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 15.3+/−1.2 nm, and strikingly uniform. Measurements of mass-per-unit-area from the STEM images yielded values of approx. 7.0+/−0.8 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.

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