Proteomic analysis of desmosomes reveals novel components required for epidermal integrity

Abstract

Desmosomes are cell-cell adhesions necessary for the maintenance of tissue integrity in the skin and heart. While the core components of desmosomes have been identified, peripheral components that modulate canonical or noncanonical desmosome functions still remain largely unexplored. Here we used targeted proximity labeling approaches to further elaborate the desmosome proteome in epidermal keratinocytes. Quantitative mass spectrometry analysis identified all core desmosomal proteins while uncovering a diverse array of new constituents with broad molecular functions. By individually targeting the inner and outer dense plaques, we defined proteins enriched within these subcompartments. We validated a number of these novel desmosome-associated proteins and find that many are membrane proximal proteins that show a dependence on functional desmosomes for their cortical localization. We further explored the mechanism of localization and function of two novel desmosome-associated adaptor proteins enriched in the desmosome proteome, Crk and Crk-like (CrkL). These proteins interacted with Dsg1 and rely on Dsg1 and desmoplakin for robust cortical localization. Epidermal deletion of both Crk and CrkL resulted in perinatal lethality with defects in desmosome morphology and keratin organization, thus demonstrating the utility of this dataset in identifying novel proteins required for desmosome-dependent epidermal integrity.

FIGURE 1:

Development of desmosome-BioID in keratinocytes. (A) Schematic of doxycyline-inducible BioID approach for targeting desmosomes. BirA, biotin ligase; TRE-CMV, tet-response element promoter; LTR, Long terminal repeat; PGK, 3-phosphoglycerate kinase promoter; rtTA-2A-Puro, reverse tetracycline-controlled transactivator-2A peptide-Puromycin fusion protein; Dsp, desmoplakin; Dsc, desmocollins; Dsg, desmogleins; Pkg, plakoglobin. (B) Validation of Dsp-BirA constructs in mouse keratinocytes immunolabeled for HA-tagged-BirA and Dsp. HA, Hemagglutinin fusion tag; Scale bar, 5 μm. (C) Immunofluorescent labeling of Dsp^CC-BirA expressing keratinocytes after overnight incubation in Ca²⁺ and exogenous biotin (50 μM). Scale bar, 5μm. Note that the anti-desmoplakin antibody used is to the C-terminus of the protein and does not bind to the BirA fusion proteins. (D) Western blot analysis displays construct expression and biotinylated fraction in biotin-fed, Ca²⁺ induced Dsp-BirA keratinocyte lysates. Membranes were probed with anti-HA and streptavidin-HRP in order to identify Dsp-BirA expression and biotinylation spectrum, respectively. (E) Western blot analysis of junctional components after enrichment of biotinylated fractions from Dsp-BirA lysates. This is representative of three independent experiments.

FIGURE 2:

Network graph of the desmosome proteome. (A) Outline of desmosome-BioID protocol for proteomic evaluation of desmosomes in keratinocytes. (B) String diagram of top hits from desmosome BioID analyses. Node titles, sizes, and colors represent gene names; total abundances; and Dsp^CC vs. Dsp^N abundance ratios, respectively; edge connections indicate protein interactions identified by STRING; edge color highlights predicted interactions with core desmosome components. (C) Network topologies of desmosome BioID hits from select protein families.

FIGURE 3:

Characterization and quantitative proteomic analysis of desmosome-BioID. (A) Revigo plot highlighting functional categorization of biological processes enriched by desmosome BioID. Node size represents relative number of proteins while edge width indicates degree of similarity between connecting GO term nodes. (B) Cross-correlation plot compares statistical fold enrichment of protein hits from Dsp^IDP vs. Dsp^ODP analyses. Each point represents a protein with an associated p value indicating enrichment in either Dsp^IDP or Dsp^ODP conditions, or neither. (C) GO term graph of molecular functions for proteins enriched in Dsp^IDP. (D) GO term graph of molecular functions for proteins enriched in Dsp^ODP.

FIGURE 4:

Validation of select desmosome BioID protein targets identify novel desmosome-associated proteins. Wild-type (A–J) and Dsp –/– (A’–J’) keratinocytes were fixed and costained for desmosomes (red) and candidate proteins, either endogenous or GFP tagged, as indicated. Line scan analyses across desmosomes, depicted in the inset, were used to profile relative enrichment of selected proteins near desmosomes verses the cytoplasm. Corresponding colocalization analyses of these hits are shown in Table 1 which also lists the protein names associated with each gene. Scale bars, 5 μm.

FIGURE 5:

Adapter molecules Crk and CrkL are novel desmosome constituents. (A) Domain enrichment analysis of desmosome BioID using SMART. (B) Schematic representation of domains in Crk and CrkL and graphs displaying fold enrichment of Crk and CrkL in Dsp-BirA proteomes over Cyto-BirA. (C) Desmosome (Dsp, red) and CrkL (green) staining in Wt, Dsp –/–, Keratin –/–, and p120-catenin –/– keratinocytes after 24hr Ca²⁺ incubation. (D) Staining for Crkl (green) and β-catenin (red) in cultured keratinocytes after 24h Ca²⁺ incubation. (E) Immunofluorescent staining of desmosomes (Dsp, red) and GFP (green) in Ca²⁺ induced mouse keratinocytes transfected with full length Crk-GFP. (F) Time course of localization after calcium-induced desmosome formation. Immunofluorescent staining of desmosomes (Dsp, red) and CrkL (green) in mouse keratinocytes before and after Ca²⁺ induction. (G) Pull-down assays from mouse keratinocyte lysates using recombinant Crk constructs reveal associations between Crk and desmoglein 1. This is representative of two independent replicates.

FIGURE 6:

Tissue-specific loss of Crk and CrkL results in acantholysis and altered desmosome morphologies in the epidermis. (A) Genetic strategy for epidermal targeting of Crk and CrkL. (B) Western blot analysis of isolated epidermis from control and dKO mice. Arrow points to band representing loss of CrkL protein. (C) Representative images of control and dKO newborn pups. Note compromised patches of epidermal tissues designated by arrow. (D) H&E (left) and transmission electron microscopy (right) images of control and mutant skin. Note intercellular detachments designated by arrows. Scale bars, left, 30 μm; right, 500 nm. (E) Quantitative analysis of intercellular separations from (left) H&E (n=4 mice) and (right) transmission electron microscopy (n = 15 regions, 2 mice). (F) Left: high magnification transmission electron micrographs of control and Crk/CrkL dKO epidermis. Right: quantitation of disrupted desmosome organization in the control and Crk/CrkL dKO skin. Scale bars, 100 nm (n = 15 regions, 2 mice). (G) Top: representative immunofluorescent stains of desmoglein 1, desmoplakin, and plakoglobin in control and mutant epidermis. Dashed line delineates cell membrane area. Scale bars, 20 μm. Bottom: line scan analysis of plakoglobin stains across cell–cell interfaces, Shaded regions represent standard deviation from mean. (n = 20 scans, 3 mice). (H) Representative immunofluorescent stains of differentiation related markers keratin 10 (red) and keratin 5/14 (green) in control and dKO epidermis. Inset: Note the perinuclear accumulation of keratin in suprabasal cells of mutant mice. Scale bars, 20 μm.