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• W2890850862 abstract "Epidermal lamellar granules transport various lipids, proteins, and protein inhibitors from the trans-Golgi network to the extracellular space, and play an important role in skin barrier formation. We elucidated the 3-dimensional structure of lamellar granules and the trans-Golgi network in normal human skin by focused ion beam scanning electron microscopy. Reconstructed focused ion beam scanning electron microscopy 3-dimensional images revealed that the overall lamellar granule structure changed from vesicular to reticular within the second layer of the stratum granulosum. Furthermore, the trans-Golgi network was well developed within this layer and spread through the cytoplasm with branched, tubular structures that connected to lamellar granules. Our study reveals the unique overall 3-dimensional structure of lamellar granules and the trans-Golgi network within the cells of the epidermis, and provides the basis for an understanding of the skin barrier formation. Epidermal lamellar granules transport various lipids, proteins, and protein inhibitors from the trans-Golgi network to the extracellular space, and play an important role in skin barrier formation. We elucidated the 3-dimensional structure of lamellar granules and the trans-Golgi network in normal human skin by focused ion beam scanning electron microscopy. Reconstructed focused ion beam scanning electron microscopy 3-dimensional images revealed that the overall lamellar granule structure changed from vesicular to reticular within the second layer of the stratum granulosum. Furthermore, the trans-Golgi network was well developed within this layer and spread through the cytoplasm with branched, tubular structures that connected to lamellar granules. Our study reveals the unique overall 3-dimensional structure of lamellar granules and the trans-Golgi network within the cells of the epidermis, and provides the basis for an understanding of the skin barrier formation. Lamellar granules (LGs), also known as lamellar bodies, are organelles unique to epidermal keratinocytes that play a crucial role in skin barrier formation and desquamation. LGs contain glucosylceramides and other lipids, proteases, protease inhibitors, and various other proteins (Hayward, 1979Hayward A.F. Membrane-coating granules.Int Rev Cytol. 1979; 59: 97-127Crossref PubMed Scopus (65) Google Scholar, Madison et al., 1998Madison K.C. Sando G.N. Howard E.J. True C.A. Gilbert D. Swartzendruber D.C. et al.Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the golgi.J Investig Dermatol Symp Proc. 1998; 3: 80-86Abstract Full Text PDF PubMed Scopus (82) Google Scholar, Odland and Holbrook, 1981Odland G. Holbrook K. The lamellar granules of epidermis.Curr Probl Dermatol. 1981; 9: 29-49Crossref PubMed Google Scholar). LGs are observed as round or oblong, membrane-delimited, lamellate structures in the stratum granulosum (SG) and stratum spinosum of the epidermis under transmission electron microscopy (TEM). Previous studies of LGs have proposed several structural models. Freeze-fracture imaging studies have shown that LGs contain lamellar granule disks, and that these internal lipids are extruded into the extracellular space, where they give rise to lamellar sheets (Landmann, 1986Landmann L. Epidermal permeability barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freeze-fracture study.J Invest Dermatol. 1986; 87: 202-209Abstract Full Text PDF PubMed Scopus (168) Google Scholar). In Landmann’s model, a barrier exists between intracellular LGs and the extracellular space. However, in the “membrane-folding” model proposed by Norlen, the trans-Golgi network (TGN), LGs, and the intercellular space between the SG and stratum corneum are regarded as one continuous membrane structure, with no barrier existing between the cell exterior and this membrane network (Norlen, 2001Norlen L. Skin barrier formation: the membrane folding model.J Invest Dermatol. 2001; 117: 823-829Abstract Full Text Full Text PDF PubMed Google Scholar, Norlen et al., 2003Norlen L. Al-Amoudi A. Dubochet J. A cryotransmission electron microscopy study of skin barrier formation.J Invest Dermatol. 2003; 120: 555-560Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We have previously proposed a “membrane trafficking” model where the contents of LGs are transported from the TGN through LGs to the intercellular space, based upon studies using immuno-electron microscopy (Ishida-Yamamoto et al., 2005Ishida-Yamamoto A. Deraison C. Bonnart C. Bitoun E. Robinson R. O'Brien T.J. et al.LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.J Invest Dermatol. 2005; 124: 360-366Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In this model, LGs exist as branched tubular structures as well as oval structures. LG abnormalities have been reported in certain skin diseases, such as atopic dermatitis; Harlequin ichthyosis; cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma syndrome; arthrogryposis-renal dysfunction-cholestasis syndrome; and autosomal recessive keratoderma-ichthyosis-deafness syndrome (Akiyama et al., 2005Akiyama M. Sugiyama-Nakagiri Y. Sakai K. McMillan J.R. Goto M. Arita K. et al.Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J Clin Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (295) Google Scholar, Gruber et al., 2017Gruber R. Rogerson C. Windpassinger C. Banushi B. Straatman-Iwanowska A. Hanley J. et al.Autosomal recessive keratoderma-ichthyosis-deafness (ARKID) syndrome is caused by VPS33B mutations affecting Rab protein interaction and collagen modification.J Invest Dermatol. 2017; 137: 845-854Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, Hershkovitz et al., 2008Hershkovitz D. Mandel H. Ishida-Yamamoto A. Chefetz I. Hino B. Luder A. et al.Defective lamellar granule secretion in arthrogryposis, renal dysfunction, and cholestasis syndrome caused by a mutation in VPS33B.Arch Dermatol. 2008; 144: 334-340Crossref PubMed Scopus (39) Google Scholar, Igawa et al., 2017Igawa S. Kishibe M. Minami-Hori M. Honma M. Tsujimura H. Ishikawa J. et al.Incomplete KLK7 secretion and upregulated LEKTI expression underlie hyperkeratotic stratum corneum in atopic dermatitis.J Invest Dermatol. 2017; 137: 449-456Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, Sprecher et al., 2005Sprecher E. Ishida-Yamamoto A. Mizrahi-Koren M. Rapaport D. Goldsher D. Indelman M. et al.A mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma.Am J Hum Genet. 2005; 77: 242-251Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, Yamanaka et al., 2007Yamanaka Y. Akiyama M. Sugiyama-Nakagiri Y. Sakai K. Goto M. McMillan J.R. et al.Expression of the keratinocyte lipid transporter ABCA12 in developing and reconstituted human epidermis.Am J Pathol. 2007; 171: 43-52Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To elucidate the pathological significance of LG abnormalities in these skin diseases, it is necessary to have a detailed understanding of the overall structure of LGs in normal skin. Techniques for the 3-dimensional (3D) reconstruction of ultrastructural images have advanced significantly in recent years. For example, a 3D imaging technique has been developed that uses a scanning electron microscope equipped with an ultramicrotome within the imaging chamber (Denk and Horstmann, 2004Denk W. Horstmann H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.PLoS Biol. 2004; 2: e329Crossref PubMed Scopus (1130) Google Scholar). This technique enables the acquisition of image stacks by the sequential slicing and imaging of the specimen’s surface, and also improves on image resolution by using lower accelerating voltages. Later, an alternative sectioning approach that uses a focused ion beam was conducted with yeast (Heymann et al., 2006Heymann J.A.W. Hayles M. Gestmann I. Giannuzzi L. Lich B. Subramaniam S. Sitespecific 3D imaging of cells and tissues with a dual beam microscope.J Struct Biol. 2006; 155: 63-73Crossref PubMed Scopus (269) Google Scholar) and mice samples (Knott et al., 2008Knott G. Marchman H. Wall D. Lich B. Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling.J Neurosci. 2008; 28: 2959-2964Crossref PubMed Scopus (495) Google Scholar). Focused ion beam scanning electron microscopy (FIB-SEM), which employs a focused gallium ion beam for sample surface milling and a back scattered electron detector to image the milled surface, enables a large series of images to be acquired. Serial stacked images enable the reconstruction of the 3D ultrastructure of biological tissues. The FIB-SEM technique has been rapidly adapted for use in the morphological study of various tissues and cells (Ichimura et al., 2015Ichimura K. Miyazaki N. Sadayama S. Murata K. Koike M. Nakamura K. et al.Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.Sci Rep. 2015; 5: 8993Crossref PubMed Scopus (59) Google Scholar, Xu et al., 2017Xu C.S. Hayworth K.J. Lu Z. Grob P. Hassan A.M. Garcia-Cerdan J.G. et al.Enhanced FIB-SEM systems for large-volume 3D imaging.Elife. 2017; 6Crossref Scopus (157) Google Scholar). SEM analysis of these serial sectioned block faces generates high resolution and back scattered images that are comparable with TEM micrographs. In a recent study employing FIB-SEM, den Hollander et al., 2016den Hollander L. Han H. de Winter M. Svensson L. Masich S. Daneholt B. et al.Skin lamellar bodies are not discrete vesicles but part of a tubuloreticular network.Acta Derm Venereol. 2016; 96: 303-308Crossref PubMed Scopus (16) Google Scholar proposed that LGs are not discrete vesicles, but are instead part of a tubulo-reticular network. Because their findings represent a significant departure from the classical view of epidermal structure, we conducted the present study using FIB-SEM to elucidate in high detail the 3D structure of LGs and the TGN. In particular, we aimed to reveal that LGs are discrete vesicles and fused to cellular membrane at a certain level of cell layers. We first tested various fixation methods to determine optimal conditions for FIB-SEM imaging of human skin and found that ultrastructure was better preserved by adding a freeze–thaw cycle between the two fixation steps (Supplementary Figures S1, S2 online). Epidermal ultrastructure was very similar when observed by TEM and FIB-SEM, including the characteristic images of LGs fusing with the apical cell membrane within the superficial SG. The signal-to-noise ratio was slightly higher in TEM micrographs than in FIB-SEM images, but LGs, a multivesicular bodies–like structure, keratohyalin granules, keratin filaments, desmosomes, hemi-desmosomes, the cell membrane, and other major cell organelles were all easily identifiable by FIB-SEM (Supplementary Figure S1b, S3 online). 3D reconstructions of FIB-SEM image stacks revealed that overall LG structure changed from vesicular to vesiculo-reticular as granular cell differentiation progressed. Figure 1 is a representative 3D view of a stack of serial sections taken through the three granular layers (SG1, SG2, and SG3 from the most superficial layer). The 3D reconstruction, which was cropped to 4.8 × 5.5 × 4.5 μm3, contains five keratinocytes; two cells within the SG1 layer, two within the SG2 layer, and one within the SG3 layer (Figure 1a, Supplementary Movie S1 online). To compare the 3D structures of the LGs among the cells of the different granular layers, LGs from each given cell were assigned a specific color (LGs in SG1, yellow and gray; SG2, blue and green; SG3, deep pink, and at the cell border, deep blue [SG1] and red [SG2] in Figure 1b, 1c). Within the SG1 layer, most of the LGs were fused with the cell membrane and had an overall reticular structure (shown in yellow and gray in Figure 1d, 1g). Only a few cytoplasmic LGs demonstrated a vesicular structure in this layer (arrowheads in Figure 1d). Within the SG2 layer, a subpopulation of LGs were observed in the cytoplasm as vesicles (arrowhead in Figure 1e), while other LGs exhibited a reticular structure and had fused with the cell membrane at the apical surface (arrow in Figure 1e, 1h). Within the SG3 layer, LGs resided in the cytoplasm as vesicles (Figure 1f). In the projection view, taken from the top of the image stack, it can be seen that the reticular LG network in the SG1 layer was fragmented and that spaces were present between LGs and corneodesmosomes (shown in purple and pale blue in Figure 1g). In contrast, within the SG2 layer, the reticular network of LGs at the apical surface of cells was not fragmented and instead tightly surrounded desmosomes (shown in brown and orange in Figure 1h). We next investigated the relationship between overall LG structure and cell position within the granular layer. Figure 2 is a representative TEM image showing that both intracellular (black arrowheads) and extracellular (black arrows) LGs were associated with an SG2 cell. In this SG2 cell, the extracellular LGs were found beneath an SG1 cell (SG1b), which otherwise had no intracellular LGs. Secreted LGs were not found beneath the second SG1 cell (SG1a), which had both intracellular (white arrowheads) and apically secreted LGs (white arrows). FIB-SEM–based image reconstruction provided us with 3D information regarding LG structure relative to organelle position within the granular cells. In the image plane shown in Figure 3a, the cell indicated with an arrow is located within the SG3 layer. However, the 3D reconstruction demonstrates that the same cell also occupies the SG2 layer (an arrow in Figure 3b, 3c). Interestingly, LGs were not found within the SG3 zone occupied by this cell, while LGs fused to the apical membrane of the cell were found directly beneath an overlying SG1 cell (asterisk in Figure 3c, see also Supplementary Movie S2 online).Figure 3Overall 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To clarify the relationship between LGs and the TGN, we next investigated the overall structure of the TGN within granular cells. To identify the TGN, the Golgi apparatus was used as a landmark, and empty tubulo-vesicular structures extending from the trans side of the Golgi apparatus were regarded as TGN. FIB-SEM images showed that the Golgi apparatus, with typical cisternae, was located adjacent to the nucleus (Figure 4a, 4b ). TGN and endoplasmic reticulum (ER) were morphologically differentiated as follows. The TGN appeared as vesicular structures with electron-lucent content (arrowheads in Figure 4b), whereas the ER showed membranous or thin tubular structures continuous with the nuclear membrane (representative pictures of TGN and ER in SG and stratum basale are shown in Supplementary Figure S3). Distinction between rough ER and smooth ER was not possible using this technique. A representative 3D reconstruction of serial sectioned images shown in Figure 4 reveals five Golgi stacks at the basolateral side of an SG2 layer cell within a space of 4.2 × 4.8 × 2.1 μm3 (Figure 4c, 4f, Supplementary Movie S3 online). The TGN spreads extensively from the trans side of the Golgi apparatus out toward the cell periphery. Next, we investigated the relationship between LGs and the TGN by FIB-SEM (Figure 5a , 5b and Supplementary Movie S4 online show 3D). To compare the 3D structures of LGs and the TGN among the cells of the different granular layers, LGs from each given cell were assigned a specific color (LGs in SG1, pale yellow; SG2, blue; SG3, deep pink; stratum spinosum, brown and gray; TGN in SG2, yellow; SG2, green; stratum spinosum, light brown and beige in Figure 5). The TGN spread throughout the cytoplasm of cells in the SG2 and SG3 layers within a space of 5.4 × 13.0 × 3.0 μm3 (Figure 5c), and extended underneath the reticular LGs, which are shown in blue, within cells of the SG2 layer (shown in yellow in Figure 5d). Serial section images showed multiple connection points between the TGN and LGs (indicated by arrows with four colors in Figure 5e–5i). In the magnified 3D images shown in Figure 5f–5i and in Supplementary Movie S5 (online), it can be seen that the TGN branches out and connects to four LGs. We have succeeded in obtaining highly detailed 3D images of LG and TGN ultrastructure within cells of the human epidermis using FIB-SEM. FIB-SEM is a recently developed technology and its application in skin biology is limited (den Hollander et al., 2016den Hollander L. Han H. de Winter M. Svensson L. Masich S. Daneholt B. et al.Skin lamellar bodies are not discrete vesicles but part of a tubuloreticular network.Acta Derm Venereol. 2016; 96: 303-308Crossref PubMed Scopus (16) Google Scholar). The 3D ultrastructure of telocytes, a distinctive stromal cell of the dermis, was demonstrated by FIB-SEM (Cretoiu et al., 2015Cretoiu D. Gherghiceanu M. Hummel E. Zimmermann H. Simionescu O. Popescu L.M. FIB-SEM tomography of human skin telocytes and their extracellular vesicles.J Cell Mol Med. 2015; 19: 714-722Crossref PubMed Scopus (58) Google Scholar). Telocytes have an elongated or triangular cell body and thin, moniliform, bipolar, or multipolar cytoplasmic processes. These FIB-SEM images demonstrated the characteristics of telocytes in terms of their overall cellular structure. When first analyzing samples in this study, we applied a fixation method that has previously been used for other tissues, but it did not give us satisfactory ultrastructure, possibly because of the tightly packed nature of epidermal tissue. We then found that a freeze–thaw step before the second fixation could improve results, possibly by increasing heavy metal penetration. The beneficial effects of a freeze–thaw cycle in terms of enhanced image contrast has also been reported for pre-embedding immuno-electron microscopy (Nitsch and Klauer, 1989Nitsch R. Klauer G. Cryostat sections for coexistence studies and preembedding electron microscopic immunocytochemistry of central and peripheral nervous system tissue.Histochemistry. 1989; 92: 459-465Crossref PubMed Scopus (13) Google Scholar). We found that the overall structure of LGs changed substantially as cells transit the layers of the SG, demonstrating a vesicular-to-reticular morphological change as cells progress toward the skin’s surface. LGs of the SG3 layer had a dispersed vesicular morphology, while those of the SG2 layer had a more vesiculo-reticular morphology, and those of the SG1 layer a more reticular morphology (Figure 1 and Supplementary Movie S1). This suggests that LGs mature along with keratinocyte differentiation, and that the changes in LG morphology associated with the SG2 layer represent an important step preceding LG fusion with the cell membrane. It is possible that granular cells regulate LG maturation, maintaining pools of both vesicular and reticular granules to control their step-by-step secretion into the intercellular space. Our observation that SG2 cells retain LGs that can also be observed within the extracellular space is consistent with Landmann’s model, but not with that proposed by den Hollander et al., 2016den Hollander L. Han H. de Winter M. Svensson L. Masich S. Daneholt B. et al.Skin lamellar bodies are not discrete vesicles but part of a tubuloreticular network.Acta Derm Venereol. 2016; 96: 303-308Crossref PubMed Scopus (16) Google Scholar, where physical continuity between LGs and the outside space is suggested. This difference may be because of the different methods used to assign LGs during image data analysis and observed region. We assigned LGs based on electron density and structure semi-automatically, while den Hollander et al., 2016den Hollander L. Han H. de Winter M. Svensson L. Masich S. Daneholt B. et al.Skin lamellar bodies are not discrete vesicles but part of a tubuloreticular network.Acta Derm Venereol. 2016; 96: 303-308Crossref PubMed Scopus (16) Google Scholar identified LGs automatically using a watershed algorithm. And LGs were discriminated from a multivesicular bodies–like structure, which was located at basolateral side of SG1 layer, by their structures in our FIB-SEM images (Supplementary Figure S3). Furthermore, their observation by cryo-electron microscopy of vitreous sections was limited to a relatively narrow range (approximately 4 μm2) at the interface between SG1 and stratum corneum. Our results also suggest that granular cells coordinate secretion of the contents of their LGs with their relative position within the granular layer, because LG fusion with the plasma membrane was observed at the SG1–SG2 cell interface, but not at the SG2–SG3 cell interface (Figures 2 and 3c). We also revealed the overall structure of LGs within the intercellular space (Figure 1g, 1h). LGs occupied the extracellular space around desmosomes and corneodesmosomes. The morphology of desmosomes and lipid lamellar sheets within the intercellular spaces between the SG and corneocytes have been characterized in detail by Fartasch et al., 1993Fartasch M. Bassukas I.D. Diepgen T.L. Structural relationshi between epidermal lipid lamellae, lamellar bodies and desmosomes in human epidermis: an ultrastructural study.Br J Dermatol. 1993; 128: 1-9Crossref PubMed Scopus (113) Google Scholar, and our images are consistent with theirs. These reconstructed images support the “bricks and mortar” model of the stratum corneum (Elias, 1983Elias P.M. Epidermal lipids, barrier function, and desquamation.J Invest Dermatol. 1983; 80 (44s–9s)Google Scholar). The reticular network–like structure of LGs at the interface between the SG1 and stratum corneum layers enables the efficient, targeted delivery of cargo within this extracellular space. For example, corneodesmosin and lympho-epithelial Kazal-type–related inhibitor are both released from LGs and target desmosomes and corneodesmosomal proteases, respectively (Ishida-Yamamoto et al., 2004Ishida-Yamamoto A. Simon M. Kishibe M. Miyauchi Y. Takahashi H. Yoshida S. et al.Epidermal lamellar granules transport different cargoes as distinct aggregates.J Invest Dermatol. 2004; 122: 1137-1144Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, Ishida-Yamamoto et al., 2005Ishida-Yamamoto A. Deraison C. Bonnart C. Bitoun E. Robinson R. O'Brien T.J. et al.LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.J Invest Dermatol. 2005; 124: 360-366Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Our FIB-SEM tomography 3D imaging has enabled the overall morphological structure of the TGN within cells of the epidermis to be clearly observed. We were able to distinguish the TGN from LGs by heavy metal staining. The TGN spread extensively into the cytoplasm with branched and tubular structures emanating from the Golgi apparatus located close to the nucleus. The TGN was connected to vesicular LGs close to the apical surface of cells of the SG2 layer. Previous studies have shown that the TGN is derived from the membrane of the trans cisternae of the Golgi stack and contributes to epithelial cell polarity (Fiedler et al., 1997Fiedler K. Kellner R. Simons K. Mapping the protein composition of trans-Golgi network (TGN)-derived carrier vesicles from polarized MDCK cells.Electrophoresis. 1997; 14: 2613-2619Crossref Scopus (18) Google Scholar). Rab11, SNAP29, ABCA12, VIPAR, VPS33B, Tmem79, and caveolin have been identified as molecules associated with membrane trafficking to LGs (Fuchs-Telem et al., 2011Fuchs-Telem D. Stewart H. Rapaport D. Nousbeck J. Gat A. Gini M. et al.CEDNIK syndrome results from loss-of-function mutations in SNAP29.Br J Dermatol. 2011; 164: 610-616PubMed Google Scholar, Ishida-Yamamoto et al., 2007Ishida-Yamamoto A. Kishibe M. Takahashi H. Iizuka H. Rab11 is associated with epidermal lamellar granules.J Invest Dermatol. 2007; 127: 2166-2170Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Li et al., 2011Li Q. Frank M. Akiyama M. Shimizu H. Ho S.Y. Thisse C. et al.Abca12-mediated lipid transport and Snap29-dependent trafficking of lamellar granules are crucial for epidermal morphogenesis in a zebrafish model of ichthyosis.Dis Model Mech. 2011; 4: 777-785Crossref PubMed Scopus (23) Google Scholar, Reynier et al., 2016Reynier M. Allart S. Gaspard E. Moga A. Goudouneche D. Serre G. et al.Rab11a is essential for lamellar body biogenesis in the human epidermis.J Invest Dermatol. 2016; 136: 1199-1209Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, Rogerson and Gissen, 2018Rogerson C. Gissen P. VPS33B and VIPAR are essential for epidermal lamellar body biogenesis and function.Biochim Biophys Acta. 2018; 1864: 1609-1621Crossref Scopus (23) Google Scholar, Sando et al., 2003Sando G.N. Zhu H. Weis J.M. Richman J.T. Wertz P.W. Madison K.C. Caveolin expression and localization in human keratinocytes suggest a role in lamellar granule biogenesis.J Invest Dermatol. 2003; 120: 531-541Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Sasaki et al., 2013Sasaki T. Shiohama A. Kubo A. Kawasaki H. Ishida-Yamamoto A. Yamada T. et al.A homozygous nonsense mutation in the gene for Tmem79, a component for the lamellar granule secretory system, produces spontaneous eczema in an experimental model of atopic dermatitis.J Allergy Clin Immunol. 2013; 132 (1111–20.e4)Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Smith et al., 2012Smith H. Galmes R. Gogolina E. Straatman-Iwanowska A. Reay K. Banushi B. et al.Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome.Hum Mutat. 2012; 33: 1656-1664Crossref PubMed Scopus (61) Google Scholar, Sprecher et al., 2005Sprecher E. Ishida-Yamamoto A. Mizrahi-Koren M. Rapaport D. Goldsher D. Indelman M. et al.A mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma.Am J Hum Genet. 2005; 77: 242-251Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). However, the overall structure of the TGN remains poorly understood. Morphological observations using labeled lipase cytochemistry have demonstrated that nascent LGs are generated from the TGN (Elias et al., 1998Elias P.M. Cullander C. Maruo T. Rassner U. Komuves L. Brown B.E. et al.The secretory granular cell: the outermost granular cell as a specialized secretory cell.J Investig Dermatol Symp Proc. 1998; 3: 87-100Abstract Full Text PDF PubMed Scopus (122) Google Scholar). LGs have also been shown to be generated from the TGN in response to extracellular stimuli (Rassner et al., 1999Rassner U. Feingold K.R. Crumrine D.A. Elias P.M. Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies.Tissue Cell. 1999; 31: 489-498Crossref PubMed Scopus (61) Google Scholar). We have previously observed TGN substructures (suck-like protrusions from the Golgi apparatus) by cryo-immuno–TEM and proposed the LG-trafficking model, in which various LG cargos are transported independently through the TGN and LGs (Ishida-Yamamoto et al., 2004Ishida-Yamamoto A. Simon M. Kishibe M. Miyauchi Y. Takahashi H. Yoshida S. et al.Epidermal lamellar granules transport different cargoes as distinct aggregates.J Invest Dermatol. 2004; 122: 1137-1144Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The TGN has been shown to exhibit diverse 3D structural characteristics, depending on cell type, in studies using thick sections with a high-voltage electron microscope (Clemont et al., 1995Clemont Y. Rambourg A. Hermo L. Trans-Golgi Network (TGN) of different cell types: three- dimensional structural characteristics and variabiiity.Anat Rec. 1995; 242: 289-301Crossref PubMed Scopus (81) Google Scholar). The branched, tubular structures of the TGN observed in our present study would be well-suited for the stepwise selection and transportation of LG cargo that must change along with cell differentiation. It seems that the development of the TGN and ER are closely associated with LG production, because the TGN is well developed and exhibits complicated branched tubular structures in the granular and upper spinous layers, but is poorly developed in the basal layer. In contrast, the ER was well development in the basal layer, while it was poorly developed in the granular layer (Supplementary Figure S4 online). The findings presented in this study should contribute to a better understanding of the membrane trafficking mechanisms of epidermal cells, and also the pathological mechanisms underlying skin conditions associated with defective LGs. A challenge for the future will be to investigate the trafficking mechanisms associated with discrete LG cargos. Further studies that examine how various cargos are selected and sorted within the TGN, how their subsequent trafficking route to LGs is determined, and how the LGs themselves are generated, should be conducted using a combination of both molecular and morphological techniques. In the future, cryo–FIB-SEM using high-pressure freezing and freeze substitution of the heavy metal staining process, which preserve membrane structure, may provide more accurate images of TGN. Also, the alternation of LGs will be analyzed in skin diseases that may alter lipid trafficking. The future development of structural and functional studies that employ 3D skin models in which LG transportation can be inhibited would greatly improve our understanding of the relevance of LG trafficking in skin disease pathologies. This study was approved by the Shiseido Committee on Human Ethics. Human skin tissues were obtained from 42-, 52-, 56-, and 68-year-old male scalp skin, and from 69-year-old female eyelid skin obtained following plastic surgery, in accordance with the Declaration of Helsinki Principles. Written informed consent was obtained from all participants. Samples were prepared according to a previously reported protocol (Holcomb et al., 2013Holcomb P.S. Hoffpauir B.K. Hoyson M.C. Jackson D.R. Deerinck T.J. Marrs G.S. et al.Synaptic inputs compete during rapid formation of the calyx of Held: a new model system for neural development.J Neurosci. 2013; 33: 12954-12969Crossref PubMed Scopus (86) Google Scholar). Briefly, specimens were cut into small blocks and immediately fixed in 4% paraformaldehyde, incubated overnight in 30% sucrose, and then embedded in OCT compound (Sakura Finetek, Tokyo, Japan). After cryosectioning using a MICROM HM550 cryo-microtome (Thermofisher Scientific, Waltham, MA), the specimens were rinsed in cacodylate buffer (0.1M, pH 7.4) and then fixed with 2.5% glutaraldehyde/2% paraformaldehyde in cacodylate buffer for 3 hours at 4°C. After rinsing in cacodylate buffer, specimens were fixed again with 1.5% potassium ferrocyanide/2% OsO4 in cacodylate buffer for 1 hour at 4°C. Specimens were then rinsed with distilled water, treated with 1% thiocarbohydrazide solution for 1 hour at room temperature, rinsed again, and fixed in 2% OsO4 solution for 1 hour at 4°C. After rinsing with distilled water, the specimens were treated with 1% uranyl acetate solution overnight at 4°C and then stained en bloc with Walton’s lead aspartate for 30 minutes at room temperature. After dehydration in an ethyl alcohol series, specimens were embedded in epoxy resin (Epon 812, TAAB Laboratories, Berks, UK), and polymerized. The resin block was trimmed using an EM UC7 ultramicrotome (Leica Microsystems, Vienna, Austria), and 80-nm ultrathin sections were then prepared for imaging using a Hitachi H-7100 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan). The resin block was mounted on a scanning electron microscope stub with glue, and coated with a layer of platinum/palladium using an MC1000 ion sputter coater (Hitachi High-Technologies). The resin block was placed into the Helios660 FIB-SEM system (FEI, Eindhoven, Netherlands), which was equipped with a scanning electron microscope and a focused beam of gallium ions. The field emission scanning electron beam was used to locate the site of the granular layer and a protective layer of platinum was deposited. The 30-kV gallium ion beam (FIB) was used to mill the block surface to expose the initial imaging face. A 30 nm-deep layer of the surface was then milled with the FIB. The SEM image was generated from backscattered electrons with the following condition: acceleration voltage; 2 kV, beam current; 0.2 nA, resolution; 3,072 × 2,048 pixels, dwell time; 6 microseconds. The contrast of the acquired images was inverted. Serial images were acquired by repeated cycles of milling and imaging using Auto Slice & View software (FEI). The resultant image stack was processed using Avizo ver. 9.2 software (FEI). Alignment of the image stack enabled the 3D reconstruction of LGs, TGN, Golgi apparatus, desmosomes, and corneodesmosomes, which were then semi-automatically segmented based on their morphological characteristics (Supplementary Figure S3). Multivesicular bodies were elucidated from LGs by their lower electron density and different internal structure (Supplementary Figure S3e, S3f). The authors state no conflict of interests. We thank Kazunori Toida (Department of Anatomy, Kawasaki Medical School, Japan), Tetsuji Hirao (Cosmetic Science Laboratory, Chiba Institute of Science, Japan), and Takuma Kanesaki (Thorlabs Japan Inc) for helpful discussions and advice. This work was supported by MEXT KAKENHI, grant number 17K10203 to AI-Y. 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• W2890850862 title "Marked Changes in Lamellar Granule and Trans-Golgi Network Structure Occur during Epidermal Keratinocyte Differentiation" @default.
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• W2890850862 doi "https://doi.org/10.1016/j.jid.2018.07.043" @default.
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