Journal: Journal of Cell Science

Article Title: GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region

doi: 10.1242/jcs.261100

Figure Lengend Snippet: GIPC3 interaction networks identified through immunoaffinity purification and protein mass spectrometry. (A) Flow chart for anti-GIPC3 immunoaffinity purification from crude chick stereocilia extracts. F/T, flow through. (B–E) Comparison of abundance (riBAQ) of proteins detected in DSP1 total or DTSSP total (starting S7 extract; plotted on X -axis) compared to the immunoprecipitates ( Y -axis) for mouse IgG control (B,C) and 10G5 anti-GIPC3 (D,E) experiments. Panels B and D show results with the DSP-crosslinked starting extract, whereas panels C and E show results with DTSSP crosslinking. Each point represents the average abundance of that protein in two experiments (biological replicates); symbol colors were arbitrarily chosen. Red dashed line is the unity line (equal riBAQ in total and IP). Key proteins are called out. Mouse IgG protein from immunoprecipitation is highlighted in gray. (F) Gene ontology analysis (cellular component) with DAVID of the top 50 proteins from the DTSSP 10G5 eluate. Red, actin-associated components; orange, intermediate filament-associated components; blue, microtubule-associated components; gray, other components. (G) Overlap of protein interaction networks of key proteins from the DTSSP 10G5 eluate (GIPC3, APPL2, MYO6, MYO18A, MYH9 and MYH10). BioGRID-defined protein networks for APPL2, MYO6, MYO18A, MYH9 and MYH10 were compared with the top 100 proteins from the DTSSP 10G5 eluate. Only proteins identified as interactors of two or more of the key proteins were included. Proteins in bold were present in the top 100 proteins from the DTSSP 10G5 eluate.

Article Snippet: Other primary antibodies used were: Atlas Antibody anti-MYO18A (Cat# HPA021121, RRID:AB_1854250; dilution of 1:200 for immunostaining) from Sigma-Aldrich (St Louis, MO, USA); Proteintech (Rosemont, IL, USA) anti-MYO18A (Cat# 14611-1-AP, RRID:AB_2201447; dilution of 1:100); Proteintech anti-APPL2 (Cat# 14294-1-AP, RRID:AB_2878041; dilution of 1:100); Thermo Fisher Scientific (Waltham, MA, USA) anti-TJP1 (also known as ZO1; Cat# 33-9100, RRID:AB_2533147; dilution of 1:200); Santa Cruz Biotechnology (Dallas, TX, USA) anti-LMO7 (Cat# sc-376807, RRID:AB_2892126; dilution of 1:100); Proteintech anti-TARA (TRIOBP; Cat# 16124-1-AP, RRID:AB_2209237; dilution of 1:200); Proteintech anti-ACTN4 (Cat# 19096-1-AP, RRID:AB_10642150; dilution of 1:100); and Biolegend anti-MYH9 (Cat# 909802, RRID:AB_2734686; dilution of 1:200).

Techniques: Immunoaffinity Purification, Mass Spectrometry, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region

doi: 10.1242/jcs.261100

Figure Lengend Snippet: MYO18A is located in the hair cell apical domain. (A) Top, sequence logo for binding of ligands to GIPC1 PDZ domain; bottom, C-terminal ten amino acids of APPL2, MYO18A, ACTN1 and ACTN4. (B) Immunolocalization of MYO18A in P14.5 mouse cochlea; slices from a three-dimensional image stack. Transects for other image axes are shown in yellow; the X and Y transects in the main X-Y image show the locations for the Y-Z and X-Z images. Arrow indicates concentration of MYO18A immunoreactivity below the IHC cuticular plate. IHC, inner hair cell; IPC, inner pillar cell; OPC, outer pillar cell; OHC, outer hair cell. Immunolocalization experiments for MYO18A were performed more than five times. (C,D) MYO18A immunoreactivity in P15.5 IHCs using lattice SIM imaging. (C) Image showing four IHCs at the stereocilia/cuticular plate level. (D) Image showing a single IHC (labeled with asterisk in C) at the cuticular plate level (different plane than in C). Arrows delineate the gap between cuticular plate actin and the circumferential actin belt. (E,F) MYO18A immunoreactivity in P14.5 IHCs from folded cochleas using Airyscan imaging. E is from a Gipc3 KO /+ mouse and F is from a Gipc3 KO/KO mouse. (G) NanoSPD of MYO18A–GIPC3. Example of filopodial targeting of mCherry–GIPC3 by GFP–FL-MYO18A, mediated by MYO10 NANOTRAP . (H) Expression of GFP–FL-MYO18A and mCherry–FL-GIPC3 constructs in HeLa cells (no MYO10 NANOTRAP expressed). Arrows indicate large cytoplasmic aggregates containing GFP and mCherry. squeezing. Images in C–H representative of at least three repeats. (I) GFP–MYO18A constructs. ‘Motor’, actin- and ATP-binding domains are homologous to myosin motor domains in active myosins; IQ, isoleucine/glutamine calmodulin-binding; PBM, PDZ-binding motif. (J) mCherry–GIPC3 constructs. mCh, mCherry; GH1, GIPC-homology 1; GH2, GIPC-homology 2. (K) Prey (mCh–GIPC3) fluorescence with GFP–MYO18A constructs or GFP control. Mean±s.d. plotted in K and L. One-way ANOVA comparisons to no-bait condition with Dunnett correction for multiple comparisons. Sample sizes ( n ) were GFP–FL-MYO18A (105 filopodia), GFP–IQ-PBM-MYO18A (109), GFP–CC-PBM-MYO18A (116), GFP–C-PBM-MYO18A (129), GFP–ΔPBM-MYO18A (127), GFP (208). (L) Prey (mCh–GIPC3 constructs or mCh–MYO6) fluorescence with GFP-MYO18A constructs or GFP control. One-way ANOVA comparisons to no-bait condition with Dunnett correction for multiple comparisons. Sample sizes ( n ) were mCh–D1-GIPC3 (128 filopodia for GFP–FL-MYO18A and 88 for GFP alone), mCh–D3-GIPC3 (180 and 47) and mCh–MYO6 (107 and 93). Panel widths: B, 37.5 µm for X-Y plot (same scale applies to Y-Z and X-Z panels); C, 50 µm; D, 12 µm; E-F, 45 µm; G,H, 15 µm.

Article Snippet: Other primary antibodies used were: Atlas Antibody anti-MYO18A (Cat# HPA021121, RRID:AB_1854250; dilution of 1:200 for immunostaining) from Sigma-Aldrich (St Louis, MO, USA); Proteintech (Rosemont, IL, USA) anti-MYO18A (Cat# 14611-1-AP, RRID:AB_2201447; dilution of 1:100); Proteintech anti-APPL2 (Cat# 14294-1-AP, RRID:AB_2878041; dilution of 1:100); Thermo Fisher Scientific (Waltham, MA, USA) anti-TJP1 (also known as ZO1; Cat# 33-9100, RRID:AB_2533147; dilution of 1:200); Santa Cruz Biotechnology (Dallas, TX, USA) anti-LMO7 (Cat# sc-376807, RRID:AB_2892126; dilution of 1:100); Proteintech anti-TARA (TRIOBP; Cat# 16124-1-AP, RRID:AB_2209237; dilution of 1:200); Proteintech anti-ACTN4 (Cat# 19096-1-AP, RRID:AB_10642150; dilution of 1:100); and Biolegend anti-MYH9 (Cat# 909802, RRID:AB_2734686; dilution of 1:200).

Techniques: Sequencing, Binding Assay, Concentration Assay, Imaging, Labeling, Expressing, Construct, Fluorescence