Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1
- Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, United States
- Department of Cell and Developmental Biology, University of Michigan Medical School, United States
- Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, United States
- Department of Ophthalmology, Johns Hopkins School of Medicine, United States
- Department of Dermatology, University of Michigan Medical School, United States
- Rogel Cancer Center, Michigan Medicine, University of Michigan, United States
Figures
Figure 1
Decreased K14-dependent disulfide-bonded species and thickened epidermis in Krt14 C373A mouse skin.
(A) Location of cysteine (C) residues in mouse K14 protein (C4, C18, C36, C58, C373, C395), in which N-terminal head and C-terminal tail domains are flanking the central α-helical rod domain (coils …
Figure 2 with 1 supplement
Alternations in morphology and barrier status in Krt14 C373A skin.
(A) Toluidine blue-stained sections (1 mm thick from epoxy-embedded skin of young adult WT and Krt14 C373A mice. (B) Quantification of whole epidermal thickness (living epidermal layers and stratum …
Figure 2—figure supplement 1
Analysis of purified cornified envelopes from ear skin.
(A) Representative microscopic images of cornified envelopes isolated (CE) from WT and Krt14 C373A ear skin. Bar, 100 µm. (B) Quantitation of surface area, circumference, and aspect ratio of CEs. N =…
Figure 3 with 2 supplements
Altered tissue homeostasis and dysregulated keratinocyte differentiation in Krt14 C373A skin.
(A) Indirect immunofluorescence for Edu in tail skin section from WT and Krt14 C373A at 2 hr, 1 d, and 3 d after treatment with thymidine analog EdU. Nuclei as stained with DAPI (blue). (B) …
Figure 3—figure supplement 1
Analysis of terminal differentiation in mouse back skin tissue.
(A-C) Cryosections of back skin tissue from young adult WT and Krt14 C373A were subjected to dual indirect immuno-fluorescence using antibodies to (A) K14 and K10; (B) K14 and filaggrin; and (C) K14 …
Figure 3—figure supplement 2
Ultrastructural changes and abnormal nuclei in Krt14 C373A keratinocytes.
(A) Transmission electron micrograph of ear skin epidermis from WT and Krt14C373A mice. (B) High magnification images of representative cells in frame a. Dotted lines mark the dermo-epidermal …
Figure 4 with 1 supplement
14-3-3σ interacts with K14, and abnormal localization 14-3-3σ and YAP in Krt14 C373A epidermis.
(A) Top nine most abundant non-keratin entries from a mass spectrometry screen for proteins interacting with K14 in WT newborn skin keratinocytes (primary culture,1 mM calcium, 4 days). Spectral …
Figure 4—figure supplement 1
Additional analyses of newborn skin keratinocytes in primary culture.
(A) Western blot analysis of total protein extracts from WT newborn keratinocytes in primary culture, in the absence (0 hr) and presence of calcium for 15 hr and 4 days (4d) after addition of …
Figure 5 with 1 supplement
Localization and interaction of 14-3-3σ and YAP activity in Krt14 C373A keratinocytes.
(A) Indirect immunofluorescence microscopy for YAP (red) and K14 (green) in WT and Krt14 C373A newborn skin keratinocytes in primary culture in the absence and presence of 1 mM calcium (for 4 d). …
Figure 5—figure supplement 1
Localization of YAP is specifically regulated by cysteine residue 367 in human K14.
Newborn skin keratinocytes from Krt14 null mice were seeded in primary culture, transiently transfected with either GFP-K14 WT, GFP-K14CF, GFP-K14C367A or GFP-K14CF-C367 constructs, and cultured in …
Figure 6
Altered mechanics in Krt14 C373A epidermis and keratinocytes in culture.
(A-C) Studies involving tail skin sections from young adult WT and Krt14 C373A mice. A. Indirect immuno-fluorescence microscopy for α-catenin, desmoglein one and lamin A/C. Dashed lines depict the …
Figure 7 with 1 supplement
Model depicting the role of keratin-dependent disulfide bonding in integrating signaling and mechanical cues as keratinocytes initiate terminal differentiation in epidermis.
(A) Left to right: the stage of epidermal differentiation, keratin expression, epidermal morphology, and state of keratin filament organization are related to 14-3-3 binding, YAP1 subcellular …
Figure 7—figure supplement 1
Genomic context, gene expression, chromatin organization, and presence of TEAD binding sites in gene loci of interest.
This information was obtained from the ENCODE project (www.encodeproject.org) and applies to human fore-skin keratinocytes cultured in the presence or absence of calcium (see Materials and methods …
Additional files
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Source data 1
Raw data used for quantitation.
- https://cdn.elifesciences.org/articles/53165/elife-53165-data1-v2.xlsx
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Supplementary file 1
Mass spectrometric screen for proteins interacting with wildtype K14 in WT newborn skin keratinocyte in primary culture.
List of all proteins with at least 35 spectral counts (cumulative over three biological replicates). Gene symbols are provided at left. WT1, WT2, WT3, WT4 and WT5 represent different regions of silver stained protein electrophoretic gels subjected to MS analysis (data not shown). Keratin protein entries dominate this screen, as expected, and are listed using blue lettering. 14-3-3 protein isoforms are listed using red lettering. The top non-keratin protein from this screen is 14-3-3sigma.
- https://cdn.elifesciences.org/articles/53165/elife-53165-supp1-v2.xlsx
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Supplementary file 2
Summary of data relating genomic context, gene expression, chromatin organization, and presence of TEAD binding sites in gene loci of interest.
This summary account applies to human foreskin keratinocytes cultured in the absence (day 0) or presence of calcium for 6 days (see Figure 7—figure supplement 1) and is derived from the ENCODE project. Ratings for gene expression levels (low, moderate (med), high or very high) and promoter accessibility (low, moderate (med) or high are reported for keratin genes known to be expressed in progenitor (KRT14, KRT15, KRT5) and differentiating keratinocytes (KRT10, KRT1, KRT2) in epidermis, and for additional genes that are relevant to our study (SFN, ITGB1, CCN1, TP63, YAP1, and TEAD1). The presence of consensus TEAD binding sites in the proximal promoter of these genes (either 1–2 sites, shown as ‘+”, or >3 sites, shown as ‘+++””) is also reported. See Discussion for further information.
- https://cdn.elifesciences.org/articles/53165/elife-53165-supp2-v2.ai
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Supplementary file 3
List of oligonucleotide primers used in this study.
- https://cdn.elifesciences.org/articles/53165/elife-53165-supp3-v2.xlsx
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Supplementary file 4
Key Resources Table.
- https://cdn.elifesciences.org/articles/53165/elife-53165-supp4-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53165/elife-53165-transrepform-v2.docx
