Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia

Abstract

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

Figure 1

Detection of epitopes of 3-d CEs by immunogold electron microscopy. The epitopes were tested with 10-nm gold particles, and were (A) desmoplakin, (B) envoplakin, (C) periplakin, (D) involucrin, (E) keratin 1, (F) keratin 10, (G) SPR1, (H) loricrin, (I) plectin, and (J) annexin I. Bars, 100 nm.

Figure 2

Detection of epitopes on 6- to 7-d CEs by immunogold electron microscopy. The epitopes were tested with 10-nm gold particles and were (A) desmoplakin, (B) envoplakin, (C) periplakin, (D) involucrin, (E) keratin 1, (F) keratin 10, (G) SPR1, (H) loricrin, (I) plectin, and (J) annexin I. Bars, 100 nm.

Figure 3

Use of double-labeling immunogold electron microscopy to monitor distributions of epitopes in 2-d (A), 3-d (B, D, and F), 5-d (C), or 6- to 7-d (E and G) CEs. Combinations used were (A–C) desmoplakin (5-nm particles) and involucrin (15 nm), (D and E) envoplakin (5 nm) and involucrin (15 nm), and (F and G) envoplakin (5 nm) and desmoplakin (15 nm). Bars, 100 nm.

Figure 4

Use of double-labeling immunogold electron microscopy to monitor distribution of epitopes for SPRs (15-nm particles) and envoplakin (5 nm). (A) 2-d CEs, (B) 3-d, (C) 5-d, and (D) 7-d. Bars, 100 nm.

Figure 5

Use of double-labeling immunogold electron microscopy to monitor distribution of epitopes for envoplakin (15-nm particles) and loricrin (5 nm) in 6- to 7-d CEs. Note that the two labels occur of different sides. Bar, 100 nm.

Figure 6

New model for the early stages of CE assembly in stratified squamous epithelia. (A) Our immunogold and sequencing data indicate that a very early step in CE assembly involves the head-to-head and head-to-tail oligomerization of involucrin (red bars) at interdesmosomal regions of the plasma membrane, in close juxtaposition with the TGase 1 enzyme (green circles), envoplakin (yellow rods), and perhaps periplakin (blue ovoids). (B) subsequently in 3-d CEs, involucrin deposition elongates to the desmoplakin (green rods) component of desmosomes, and SPR (pink rods) proteins become attached. (C) By 6-d CEs, an involucrin–envoplakin–SPR continuum or scaffold has formed along the entire cell periphery. (D) In 7-d or more mature sloughed cell CEs, this scaffold serves as a platform for the subsequent addition of other reinforcement proteins, including loricrin (white circles) and other proteins (small blue circles). Also, the keratin chain composition of the keratin intermediate filaments at desmosomes changes from mostly keratins 5, 6, and 14 (dark pink rods) to keratins 1, 2e, and 10 (orange rods) located along the entire periphery. In all models, the lower side is cytoplasmic. Note that to recover recognizable CE fragments with associated desmosomal remnants after 2-d culture, many other cell peripheral proteins presumably have become cross-linked in minor amounts, but we were unable to identify these (e.g., plectin and integrins; pink ovoids) by sequencing or immunogold electron microscopy.