990
12. Raknerud N: The ultrastructure of the interfollicular epidermis of the Jlairless (hr/hr) mouse. J Ultrastr Res 52:32 - 51, 1975