(A) RPE1 cells were fixed and stained with anti-Cav1 antibodies. The rear localisation index (fold enrichment at the rear) was determined by measuring the Cav1 intensity at the cell front and the …
(A) RPE1 cells stably transfected with cavin1-A2E were fixed and stained with anti-Cav1 antibody. Note the colocalisation of Cav1 and cavin1 at the rear (arrows). The Golgi apparatus (*) is stained …
(A) Time-lapse images of RPE1 cells stably transfected with Cavin3-miniSOG-mCherry. The movie starts with an unpolarised cell state. Cavin3 becomes aligned in ‘stripes’ as the cell establishes a …
Sudden rear retractions with large rear replacements can be seen between 145 and 150 min, between 235 and 240 min, and between 705 and 715 min of the movie (related to Figure 1F).
A sudden rear retraction can be seen between 235 and 250 min of the movie (related to Figure 1—figure supplement 1D).
The cell changes its front-rear axis between 140 and 200 min of the movie, which is accompanied by a reorganisation of cavin3.
Abscission of the cytokinetic bridge can be observed at 175 min. Cavin3 rear localisation is evident and the cells start to migrate.
Caveolae become initially aligned along the long cell axis and progressively accumulate at the rear as the cell polarises and the rear retracts (160–250 min) (related to Figure 1—figure supplement 2A…
Note that cavin3 is aligned in ‘stripes’ or ‘filaments’ as the cell migrates (related to Figure 1—figure supplement 2B).
(A) Electron tomography of caveolae and caveolar networks at the rear of RPE1 cells stably transfected with Cavin3-miniSOG-mCherry. Two slices of the tomogram are shown. Note that individual …
Caveolae were specifically labelled using miniSOG (related to Figure 2B).
(A) Western blot of stable RPE1 cell lines expressing either Cav1-A2E or NES-A2E fusion proteins. The membrane was probed with anti-Cav1 antibodies. (B) Confocal fluorescence microscopy of Cav1-A2E …
Original western blots.
(A) RPE1 cells stably transfected with Cav1-A2E were fixed and stained with anti-cavin1 antibodies. Note the pronounced colocalisation and the enrichment of Cav1-A2E and cavin1 at the cell rear. …
Original western blots.
(A) Experimental set-up used for APEX2-mediated proximity biotinylation and LC-MS/MS. After proximity labelling, cells were lysed and biotinylated proteins were captured using magnetic streptavidin …
Heatmap of 111 z-scored LFQ quantified protein groups differentially enriched across the three treatments (non-treated [NT, control], hypo-osmotic shock [HYPO], and recovery from hypo-osmotic shock …
(A–D) PLA in the Cav1-A2E RPE1 cell line using anti-GFP and anti-PTRF/cavin1 (A, B), anti-FLNA (C), or anti-CTTN (D) antibodies as indicated. (E, F) PLA in RPE1 cells transfected with EHD2-A2E using …
(A, B) Representative images of PLA on Cav1-A2E-expressing RPE1 cells using anti-GFP antibodies and anti-FLNA (A) or anti-CTTN antibodies (B). (C) Control PLA of Cav1-A2E cells. Samples were probed …
(A, B) Representative images of PLA on Cav1-A2E-expressing RPE1 cells using anti-GFP antibodies and anti-HSPB1 (A) or anti-ROCK1 antibodies (B). GFP and DAPI signals used to determine cell …
(A) RPE1 cells were transfected with siRNAs against ARHGAP29, caveolin-1, or non-targeting siRNAs and analysed by western blotting 48 hr post-transfection. (B) Quantification of western blot …
Original western blots shown in Figure 6A used for the quantification of data shown in Figure 6B.
Original pMLC western blots shown in Figure 6A used for the quantification of pMLC levels shown in Figure 6B.
qPCR analysis of control and caveolin-1 siRNA-transfected RPE1 cells. Data is derived from three independent technical replicates. Error bars indicate SEM. ***p≤0.001.
(A–D) Migration tracks (A), migration speed (B), displacement (C), and mean squared displacement (D) of RPE1 cells transfected with esiRNAs against ARHGAP29 or non-targeting esiRNAs. Quantification …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (human) | hTERT RPE-1 | ATCC | ATCC CRL-4000 RRID:CVCL_4388 | Human retinal pigment epithelium |
Cell line (human) | hTERT RPE-1 Cavin1-APEX2-EGFP | Ludwig, 2020 | NA | Stable transfection |
Cell line (human) | hTERT RPE-1 Cavin3-miniSOG-mCherry | Ludwig et al., 2013 | NA | Stable transfection |
Cell line (human) | hTERT RPE-1 NES-A2E | This study | NA | Stable transfection |
Cell line (human) | hTERT RPE-1 Cav1-A2E | This study | NA | Stable transfection |
Transfected construct (human) | NES-A2E | Tan et al., 2020b | NA | Recombinant DNA |
Transfected construct (human) | Cav1-A2E | Ludwig et al., 2016 | NA | Recombinant DNA |
Chemical compound, drug | DMEM:F12 1:1 Mixture | Westburg | Cat# LO BE04-687F/U1 | NA |
Chemical compound, drug | Geneticin Selective Antibiotic (G418 Sulfate) | Fisher Scientific | Cat# 10131027 | 500 µM |
Peptide, recombinant protein | Gibco Fibronectin Bovine Protein, Plasma | Fisher Scientific | Cat# 33010018 | 1:200 |
Other | Lipofectamine 3000 Reagent | Thermo Fisher Scientific | Cat# L3000015 | transfection reagent |
Other | Dharmafect 1 | Thermo Fisher Scientific | Cat# T-2001 | transfection reagent |
Peptide, recombinant protein | Streptavidin Alexa Fluor 568 | Thermo Fisher Scientific | Cat# S11226 RRID:AB_2315774 | IF: 1:1000 |
Peptide, recombinant protein | Streptavidin-HRP | Thermo Fisher Scientific | Cat# 434323 RRID:AB_2619743 | WB: 1:10,000 |
Commercial assay or kit | Duolink In Situ Orange Starter Kit Mouse/Rabbit | Sigma-Aldrich | Cat# DUO92102-1KT | NA |
Chemical compound, drug | Biotin phenol | Iris Biotech | Cat# LS-3500 | 0.5 mM |
Chemical compound, drug | Trolox | Sigma-Aldrich | Cat# 238813 | 5 mM |
Chemical compound, drug | Glutaraldehyde (32%) | Electron Microscopy Sciences (EMS) | Cat# 16220 | 2% |
Chemical compound, drug | Paraformaldehyde (32%) | Electron Microscopy Sciences (EMS) | Cat# 100504–858 | 1–4% |
Chemical compound, drug | Diaminobenzidine (DAB) (Free-Base) | Sigma-Aldrich | Cat# D8001 | 0.5 mg/ml |
Chemical compound, drug | Durcupan ACM resin | Electron Microscopy Sciences (EMS) | Cat# 44610 | NA |
Chemical compound, drug | Osmium tetroxide (2%) | Electron Microscopy Sciences (EMS) | Cat# 19152 | 1–2% |
Chemical compound, drug | Potassium ferricyanide | Sigma-Aldrich | Cat# 702587 | 1–2% (w/v) |
Peptide, recombinant protein | Sequencing Grade Modified Trypsin | Promega Corporation | Cat# V5111 | |
Peptide, recombinant protein | Lysyl Endopeptidase, MS Grade | FUJIFILM Wako Pure Chemical Corporation | Cat# 125–05061 | |
Peptide, recombinant protein | Complete, EDTA-free Protease Inhibitor Cocktail | Roche | Cat# 11873580001 | |
Peptide, recombinant protein | Thermo Scientific Pierce Streptavidin Magnetic Beads | Fisher Scientific | Cat# 10615204 | |
Commercial assay or kit | EZQ Protein Quantitation Kit | Fisher Scientific | Cat# R33201 | |
Antibody | Rabbit monoclonal anti-PARG1 (ARHGAP29) | Invitrogen | Cat# PA5-55336 RRID:AB_2645210 | WB: 1:1000 |
Antibody | Rabbit polyclonal anti-Caveolin-1 (CAV1) | BD Bioscience/ Transduction labs | Cat# 610060 RRID:AB_397472 | WB: 1:10,000 IF/PLA: 1:500 |
Antibody | Mouse monoclonal anti-Caveolin-1 pY14 | BD Bioscience/ Transduction labs | Cat# 611339 RRID:AB_398863 | WB: 1:1000 |
Antibody | Rabbit polyclonal anti-Cavin-1 (PTRF) | Abcam | Cat# ab48824 RRID:AB_882224 | WB: 1:5000 IF/PLA: 1:500 |
Antibody | Goat polyclonal anti-EHD2 | Abcam | Cat# ab23935 RRID:AB_2097328 | WB: 1:2000 |
Antibody | Mouse monoclonal anti-Filamin 1 (E-3) (FLNA) | Santa Cruz Biotechnology | Cat# sc-17749 RRID:AB_627606 | IF/PLA: 1:100 |
Antibody | Mouse monoclonal anti-Cortactin (clone 4F11) | Merck Millipore | Cat# 05–180 RRID:AB_309647 | IF/PLA: 1:100 |
Antibody | Mouse monoclonal anti-Rock-1 (G-6) | Santa Cruz Biotechnology | Cat# sc-17794 RRID:AB_628223 | IF/PLA: 1:100 |
Antibody | Mouse monoclonal anti-HSP27 (F-4) (HSPB1) | Santa Cruz Biotechnology | Cat# sc-13132 RRID:AB_627755 | IF/PLA: 1:100 |
Antibody | Mouse monoclonal anti-GFP | Roche | Cat# 11814460001 RRID:AB_390913 | WB: 1:2000 |
Antibody | Rabbit polyclonal anti-GFP | Abcam | Cat# ab290 RRID:AB_2313768 | IF/PLA: 1:100 |
Antibody | Mouse monoclonal anti-YAP | Cell Signalling Technology | Cat# 14074 RRID:AB_2650491 | WB: 1:1000 |
Antibody | Mouse monoclonal anti-YAP pS127 | Cell Signalling Technology | Cat# 13008 RRID:AB_2650553 | WB: 1:5000 |
Antibody | Rabbit polyclonal anti-ppMLC2 (Thr18/Ser19) | Cell Signalling Technology | Cat# 3674 RRID:AB_2147464 | WB: 1:1000 |
Antibody | Mouse anti-GAPDH | Santa Cruz Biotechnology | Cat# 47724 RRID:AB_627678 | WB: 1:5000 |
Antibody | Donkey anti-Mouse IgG (H+L) Alexa Fluor 488 | Invitrogen | Cat# A-21202 RRID:AB_141607 | IF: 1:500 |
Antibody | Donkey anti-Rabbit IgG (H+L) Alexa Fluor 488 | Invitrogen | Cat# A-21206 RRID:AB_2535792 | IF: 1:500 |
Antibody | Donkey anti-Mouse IgG (H+L) Alexa Fluor 555 | Invitrogen | Cat# A-31570 RRID:AB_2536180 | IF: 1:500 |
Antibody | Donkey anti-Rabbit IgG (H+L) Alexa Fluor 555 | Invitrogen | Cat# A-31572 RRID:AB_162543 | IF: 1:500 |
Antibody | Goat anti-Rabbit IgG (H+L) Alexa Fluor 633 | Invitrogen | Cat# A-21071 RRID:AB_2535732 | IF: 1:500 |
Antibody | Goat anti-Mouse IgG (H+L) Alexa Fluor 633 | Invitrogen | Cat# A-21052 RRID:AB_2535719 | IF: 1:500 |
Antibody | Goat anti-rabbit IgG (H+L) HRP conjugate | Invitrogen | Cat# A16104 RRID:AB_2534776 | WB: 1:5000 |
Antibody | Goat anti-mouse IgG (H+L) HRP conjugate | Invitrogen | Cat# A16072 RRID:AB_2534745 | WB: 1:5000 |
Recombinant DNA reagent | Caveolin-1 siRNA ON-TARGET Plus SMART pool | Dharmacon | Cat# L-003467-00-0020 | 100 nM |
Recombinant DNA reagent | ARHGAP29 Mission esiRNA | Sigma | Cat# EHU13457 | 100 nM |
Recombinant DNA reagent | FLUC Control Mission esiRNA | Sigma | Cat# EHUFLUC | 100 nM |
Recombinant DNA reagent | EHD2 For (HindIII) | Integrated DNA Technologies | CGCAAAGCTTCTATGTTCAGCTGGCTGAAGCGG | Forward primer for cloning of EHD2-A2E |
Recombinant DNA reagent | EHD2 Rev (EcoRI) | Integrated DNA Technologies | ATACGAATTCTCTCGGCGGAGCCCTTGTGGCG | Reverse primer for cloning of EHD2-A2E |
Recombinant DNA reagent | CCN1 (NM_001554.5) | Integrated DNA Technologies | For: TGAAGCGGCTCCCTGTTTTT Rev: TGAGCACTGGGACCATGAAG | primers for qPCR |
Recombinant DNA reagent | CCN2 (NM_001901.4) | Integrated DNA Technologies | For: CACCCGGGTTACCAATGACA Rev: GGATGCACTTTTTGCCCTTCTTA | primers for qPCR |
Recombinant DNA reagent | ANKRD1 (NM_014391.3) | Integrated DNA Technologies | For: TAGCGCCCGAGATAAGTTGC Rev: GTCTGCCTCACAGGCGATAA | primers for qPCR |
Recombinant DNA reagent | YAP (NM_001130145.3) | Integrated DNA Technologies | For: ACTCGGCTTCAGGTCCTCTT Rev: GGTTCATGGCAAAACGAGGG | primers for qPCR |
Recombinant DNA reagent | ARHGAP29 (NM_001328664.2) | Integrated DNA Technologies | For: ACATCTAAAGCGGGTAGTAG Rev: AAGGGAGGAGATGGTGATAG | primers for qPCR |
Recombinant DNA reagent | CAV1 (NM_001753.5) | Integrated DNA Technologies | For: ACGTAGACTCGGAGGGACA Rev: TCGTACACTTGCTTCTCGCT | primers for qPCR |
Recombinant DNA reagent | GAPDH (NM_002046.7) | Integrated DNA Technologies | For: TCGGAGTCAACGGATTTGGT Rev: TGAAGGGGTCATTGATGGCA | primers for qPCR |
Software, algorithm | ImageJ/FIJI | NA | https://Imagej.nih.gov/ij RRID:SCR_002285 | |
Software, algorithm | MaxQuant (version 1.6.7.0) | NA | https://www.maxquant.org/ RRID:SCR_014485 | |
Software, algorithm | UniProt | NA | https://www.uniprot.org/ RRID:SCR_002380 | |
Software, algorithm | ProTIGY (version 1.1.7) | Broad Institute, Proteomics Platform | https://github.com/broadinstitute/protigy | |
Software, algorithm | Cytoscape | NA | https://cytoscape.org RRID:SCR_003032 | |
Software, algorithm | BioGRID | NA | https://thebiogrid.org RRID:SCR_007393 | |
Software, algorithm | STRING | NA | https://string-db.org RRID:SCR_005223 | |
Software, algorithm | Inkscape | NA | https://inkscape.org/ RRID:SCR_014479 | |
Software, algorithm | R (version 4.0.3) | NA | https://www.r-project.org/ RRID:SCR_001905 |
Mass spectrometry data.
Literature and database entries for the Cav1 interactome analysis.