Rab11a Is Essential for Lamellar Body Biogenesis in the Human Epidermis  Marie Reynier, Sophie Allart, Elise Gaspard, Alain Moga, Dominique Goudounèche, Guy Serre, Michel Simon, Corinne Leprince  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1199-1209 (June 2016) DOI: 10.1016/j.jid.2016.02.001 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Rab11a is strongly expressed in granular keratinocytes and partially co-localized with lamellar body markers. (a) Keratinocyte subpopulations: basal keratinocytes, lower spinous keratinocytes, upper spinous keratinocytes, and granular keratinocytes were isolated by sequential trypsination of human epidermis. RAB11A and RAB11B gene expression was analyzed by quantitative real-time reveres transcriptase-PCR in each keratinocyte subpopulation. Gene expression was quantified as ΔCt corresponding to Ct(reference gene) - Ct(target gene). Data are reported as mean ± standard deviation, n ≥ 3. Unpaired Student t test, *P < 0.05, nsP > 0.05. (b) Rab11a and Rab11b expression were analyzed by Western blot test in protein extracts of the different keratinocyte subpopulations. Cell separation was validated with the basal keratinocyte marker keratin-14 and the granular keratinocyte marker corneodesmosin. Protein extract loading was verified with anti-actin antibody. n ≥ 3. (c) Normal human skin sections were analyzed by confocal microscopy using anti-Rab11a antibody and either anti-kallikrein-6 (left panel) or anti-CDSN antibodies (right panel), considering both as lamellar body markers, with 4′,6-diamidino-2-phenylindole (DAPI) as nuclear counterstaining (blue). Image superpositions of Rab11a/KLK6 and Rab11a/CDSN are shown (merged). Scale bars = 10 μm. n ≥ 3. BK, basal keratinocytes; CDSN, corneodesmosin; Ct, cycle threshold; GK, granular keratinocytes; K-14, keratin-14; KLK6, kallikrein-6; lSK, lower spinous keratinocytes; uSK, upper spinous keratinocytes. Journal of Investigative Dermatology 2016 136, 1199-1209DOI: (10.1016/j.jid.2016.02.001) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Rab11a is associated with epidermal lamellar bodies. (a) Stimulated emission depletion microscopy analysis of a human skin section immunostained for Rab11a (red) and corneodesmosin (green) (images linearly deconvolved). Scale bar = 1 μm. n ≥ 3. (b) Graphs show the signal intensities acquired from stimulated emission depletion microscopy images along the line drawn in the high magnification box. Scale bar = 0.5 μm. Journal of Investigative Dermatology 2016 136, 1199-1209DOI: (10.1016/j.jid.2016.02.001) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Rab11a silencing leads to an alteration of stratum corneum structure and function. (a) Sections of shCtrl-RHE and shRab11a-RHE were stained with hematoxylin and eosin. One representative experiment out of three is shown. n ≥ 3. (b) ShCtrl-RHE and shRab11a-RHE were analyzed by Western blot test using antibodies directed against different epidermal proteins: loricrin, involucrin, and desmoglein-1 and -2. Protein extract loading was verified with anti-actin antibody. Signal intensity was quantified using the Gene Tools software version 4.03 (Syngene, Cambridge, UK). Quantification values are given below the Western blot. n ≥ 3. (c) ShCtrl-RHE and shRab11a-RHE were analyzed by immunofluorescence staining using antibodies directed against loricrin, involucrin, desmoglein-1 and -2, and keratin-10. Scale bars = 10 μm. n ≥ 3. (d) The outside-in epidermal barrier was assessed using a Lucifer yellow penetration assay. A Lucifer yellow solution was applied onto the stratum corneum of the RHE. Penetration of the dye was investigated by measuring its concentration in the culture medium after 0–6 hours of incubation at 37°C. Data are mean ± standard deviation, n = 2. (e) The inside-out barrier was assessed by measuring transepidermal water loss. Data are mean ± standard deviation, n ≥ 3. Unpaired Student t test, *P < 0.05. DSG1/2, desmoglien-1 and -2; IVL, involucrin; K10, keratin-10; LOR, loricrin; LY, Lucifer yellow; RHE, reconstructed human epidermis; shCtrl, control short hairpin RNA; shRab11a, Rab11a short hairpin RNA; TEWL, transepidermal water loss. Journal of Investigative Dermatology 2016 136, 1199-1209DOI: (10.1016/j.jid.2016.02.001) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Rab11a silencing induces a decrease in stratum corneum lipid content. (a) The stratum corneum of shCtrl-RHE and shRab11a-RHE were analyzed by transmission electron microscopy with focuses on the intercorneocyte spaces and lipid lamellae. Scale bars = 1 μm for upper panels and 200 nm for lower panels. n ≥ 3. (b) Cryosections of shCtrl-RHE (left panel) and shRab11a-RHE (right panel) were stained with Oil Red O to detect neutral lipids in the stratum corneum. Scale bars = 10 μm. n ≥ 3. (c) The levels of different epidermal lipids were analyzed in the stratum corneum of shCtrl-RHE and shRab11a-RHE: cholesterol (left histogram); short ceramides Cer-NS, Cer-NP, Cer-AS, Cer-NDS, and Cer-ADS (middle histogram); and acyl ceramides Cer-EOS and Cer-EOP (right histogram). Data are mean ± standard deviation, n ≥ 3. Unpaired Student t test, *P < 0.05; **P < 0.01, ***P < 0.001. Cer, ceramide; Chol, cholesterol; RHE, reconstructed human epidermis; SC, stratum corneum; shCtrl, control short hairpin RNA; shRab11a, Rab11a short hairpin RNA. Journal of Investigative Dermatology 2016 136, 1199-1209DOI: (10.1016/j.jid.2016.02.001) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Rab11a silencing induces a decrease in LB density and secretion areas. (a) LB density (white arrows) in shCtrl-RHE and shRab11a-RHE was analyzed by transmission electron microscopy. Note the high density of LBs in the cytosol of the granular keratinocytes in shCtrl-RHE and the reduced number of LBs in shRab11a-RHE. Scale bars = 100 nm. n ≥ 3. (b) Histograms show quantitative analyses of LB density in the cytoplasm of the two outermost granular keratinocytes, performed on 10 random electron micrographs. LB density was defined as the ratio of the number of LBs to the cytoplasm area (in μm2). Data are mean ± standard deviation, n ≥ 3. Unpaired Student t test, **P < 0.01. (c) LB secretion in shCtrl-RHE and shRab11a-RHE was analyzed by transmission electron microscopy. Secretion areas, filled with the secreted LB content, at the interface between granular keratinocytes and stratum corneum, were delimited with dotted lines. Scale bars = 500 nm. n ≥ 3. (d) Histograms show quantitative analyses of LB secretion areas, performed on 10 random electron micrographs. The secretion area was calculated as the ratio of the sum of the measured secretion areas (in nm2) to the length (in μm) of the granular keratinocyte-stratum corneum interface. Data are reported as mean ± standard deviation, n ≥ 3. Unpaired Student t test, *P < 0.05. LB, lamellar body; RHE, reconstructed human epidermis; shCtrl, control short hairpin RNA; shRab11a, Rab11a short hairpin RNA. Journal of Investigative Dermatology 2016 136, 1199-1209DOI: (10.1016/j.jid.2016.02.001) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Rab11a silencing leads to the mis-sorting of lamellar body components and lysosomal-dependent degradation. (a) shCtrl-RHE and shRab11a-RHE (shRab11a#1 and shRab11a#2) were analyzed by qRT-PCR for lamellar body marker gene expression: ABCA12, corneodesmosin (CDSN), kallikrein-5 (KLK5) and kallikrein-7 (KLK7). Results are shown as percentage shCtrl-RHE for the three species of mRNA. Data are mean ± standard deviation, n ≥ 3. (b) ShCtrl-RHE and shRab11a-RHE (shRab11a#1 and shRab11a#2) were analyzed by Western blot test using antibodies directed against the same lamellar body markers. Protein extract loading was verified with anti-actin antibody. n ≥ 3. (c) ShRab11a#1-RHE untreated (0) or treated with chloroquine (100 μmol/L or 200 μmol/L) were used for Western blot analysis of the same lamellar body markers. n ≥ 3. qRT-PCR, quantitative real-time reverse transcriptase-PCR; shCtrl, control short hairpin RNA; shRab11a, Rab11a short hairpin RNA. Journal of Investigative Dermatology 2016 136, 1199-1209DOI: (10.1016/j.jid.2016.02.001) Copyright © 2016 The Authors Terms and Conditions