Sequential Reorganization of Cornified Cell Keratin Filaments Involving Filaggrin- Mediated Compaction and Keratin 1 Deimination Akemi Ishida-Yamamoto,

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Sequential Reorganization of Cornified Cell Keratin Filaments Involving Filaggrin- Mediated Compaction and Keratin 1 Deimination Akemi Ishida-Yamamoto, Tatsuo Senshu, Robin A.J. Eady, Hidetoshi Takahashi, Hiroshi Shimizu, Masashi Akiyama, Hajime Iizuka Journal of Investigative Dermatology Volume 118, Issue 2, Pages 282-287 (February 2002) DOI: 10.1046/j.0022-202x.2001.01671.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The superficial epidermal layers of the normal human skin are ACP positive. Cryostat sections are labeled for ACP (fluorescein isothiocyanate) and 34βB4 (Texas red) (a) or ACP (fluorescein isothiocyanate) and filaggrin (Texas red) (b). Enucleated flat cells (bracket) are possible lower cornified cells that are ACP negative, 34βB4 positive (a), and filaggrin positive (b). Nuclei are stained with 4′,6-diamidino-2-phenylindole dihydrochloride. Scale bar: 10 μm. Journal of Investigative Dermatology 2002 118, 282-287DOI: (10.1046/j.0022-202x.2001.01671.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunoelectron microscopy shows sequential reorganization of cornified cell keratin filaments. (a) The first (1) and second (2) cornified cell layers are ACP negative, whereas the third layer (3) is positive. Note that the cytoplasm of negative cells shows a reticular pattern, whereas that of positive cells is amorphous in texture. Five nanometer gold labels have been enhanced using a silver staining method. The inset is to show these cells at lower magnification. G, granular cell. (b) Double staining with ACP (5 nm gold) and 34βB4 (10 nm gold). All the cornified cells are similarly positive to 34βB4. ACP staining is not detected in the first layer (1), only a few labels are seen in the second layer (2) and many labels in the third layer (3). These are shown in a lower magnification in the inset. G, granular cell. (c, d) Double staining with anti-filaggrin (5 nm gold) and ACP (10 nm gold). The first cornified cell (1) is filaggrin positive/ACP negative. The second cell (2) is double positive, and the third cell (3) is filaggrin negative/ACP positive. The inset in c is to show cells 1–3 in c and d at lower magnification. Scale bars: 0.1 μm; 1 μm in the inset. Journal of Investigative Dermatology 2002 118, 282-287DOI: (10.1046/j.0022-202x.2001.01671.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 BCIE cornified cells are only partially positive to ACP. Case 1 (a) and 2 (b). Semithin sections of Lowicryl K11M blocks are labeled for ACP (fluorescein isothiocyanate) and 34βB4 (R-phycoerythrin). Nuclei are stained with 4′,6-diamidino-2-phenylindole dihydrochloride. Scale bar: 50 μm. Journal of Investigative Dermatology 2002 118, 282-287DOI: (10.1046/j.0022-202x.2001.01671.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunoelectron microscopy shows that deimination and association with filaggrin are heterogeneous in BCIE keratins. More labels for ACP (a) and for filaggrin (b) (silver enhanced 5 nm gold) are seen over filamentous keratin (*) than over round amorphous keratin clumps (arrows). Scale bar: 0.5 μm. Journal of Investigative Dermatology 2002 118, 282-287DOI: (10.1046/j.0022-202x.2001.01671.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Decreased V subdomain deimination of K1 in BCIE. Proteins extracted from tape-stripped outermost cornified cells of normal skin (lane 2) and from scraped scales of three patients with BCIE (cases 3, 4, and 5) (lanes 3, 4, and 5, respectively) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotted for sequential probing with ACP (b) and AMC (d) followed by staining total proteins (a) as described in the text. Keratins in the viable layers of epidermis were loaded to lane 1 as references. Keratin types were estimated by their mobilities. The amounts of proteins loaded were about 10 μg. Superimposed color images of ACP (red) and protein (green) (c) and AMC (red) and protein (green) (e) were also included for comparison. Journal of Investigative Dermatology 2002 118, 282-287DOI: (10.1046/j.0022-202x.2001.01671.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions