Fig 1.
Intraepidermal blistering in Rpgrip1l-/- skin.
(a) In situ hybridization of Rpgrip1l in the dorsal skin of E18.5 wild-type mouse. Rpgrip1l (pink dot) is expressed in the epidermis (Epi), dermis (Der), and hair follicles (HF). Dotted line represents basement membrane. B, basal keratinocyte; SB, suprabasal keratinocyte; S, spinous keratinocyte; G, granular keratinocyte. Positive and negative control probes detect the mouse POLR2A or bacterial dapB gene, respectively. (b) Hematoxylin and eosin (H&E) staining and immunofluorescence labeling of KRT14 (green) and KRT1 (red) in dorsal skins of E18.5 control (Rpgrip1+/+) and homozygous mutants (Rpgrip1l-/-). Nuclei were labeled with DAPI (blue). Asterisks indicate intraepidermal blisters. (c) High power H&E images to demonstrate details of the blistering region. B, basal keratinocyte; SB, suprabasal keratinocyte; S, spinous keratinocyte. (d and e) H&E staining (d) and immunofluorescence labeling of KRT14 (green, e) of organotypic skin explants from E18.5 control (Rpgrip1l+/+, n = 8) and homozygous (Rpgrip1l–/–, n = 7) embryos at day 2. Nuclei were labeled with DAPI (blue). Asterisks indicate intraepidermal blisters. Scale bar, 25 μm in (a), 100 μm in (b), 10 μm in (c), 50 μm in (d, e).
Fig 2.
Expression patterns of desmosomal proteins in Rpgrip1l-/- skin.
(a) Immunofluorescence labeling of desmosomal proteins in dorsal skins of E18.5 control (Rpgrip1l+/+) and homozygous (Rpgrip1l-/-) mutants. Dotted lines illustrate epidermal-dermal junction. Asterisks and vertical lines indicate blisters between basal and suprabasal cells. (b) Quantification of the mean fluorescence intensity of corresponding panels in (a) (2 regions per specimen, n ≥ 3 mice per group;* P < 0.05, ** P < 0.01 *** P < 0.001; Student’s t-test). Scale bars, 20 μm.
Fig 3.
Ultrastructure of desmosomes in Rpgrip1l-/- mice.
(a) Transmission electron microscopy micrographs of desmosomes between basal and suprabasal keratinocytes (Basal/suprabasal), and between spinous keratinocytes (Spinous) in E18.5 control (Rpgrip1l+/+) and homozygous (Rpgrip1l-/-) mutant (n = 7 mice). Arrowheads point to the electron dense midline. B, basal keratinocytes; SB, suprabasal keratinocytes; KF, keratin filaments. (b) Quantification of the length of desmosomes between basal and suprabasal keratinocytes of control and Rpgrip1l-/- mutants (n ≥ 15 desmosomes; 3 mice per group; *** P < 0.001; Student’s t-test). Scale bars, 200 nm.
Fig 4.
RPGRIP1L-knockdown in keratinocytes compromises cell-cell adhesion.
(a) Immunofluorescence labeling of RPGRIP1L, primary cilia (ARL13B), and basal body/centriole (γ-TUB) in HaCaT cells, normal human epidermal keratinocytes (NHEK), and mouse embryonic fibroblasts (MEFs) at 48 hours after serum-starvation. Nuclei were labeled with DAPI (blue). Arrows point to centrioles or a basal body where RPGRIP1L is enriched. Bar graph represents percentage of ciliated cells (# indicates that cilium was undetectable in HaCaT cells). Scale bar, 10 μm. (b and c) Verification of RPGRIP1L-knockdown by qRT-PCR (b) and western blotting (c) in HaCaT cells. Values were normalized to mock transfection in (b). (d) Timeline of the dispase dissociation assay. (e) Dispase dissociation assay in control (Control siRNA) and RPGRIP1L-knockdown (RPGRIP1L siRNA) HaCaT cells. (f) Quantification of cell fragments (mean ± SEM, n = 3 independent experiments; ** P < 0.01; *** P < 0.001; Student’s t-test).
Fig 5.
RPGRIP1L-knockdown disrupts desmosomes in HaCaT cells.
(a) Expression of desmosomal proteins in control (Control siRNA) and RPGRIP1L-knockdown (RPGRIP1L siRNA) HaCaT cells by western blotting. Note that both DSG1 and DSG2 are expressed in HaCaT cells, and detectable by the DSG1/2 antibody. (b) Immunofluorescence labeling of desmosomal proteins in control and RPGRIP1L-knockdown HaCaT cells. Bar graphs represent plasma membrane/cytoplasm ratios of average pixel intensities of desmosomal proteins (n = 50 cells, ** P < 0.01, *** P < 0.001; Student’s t-test). (c) Transmission electron microscopy micrographs of desmosomes in control and RPGRIP1L-knockdown HaCaT cells. Arrowheads point to the electron dense midline. KF, keratin filaments. Scale bar, 10 μm in (b), 200 nm in (c).
Fig 6.
Disrupting RPGRIP1L promotes the disassembly of desmosomes.
(a) Timeline of knockdown and desmosome assembly assay. (b and c) Immunofluorescence labeling (b) and quantification (c) of DSG1/2 at 0.5, 1, and 3 hours after switching to high calcium media in control (Control siRNA) and RPGRIP1L-knockdown (RPGRIP1L siRNA) HaCaT cells. (d) Timeline of knockdown and desmosome disassembly assay. (e and f) Immunofluorescence labeling (e) and quantification (f) of DSG1/2 in control and RPGRIP1L-knockdown HaCaT cells treated with EGTA. (g and h) Immunofluorescence labeling (g) and quantification (h) of DSG3 as described for DSG1/2. Quantifications (c, f, and h) represent plasma membrane/cytoplasm ratios of the pixel intensities of DSG1/2 or DSG3 (n = 25 cells, * P < 0.05, ** P < 0.01, *** P < 0.001, NS, not statistically significant, Bonferroni’s post hoc tests. Note, when EGTA-treated cells were compared directly, RPGRIP1L-knockdown elicited a significant reduction of membrane DSG1/2 in f, P = 0.049). Scale bar, 10 μm.
Fig 7.
Disrupting RPGRIP1L promotes endocytosis of desmogleins in HaCaT cells.
(a) Timeline of RPGRIP1L-knockdown and treatment with dynasore or sucrose, and EGTA. (b) Immunofluorescence labeling of DSG1/2 in control (Control siRNA) and RPGRIP1L-knockdown (RPGRIP1L siRNA) cells treated with dynasore or sucrose, and EGTA. Nuclei were labeled with DAPI (blue). (c) Quantification of the ratios of plasma membrane/cytoplasm pixel intensities of DSG1/2 in (b) (n = 25). (d) Timeline of the cell surface DSG3 internalization assay carried out with the AK23 antibody. (e) Immunofluorescence of internalized DSG3 (labeled by the AK23 antibody) in control and RPGRIP1L-knockdown cells treated with PV IgG. Nuclei were labeled with DAPI (blue). (f) Quantification of the number of particles (DSG3) per cell (n ≥ 160 cell per group, three independent experiments). (g) Detection of cell-surface proteins (DSG3, DSC3, EGFR and CDH1) by western blotting in whole cell lysate and biotinylated (endocytosed) fraction in a biotinylation assay on control and RPGRIP1L-knockdown cells. The numbers indicate fold changes relative to control siRNA knockdowns as determined by densitometry. * P < 0.05, ** P < 0.01, *** P < 0.001 (Bonferroni’s post hoc tests). Note, when EGTA-treated cells were compared directly, RPGRIP1L-knockdown elicited a significant reduction of membrane DSG1/2 in c, P = 0.027. Scale bar, 10 μm.