1. Keratins Regulate the Adhesive Properties of Desmosomal Cadherins through Signaling 
Franziska Vielmuth, Marie-Therès Wanuske, Mariya Y. Radeva, Matthias Hiermaier, Daniela Kugelmann, Elias Walter, Fanny Buechau, Thomas M. Magin, Jens Waschke, Volker Spindler 
Journal of Investigative Dermatology 
Volume 138, Issue 1, Pages (January 2018)
DOI: /j.jid
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2. Figure 1
Apical clusters of desmosomal molecules on the cell surface of murine keratinocytes. (a, b) KtyII-WT/-KO cells were transiently transfected with Dsg3-GFP, and immunostaining of keratin 14 and membrane proteins using biotin-streptavidin was conducted. In both cell lines, Dsg3-GFP showed a dotted appearance along the cell borders and on the cell surface co-localzing with the membrane staining (white arrows). In WT cells Dsg3-GFP co-localizes with keratin 14 at both locations. Right panels reflect height-encoded three-dimensional delineation of Dsg3-GFP molecules (full-colored) and the plasma membrane (half-transparent), which confirmed localization of Dsg3 molecules in the membrane at cell borders (blue-green colored areas) and on the cell surface (green-orange areas). The white dotted line represents section for the y-plane. Images are representatives of n >3. Scale bar = 10 μm. CK14, keratin 14; Dsg, desmoglein; KO, knockout; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
Dsg3 binding properties depend on keratins. (a) Areas along the cell borders chosen for Dsg3 adhesion force mapping, with each pixel representing one force-distance curve. Each blue pixel represents one Dsg3-binding event. Black arrows mark cell borders. Scale bar = 2.5 μm. (b) Analysis of adhesion maps shows higher binding frequency in KtyII-KO cells compared with WT. n = 6 independent cell borders with 1,200 force-distance curves each.∗P < (c) Western blot analysis and biotinylation assays showed higher Dsg3 levels in keratin-deficient keratinocytes. n > 3. (d) Analysis of UF distribution indicates higher forces of single-molecule interactions in KtyII-WT cells. Distribution showed a second peak solely in KtyII-KO cells (arrow). (e) First peak of UF distribution shows significantly lower forces of single molecule interaction in KtyII-KO keratinocytes. n = 6 independent cell borders, 1,200 force-distance curves/force map. (f) Peak fitting shows linear increase of unbinding forces with logarithm of pulling speed. n > 6, >300 binding events/pulling speed. (g, h) KtyI-WT and -KO cells show comparable results to KtyII cells in terms of Dsg3 binding properties. K14 reconstitution rescues an increased number of Dsg3 binding events and impaired binding strength. n > 6 independent cell borders, 1,200 force-distance curves/force map. ∗/#P < Dsg, desmoglein; IP, immunoprecipitation; K, keratin; KO, knockout; n. s., not significant; PG, plakoglobin; UF, unbinding force; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Homophilic Dsg3 binding represents the major interaction mode in KtyII. (a) Cell-free AFM measurements probing homophilic and heterophilic interaction pairs show higher binding frequency of homophilic compared with heterophilic interactions. Homophilic interactions were significantly blocked by respective antibodies applied to the mica sheet. Dsg2-Dsg3 binding frequency was slightly increased compared with the other heterophilic interactions tested and was blocked by the Dsg2 mAb. n > 10 independent tip/sample combinations from four independent coating procedures. ∗P < 0.05 versus respective homophilic control, #P < 0.05 versus respective heterophilic control. (b, c) Dsg3 adhesion measurements at areas along the cell borders on living keratinocytes using Dsg2 mAb and Dsg3 mAb for 1 hour. Quantification shows a drastic reduction in binding frequency upon Dsg3 mAb incubation in both cell lines, whereas reduction with the Dsg2 mAb was less and significant only in KtyII-KO cells. n > 6 independent cell borders, 1,200 force-distance curves/force map. ∗P < 0.05 versus respective control. Scale bar = 2 μm. AFM, atomic force microscopy; Dsc, desmocollin; Dsg, desmoglein; KO, knockout; n.s., not significant; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Keratins and actin modulate the mobility of Dsg3 molecules. (a) Dissociation assay of KtyII-WT and -KO cells under control conditions and with addition of the actin-disturbing agent latrunculin B (Latr B) for 1 hour show that actin disruption drastically impairs cell adhesion in KO cells. n > 5, ∗P < (b) AFM adhesion measurements showed unaltered Dsg3 binding frequency after 1 hour of latrunculin B treatment. (c) First peak of UF distribution shows no significant change in latrunculin B-treated cells compared with controls. (d) Color-encoded overlay of five consecutively acquired adhesion maps (1 μm2, 20 × 20 pixels). KtyII-WT show more stable events than KtyII-KO. Latrunculin B increased mobility in KtyII-WT but not further in KtyII-KO cells. Scale bar = 200 nm. (e, f) Quantification of d by determination of a stability coefficient (see Supplementary Materials and Methods). n = 6, ∗P < (g) FRAP experiments using Dsg3-GFP. (g) Representative kymographs visualizing recovery period up to 130 seconds. Scale bar = 1 μm. (h) Quantification of the immobile fraction of FRAP experiments confirmed higher mobility in KtyII-KO compared with WT cells. Latrunculin B increased mobility in both cell lines. n > 6, ∗P > AFM, atomic force microscopy; Dsg, desmoglein; FRAP, fluorescence recovery after photobleaching; KO, knockout; Latr, latrunculin; n.s., not significant; UF, unbinding force; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
PKC inhibition increases the binding probability and membrane stability of Dsg3-meditated binding events in KtyII-KO. (a, b) Dsg3 adhesion measurements at cell border areas showed an increased Dsg3 binding frequency in keratin-deficient keratinocytes after 1 hour of treatment with Gö6976 and safingol, whereas the distribution of the binding events was not changed. n > 6 independent cell borders, 1,200 force-distance curves/force map. ∗P < Scale bar = 2 μm. (c, d) AFM mobility measurements showed increased membrane stability of Dsg3 molecules after Gö6976 treatment in KtyII-KO cells. n = 6 independent cell borders, ∗P < Scale bar = 200 nm. (e) Biotinylation assays shows that 1 hour of Gö7976 incubation did not significantly increase Dsg3 membrane levels in KtyII cells. n = 5, ∗P < AFM, atomic force microscopy; Dsg, desmoglein; IP, immunoprecipitation; KO, knockout; n.s., not significant; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
Keratin-dependent p38 MAPK signaling controls Dsg3 binding strength. (a) Keratin-deficient keratinocytes show a robust activation of p38 MAPK under basal conditions in Western blot analysis of whole lysate and in both fractions of Triton extraction. n > 4. GAPDH or desmoplakin were used as markers for the respective compartments. (b) Quantification of Western blot analysis using total p38 MAPK as reference. ∗P < (d) Dissociation assays using p38 MAPK inhibitors SB and SB for 1 hour increased intercellular adhesion in both cell lines. n > 4. (e) Quantification of UF distribution of Dsg3 adhesion measurements shows no change in UF distribution in KtyII-WT cells, whereas unbinding forces increased in KtyII-KO cells after inhibition of p38 MAPK by SB and SB n > 6 independent cell borders, 1,200 force-distance curves/force map. (f) Possible model for how keratins and actin affect Dsg3 mobility, membrane availability, and binding strength and thereby modulate cell adhesion in keratinocytes. ∗P < Dsg, desmoglein; KO, knockout; MAPK, mitogen-activated protein kinase; n.s., not significant; PKC, protein kinase C; UF, unbinding force; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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