Cornified cell envelope assembly: a model based on electron microscopic determinations of thickness and projected density

Michal Jarnik 1 Martha N. Simon 0 Alasdair C. Steven ) 1 0 Department of Biology, Brookhaven National Laboratory , Upton, NY 11973 , USA 1 Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health , Bethesda, MD 20892 , USA SUMMARY In stratifying squamous epithelia, the cornified cell envelope (CE), a peripheral layer of crosslinked protein, is assembled sequentially from precursor proteins initially dispersed in the cytoplasm. Its major component is loricrin (37 kDa in mouse), which contributes from approx. 60% to >80% of the protein mass in different tissues. Despite its importance to the mechanical resilience and impenetrability of these tissues, detailed information has not been obtained on CE structure, even on such basic properties as its thickness or uniformity across a given CE or from tissue to tissue. To address this issue, we have studied CEs isolated from three murine epithelia, namely epidermis, forestomach and footpad, by electron microscopy of metal-shadowed specimens and scanning transmission electron microscopy (STEM) of unstained specimens. The former data reveal that the cytoplasmic surface is smoothly textured whereas the extracellular surface is corrugated, and that the average thickness is The stratum corneum of stratified squamous epithelia, composed of multiple layers of flattened dead cells, forms a tough, impenetrable barrier that shields the underlying living cells from hazards of the surrounding environment. Corneocytes are the end-product of the terminal differentiation pathway of keratinocytes in these tissues. Their organelles having broken down, they consist simply of a keratin filament matrix encased within the cornified cell envelope (CE). The CE is assembled late in the pathway when it replaces the cytoplasmic membrane, and consists of a layer of cross-linked protein coated with covalently bound lipids (Swartzendruber et al., 1987). It is a resilient material that is thought to be a major contributor to the protective role of the stratum corneum. This resilience is due primarily to crosslinking of the constituent proteins by (e -g -glutamyl)-lysine isopeptide bonds (Rice and Green, 1977; Thacher and Rice, 1985), reinforced by disulfide bridges. Indeed, it has been proposed that the biomechanical properties of the CE, considered as a composite biomaterial, may be modulated by altering the frequency and nature of the cross links (Jarnik et al., 1996). 15.31.2 nm, and strikingly uniform. Measurements of mass-per-unit-area from the STEM images yielded values of approx. 7.00.8 kDa/nm2, which were remarkably consistent over all three tissues. These data imply that the mature CE has a uniquely defined thickness. To explain its uniformity, we postulate that loricrin forms a molecular monolayer, not a variable number of multiple layers. In this scenario, the packing density is one loricrin monomer per 7 nm2, and loricrin should have an elongated shape, 2.5-3.0 nm wide by approx. 11 nm long. Moreover, we anticipate that any inter-tissue variations in the mechanical properties of CEs should depend more on protein composition and cross-linking pattern than on the thickness of the protein layer deposited. Despite its functional importance, the structure and assembly of the CE remain scantily understood. A number of proteins have been identified as CE precursors (Table 1; for reviews see Watt, 1989; Hohl, 1990; Reichert et al., 1993; Simon 1994), and it appears from analysis of peptides from proteolytic digests of isolated CEs (Steinert, 1995; Steinert and Marekov, 1995; 1997) that the major constituents are now known. Several lines of evidence support the notion that CE assembly is a multi-stage process (Reichert et al., 1993; Eckert et al., 1993; Steven and Steinert, 1994). First, a backing layer of such proteins as involucrin and cystatin-A is established by the transglutaminase cross-linking enzymes (Rice et al., 1994), possibly initiating at desmosomal sites (Ishida-Yamamoto et al., 1996; Steinert and Marekov, 1997), and continuing by processive attachment of substrate proteins to each other and to putative membrane-anchoring proteins (Reichert et al., 1993) such as envoplakin (Rhrbeg et al., 1996). This layer serves as a substrate for deposition of loricrin together with other proteins, notably the SPRs (small proline-rich proteins: Kartasova and van den Putte, 1988; Jarnik et al., 1996). Loricrin is the major protein of nearly all native, i.e. tissuederived, CEs characterized to date (Hohl et al., 1993), accounting for approx. 60% to >80% of their protein mass (Tables 1, 2). Beyond this broad outline, however, little is known about the detailed structure or modes of packing of molecules in the CE, or even whether these properties are uniform over the entire CE or exhibit local variations. So far, the basic property of thickness has been defined only in terms of measurements from electron micrographs of transverse thin sections. According to Matoltsy (1977), the CE consists of two electron-dense layers, one approx. 10 nm, the other approx. 2 nm thick, separated by a thin electron-translucent layer, for a total of approx. 15 nm: other observers have reported values of 15-20 nm (e.g. Hashimoto, 1969; Steven et al., 1990). However, these values are subject to considerable uncertainty. Fixation and dehydration may be accompanied by substantial shrinkage; staining may not be stoichiometric; in situ, the CE may be coated with additional material that is not detected in sections; and departures from exactly transverse sectioning geometry may result in an artifactual increase in perceived thickness (Leapman et al., 1997). As a step towards achieving a more detailed account of their molecular architecture, we have studied the structures of isolated CEs by electron microscopy, with particular attention to thickness. The methods used, namely freeze-drying/metal shadowing (Abermann et al., 1972; Nermut, 1977; Kistler et al., 1977), and dark-field scanning transmission electron microscopy (STEM) of unstained specimens (Crewe and Wall, 1970; Wall et al., 1974), are unaffected by the shortcomings listed above. The shadowed specimens characterize the physical thickness and surface relief of the CEs, while the STEM data yield local measurements of mass-per-unit area. MATERIALS AND METHODS Isolation of cell envelopes CEs were isolated from newborn mouse epidermis and from the forestomach and footpad of adult BALB/c mice, essentially as described by Mehrel et al. (1990). The epidermis was separated from the dermis after heating skin in PBS at 65C for 30 seconds. Entire forestomachs and footpads were taken from killed mice. These tissues were thoroughly rinsed in PBS, and extracted for 10 minutes in 2% SDS-extraction buffer (EB) (100 mM Tris, pH 8.5, 2% SDS, 20 mM DTT, 5 mM EDTA), 5 ml per epidermis or corresponding amounts for the other isolates, on a boiling water bath with vi (...truncated)