Probes for EGF

Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury.

Introduction Protein-protein interactions form the basis most physiological and pathophysiological mechanisms, but studying them at the tissue level is challenging, especially when they are transient, weak or involve low-abundance molecules in complex tissues. The problem is compounded when using antibody-based approaches, due to the paucity of rigorously validated antibodies for in situ studies. To circumvent these limitations, novel in situ hybridization (ISH) techniques present a number of advantages: 1) simple design of highly sensitive and specific probes for any species; 2) rapid probe synthesis; 3) uniform staining protocol across all probes; 4) efficient and reliable multiplexing, including in combination with antibodies or alternative staining approaches. Methods We are using RNAscope (ACD/Bio-Techne) ISH to examine the role of interactions involving the epithelial adapter protein Dok-4 injury models in wild type (WT) and Dok-4 null (KO) mice obtained by CRISPR/CAS9-induced frame shift. Novel interactions were identified by yeast two-hybrid and BioID method. For ISH, paraffin-embedded tissue was stained according to standard protocol either with chromogenic (DAB) method or multiplex fluorescence method. Dok-4 RNAscope probes were obtained from ACD and designed to be either overlapping or not with the short Dok-4 mRNA mismatch present in the KO mice. This allowed us to validate the specificity of the assay on one hand and to pinpoint the expected sites of Dok-4 protein expression (in WT) and deficiency (in KO). For EdU staining, EdU (ClickChemistry) was administered 2 hours before euthanasia and detected with Azide-Fluor 488 (Thermo). Results In both kidney and intestine, multiplex ISH demonstrated overlapping Dok-4 and EGFR mRNA expression. Partner identification could be also multiplexed with ISH probes for nephron segment identity (UMOD) or cellular injury (KIM1). Additionally, ISH could be multiplexed with validated immunofluorescence antibodies, such as to the proximal tubule marker AQP1, and with a marker of proliferation (EdU staining). In a mouse model of irradiation enteritis, directionally opposed but overlapping epithelial mRNA gradients of Dok-4 (dominant in differentiated villi) and its novel partner EGF receptor (dominant in undifferentiated crypts) could be demonstrated. Notably, increased EdU staining found 3 days after irradiation was concentrated in this area of Dok-4/EGFR overlap. Conclusions Multiplex ISH is a highly sensitive, specific and flexible approach to complement proteomics and biochemical interaction studies. Multiplexing for partner molecules can confirm the in vivo relevance of protein-protein interactions defined and validated in more rudimentary systems. Multiplexing can also be done with markers of nephron segment identity, tissue injury or repair, and more. Notably, in transgenic models where mRNA is minimally altered (such as with CRISPR/CAS9 methods), ISH can allow more focused phenotypic studies by delineating the predominant sites of targeted protein deficiency at the single cell level.