(A) BioID2 constructs used to generate stably transduced lines from primary Krt14-rtTA mouse keratinocytes. TRE, tetracycline regulatory element used with a minimal promoter to drive expression of MYC-BioID2-GFP or MYC-BioID2-RHOU proteins. PGK, constitutively active promoter, used to drive expression of H2B-RFP to mark transduced cells. (B) Immunoblot of lysates from Krt14rtTA+ keratinocytes transduced with Tre-Myc-BioID2-GFP-pgk-H2B-RFP or Tre-Myc-BioID2-Rhou-pgk-H2B-RFP lentiviruses and then treated for 2 days with doxycycline. Immunoblots were probed with MYC, GFP and RHOU antibodies. (C) Cloud analysis representing RHOU’s proximity interactome. Font size represents frequency of most abundant proteins in the interactome. (D) Gene ontology (GO) analysis reveals a significant enrichment for ‘cell-cell adherens junction’ and ‘focal adhesion’ proteins in RHOU’s proximity interactome. (E) Of the three PAK family members, PAK2 is the most highly expressed at both protein (left) and transcript (right) levels in primary keratinocytes and epidermis. (Left): Immunoblot of lysates from Krt14rtTA+ mouse keratinocytes showing the higher expression of PAK2. Immunoblots were probed with a PAN PAK1/2/3, a specific PAK1 and a specific PAK2 antibody. (Right): Transcriptome profiling from E14.5 transduced epidermal cells revealed Pak2 as the most highly expressed Pak1/2/3 gene. (F) Overexpression of RHOU promotes PAK2 activation and decreases phosphorylation of MLC2. (Left): Primary keratinocytes were transfected with either empty vector or Myc-Rhou. Immunoblot of lysates were probe with MYC, pPAK1Ser144/pPAK2Ser141, PAK2, pMLC2, MLC2 and TUBULIN antibodies. (Right): Quantification of protein lysates. Data are represented as SEM from n = 3 experiments. (G) Depletion of RHOU reduces the activation of PAK2 and promotes the phosphorylation of MLC2. Primary keratinocytes were transfected with shScr, shRhou-504 or shRhou-505. Immunoblots of lysates were probed with RHOU, pPAK1Ser144/pPAK2Ser141, PAK2, pMLC2 and TUBULIN antibodies. (Right): Quantification of protein lysates. Data are represented as SEM from n = 3 experiments for PAK2 level of phosphorylation and n = 4 for pMLC2 level of phosphorylation. (H) ARHGEF7 co-immunoprecipitates (co-IP) with MYC-RHOU in primary keratinocytes. Immunoblot of lysates and co-IP transfected with either an empty vector or Myc-Rhou. Immunoblots were probed with MYC and ARHGEF7 antibodies. (I) Immunofluorescence showing the co-localization of MYC-BioID2-RHOU with ARHGEF7 and PAK2 at focal adhesions in primary keratinocytes. Scale bars, 10 μm (J–L) Similarities between RHOU, PAK2 and ARHGEF7 deficiency phenotypes in skin. (J) Planar views from whole-mount immunofluorescence of transduced E15.5 headskin epidermis. P-CADHERIN marks adherens junctions; Phalloidin marks F-ACTIN; RFP verifies transduction. Shown are representative images from the midplanes of the basal cell layers. Representation from n = 3 embryos. Scale bars, 10 μm. (K) Quantifications of the numbers of placodes, germs and pegs from the different staggered HF waves in shScr, shPak2-209 and shArhgef7-966 transduced E16.5 head skin. Error bars represent SEM from shScr for Pak2 n = 5, shPak2-209 n = 5, shScr for Arhgef7 n = 4 and shArhgef7 n = 4 embryos. Normal distribution of the data was tested using the Shapiro-Wilk test. Parametric independent unpaired two-tailed t-test was used except for the comparison of shScr germ vs shArhgef7 germ and shScr peg vs shArgef7 peg for which a Mann-Whitney test was used. (L) Planar view of P-CADHERIN immunofluoresence of transduced hair peg imaged at the midplane and showing the loss of planar polarized distribution in the absence of RHOU, PAK2 and ARHGEF7. Representation from n = 3 embryos. Scale bars, 10 μm.