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) .

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