A Role for DLK in Microtubule Reorganization to the Cell Periphery and in the Maintenance of Desmosomal and Tight Junction Integrity  Carolyne Simard-Bisson, Julie Bidoggia, Danielle Larouche, Sylvain L. Guérin, Richard Blouin, Syu-Ichi Hirai, Lucie Germain  Journal of Investigative Dermatology  Volume 137, Issue 1, Pages 132-141 (January 2017) DOI: 10.1016/j.jid.2016.07.035 Copyright © 2016 The Authors Terms and Conditions

Figure 1 DLK overexpression in NHKs enhances MT cortical distribution and stabilization. NHKs cultured on plastic substrates transduced or not (control, Ctl) with adenoviral vectors containing GFP (AdGFP) or DLK and GFP (AdDLK). (a) α-Tubulin (α-tub) immunolabeling (red; top), GFP (green; middle), and merged signals (bottom). Arrows indicate cortical MTs in an AdDLK-transduced NHK differentiated into a corneocyte. (b) DLK, α-tub and acetylated α-tub (Actub) in total protein and cytoskeletal insoluble fraction (CIF). Numbers indicate molecular weight (kDa). Loading controls: actin and Ponceau’s red staining. (c) Actub immunolabeling (red; top), GFP (green, middle), and merged signals (bottom). Arrows indicate cortical Actub. (d) α-Tub staining (red) after nocodazole treatment (10 μM, 4 hours). Magnification (×9) of framed zones is in superior corners. Nuclei are in blue (Hoechst). N = 3. Bars: 25 μm. DLK, dual leucine zipper-bearing kinase; GFP, green fluorescent protein; MT, microtubules; NHK, normal human keratinocyte. Journal of Investigative Dermatology 2017 137, 132-141DOI: (10.1016/j.jid.2016.07.035) Copyright © 2016 The Authors Terms and Conditions

Figure 2 MT cortical distribution is altered in DLK-depleted TES. Analyses of normal human skin (normal skin), TES not exposed to a lentiviral vector (TES without LV), and TES exposed to a LV containing a scramble (shCtl TES) or a DLK shRNA sequence (shDLK TES). (a) DLK immunoperoxydase staining (brown) and Harris’ hematoxylin counterstaining. Arrows indicate DLK staining at the granular layer of normal skin, TES without LV and shCtl TES. (b) Confocal microscopy pictures of F-actin (red) and α-tubulin (α-tub, green). F-actin staining was used to delineate the cell periphery. Higher magnification of α-tubulin staining (c) and merged signals (d) of framed areas in (b). Pictures are representative of four independent experiments. Bars: (a, b) 50 μm and (d) 20 μm. Ctl, control; DLK, dual leucine zipper-bearing kinase; MT, microtubule; shRNA, short hairpin RNA; TES, tissue-engineered skin. Journal of Investigative Dermatology 2017 137, 132-141DOI: (10.1016/j.jid.2016.07.035) Copyright © 2016 The Authors Terms and Conditions

Figure 3 DLK regulates the cortical distribution of proteins involved in MT organization. (a) LIS1 staining (green) in TESs not exposed to lentiviral vector (TES without LV) or exposed to LV containing a scramble (shCtl TES) or DLK shRNA sequence (shDLK TES). Arrows indicate cortical LIS1 in TES without LV and shCtl TES. (b) Western blot analyses of DLK and LIS1 in total protein extracts and in cytoskeleton-enriched insoluble fractions (CIFs) of nontransduced (ctl), AdGFP- or AdDLK-transduced NHK. Numbers indicate molecular weight (kDa). Actin and Ponceau’s red stain (Ponceau) correspond to loading controls. (c) HSP27 staining (green) in TES without LV, and shCtl and shDLK TES. (d) Differential interference contrast (DIC) pictures of fields shown in (c) illustrate the cell periphery. Nuclei are in blue (Hoechst). Bars: 50 μm. AdGFP, adenoviral vectors containing GFP; Ctl, control; DLK, dual leucine zipper-bearing kinase; GFP, green fluorescent protein; HSP27, heat shock protein 27; LIS1, lissencephaly-1 protein; MT, microtubules; NHK, normal human keratinocyte; shRNA, short hairpin RNA; TES, tissue-engineered skin. Journal of Investigative Dermatology 2017 137, 132-141DOI: (10.1016/j.jid.2016.07.035) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Desmosomes and tight junctions are altered in shDLK TESs. TESs unexposed to lentiviral vector (TES without LV) or exposed to LV containing a scramble (shCtl TES) or a DLK shRNA sequence (shDLK TES) analyzed for (a) adherens junctions (β-catenin; green), (b) tight junctions (claudin 4; red), and (c) desmosomes (desmoglein 1; green). Nuclei are in blue (Hoechst). (d) TEM pictures of TES without LV, shCtl and shDLK TES. (e) Higher magnification of boxes in (d). Arrows show desmosome-like junctions and asterisks intercellular spaces in shDLK TES. Mean and standard variation of the (f) length and (g) number of desmosome-like junctions in controls (combined TES without LV and shCtl TES (ctl)) and shDLK TES. * P ≤ 0.05 (Student’s t-test). Bars: (a–c) 50 μm, (d) 2 μm, and (e) 0.5 μm. DLK, dual leucine zipper-bearing kinase; DSG-1, desmoglein 1; TEM, transmission electron microscopy; shRNA, short hairpin RNA; TES, tissue-engineered skin. Journal of Investigative Dermatology 2017 137, 132-141DOI: (10.1016/j.jid.2016.07.035) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Nocodazole treatment of TESs impairs MT reorganization to the cell periphery. Analyses of untreated TES (left panel) and of TES treated with 1% DMSO (center panel) or 1 μM nocodazole (right panel) between days 12 and 14 of culture at the air-liquid interface. (a) F-actin (red) and α-tubulin (α-tub; green) immunolabeling. (b) Higher magnification of the framed areas from panel a showing α-tubulin (green) staining only. The arrow points out the cell periphery. (c) Higher magnification of the framed areas from panel a showing both the α-tub (green) and F-actin (red) staining. The arrow points out the cell periphery. Nuclei are in blue (Hoechst) in (a) and (c). Results are representative of three independent experiments. Bars: (a) 50 μm and (b, c) 20 μm. MT, microtubules; TES, tissue-engineered skin. Journal of Investigative Dermatology 2017 137, 132-141DOI: (10.1016/j.jid.2016.07.035) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Nocodazole treatment of TESs alters desmosomes and tight junctions. Untreated TES (left panels) and TES treated with 1% DMSO (center panels) or 1 μM nocodazole (right panels) during 2 days of culture at the air-liquid interface were analyzed for (a) adherens junctions (β-catenin; green), (b) tight junctions (claudin 4; red), and (c) desmosomes (desmoglein 1; green). Nuclei are stained with Hoechst (blue). (d) TEM pictures. Dashed lines indicate dermoepidermal junctions. (e) Higher magnification of boxes shown in (d). Arrowheads indicate desmosome-like junctions. (f) Higher magnification of boxes shown in (e). Results are representative of at least two independent experiments. Bars: (a–c) 50 μm, (d) 25 μm, (e) 2.5 μm, and (f) 500 nm. DSG-1, desmoglein 1; TEM, transmission electron microscopy; TES, tissue-engineered skin. Journal of Investigative Dermatology 2017 137, 132-141DOI: (10.1016/j.jid.2016.07.035) Copyright © 2016 The Authors Terms and Conditions