Marked Changes in Lamellar Granule and Trans-Golgi Network Structure Occur during Epidermal Keratinocyte Differentiation Haruyo Yamanishi, Tsutomu Soma,

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Marked Changes in Lamellar Granule and Trans-Golgi Network Structure Occur during Epidermal Keratinocyte Differentiation Haruyo Yamanishi, Tsutomu Soma, Jiro Kishimoto, Toshihiko Hibino, Akemi Ishida-Yamamoto Journal of Investigative Dermatology Volume 139, Issue 2, Pages 352-359 (February 2019) DOI: 10.1016/j.jid.2018.07.043 Copyright © 2018 The Authors Terms and Conditions

Figure 1 The overall 3D structure of LGs changes markedly through the course of granular cell differentiation. (a–c) 3D reconstruction of the granular layers SG1–3 and surface rendering images of associated LGs. (d–f) Projection views of LGs taken from the front of the surface rendering image shown in panel c. (g, h) Projection views of LG-associated corneodesmosomes and desmosomes of the SG1 and SG2 layers. Plaques shown in panels g and h were segmented at an angle from the top of panels d and e, respectively (corneodesmosomes, purple and pale blue; desmosomes, brown and orange; reticular LGs, white arrows; vesicular LGs, white arrow heads). Images shown are from scalp skin of a 52-year-old male. 3D, 3-dimensional; LG, lamellar granule; SG, stratum granulosum. Scale bars = 1 μm. Journal of Investigative Dermatology 2019 139, 352-359DOI: (10.1016/j.jid.2018.07.043) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Overall LG structure in the SG2 layer is dependent upon LG status in the SG1 layer. A TEM image showing two cells in the SG1 layer (SG1a and SG1b) and one cell in the SG2 layer. The LGs associated with the SG1 and SG2 cells demonstrate a different pattern of localization (LGs at the SC–SG1 and the SG1–SG2 border are indicated by white and black arrows, respectively. SG1 and SG2 intracellular LGs are indicated by white and black arrowheads, respectively). Images are from eyelid skin from a 69-year-old female. LG, lamellar granule; SC, stratum corneum; SG, stratum granulosum; TEM, transmission electron microscopy. Scale bar = 500 nm. Journal of Investigative Dermatology 2019 139, 352-359DOI: (10.1016/j.jid.2018.07.043) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Overall LG structure in the SG2 layer is dependent upon cell position within the granular layer. (a) 3-Dimensional reconstruction of the granular layers. (b) A surface rendering image of the cell indicated by the arrow in panel a. In this imaging plane, the portion of the cell observed occupies the SG3 layer. LGs are vesicular and are retained in the cytoplasm (cell outline, purple; LGs, green). (c) The same cell as shown in panel b, but at an imaging plane located within the SG2 layer, toward the back of the tissue block. Some LGs are fused with the apical cell membrane (asterisk). Images are from scalp skin of a 52-year-old male. LG, lamellar granule; SG, stratum granulosum. Scale bars = 1 μm. Journal of Investigative Dermatology 2019 139, 352-359DOI: (10.1016/j.jid.2018.07.043) Copyright © 2018 The Authors Terms and Conditions

Figure 4 The Golgi apparatus is found adjacent to the nucleus in cells of the SG2 layer. (a) A FIB-SEM image, which is the 59th image of the 71 serial sections used to generate the images in panels c–f, shows the Golgi apparatus (asterisk) near to the nucleus (N). (b) A magnified view of panel a. The TGN is indicated with arrowheads. (c) An image stack reconstructed from the serial sections. (d) A reconstructed projection view of organelles in the SG2 cell layer. (e) A surface rendering image of the organelles shown in panel d. (f) A magnified view of panel e. LGs, blue; TGN, yellow; Golgi apparatus, green; nucleus, gray. Images are from the scalp skin of a 56-year-old male. FIB-SEM, focused ion beam scanning electron microscopy; LG, lamellar granule; SG, stratum granulosum; TGN, trans-Golgi network. Scale bars = 1 μm (a–e) and 500 nm (f). Journal of Investigative Dermatology 2019 139, 352-359DOI: (10.1016/j.jid.2018.07.043) Copyright © 2018 The Authors Terms and Conditions

Figure 5 3-Dimensional structure and distribution of LGs and the TGN. (a-c) Projection views of LGs and the TGN. (d) A magnified view of LGs and the TGN in the SG2 layer extracted from panel c. (e) Serial section images of the SG2 layer incorporating the region containing the LGs and TGN highlighted by the asterisk in panel d. Arrows show connections between LGs and the TGN. (f, g) Surface rendering views of LGs and the TGN reconstructed from panel e. (h, i) Surface rendering views of LGs and the TGN from panels f and g, each from a different angle. Images are from scalp skin of a 56-year-old male. LG, lamellar granule; SG, stratum granulosum; TGN, trans-Golgi network. Scale bars = 2 μm (a–c), 1 μm (d), and 400 nm (e–i). Journal of Investigative Dermatology 2019 139, 352-359DOI: (10.1016/j.jid.2018.07.043) Copyright © 2018 The Authors Terms and Conditions