Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells

  1. Eleanor Martin
  2. Rossana Girardello
  3. Gunnar Dittmar  Is a corresponding author
  4. Alexander Ludwig  Is a corresponding author
  1. School of Biological Sciences, Nanyang Technological University, Singapore
  2. NTU Institute of Structural Biology (NISB), Nanyang Technological University, Singapore
  3. Proteomics of Cellular Signaling, Luxembourg Institute of Health, Luxembourg
  4. Department of Life Sciences and Medicine, University of Luxembourg, Luxembourg
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 12 supplements
Dynamics of caveolae at the rear of migrating RPE1 cells.

(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 …

Figure 1—figure supplement 1
Characterisation of caveolae rear localisation in RPE1 cells.

(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 …

Figure 1—figure supplement 2
Caveolae become transiently aligned along the front/rear axis in migrating RPE1 cells.

(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 …

Figure 1—video 1
Time-lapse microscopy of a persistently migrating RPE1 cell stably expressing cavin1-APEX2-EGFP (related to Figure 1C).
Figure 1—video 2
Time-lapse microscopy of a persistently migrating RPE1 cell stably expressing cavin3-miniSOG-mCherry (related to Figure 1D).
Figure 1—video 3
Time-lapse microscopy of a persistently migrating RPE1 cell stably expressing cavin3-miniSOG-mCherry (related to Figure 1—figure supplement 1C).
Figure 1—video 4
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry persistently migrating in a 3D collagen matrix (related to Figure 1E).
Figure 1—video 5
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry.

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).

Figure 1—video 6
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry.

A sudden rear retraction can be seen between 235 and 250 min of the movie (related to Figure 1—figure supplement 1D).

Figure 1—video 7
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry undergoing a complete reversal of front-rear polarity.

The cell changes its front-rear axis between 140 and 200 min of the movie, which is accompanied by a reorganisation of cavin3.

Figure 1—video 8
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry undergoing cell division.

Abscission of the cytokinetic bridge can be observed at 175 min. Cavin3 rear localisation is evident and the cells start to migrate.

Figure 1—video 9
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry undergoing front-rear polarisation.

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

Figure 1—video 10
Time-lapse microscopy of an RPE1 cell stably expressing cavin3-miniSOG-mCherry.

Note that cavin3 is aligned in ‘stripes’ or ‘filaments’ as the cell migrates (related to Figure 1—figure supplement 2B).

Figure 2 with 1 supplement
Electron tomography of caveolae at the RPE1 cell rear.

(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 …

Figure 2—video 1
Electron tomogram and surface rendering of caveolar clusters and networks at the rear of RPE1 cells stably expressing cavin3-miniSOG-mCherry.

Caveolae were specifically labelled using miniSOG (related to Figure 2B).

Figure 3 with 2 supplements
Proximity labelling of caveolae in RPE1 cells stably expressing Cav1-APEX2-EGFP.

(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 …

Figure 3—figure supplement 1
Characterisation of the Cav1-A2E RPE1 cell lines.

(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. …

Figure 3—video 1
Time-lapse microscopy of a persistently migrating RPE1 cell stably expressing Cav1-APEX2-EGFP (related to Figure 3—figure supplement 1B).
Figure 4 with 1 supplement
The caveolin-1 interactome is enriched in cortical actin regulators and focal adhesion proteins and is highly sensitive to membrane tension.

(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 …

Figure 4—figure supplement 1
Quantitative proximity proteomics of Cav1-APEX2-EGFP in response to hypo-osmotic shock.

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 …

Figure 5 with 2 supplements
Proximity ligation assays (PLA) reveal dynamic interactions between caveolae and the cortical actin cytoskeleton during changes in membrane tension.

(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 …

Figure 5—figure supplement 1
Proximity ligation assays (PLA) in Cav1-A2E RPE1 cells using CTTN and FLNA antibodies.

(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 …

Figure 5—figure supplement 2
Proximity ligation assays (PLA) in Cav1-A2E RPE1 cells using ROCK1 and HSPB1 antibodies.

(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 …

Figure 6 with 5 supplements
Evidence for a functional connection between caveolae and ARHGAP29.

(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 …

Figure 6—source data 1

Original western blots shown in Figure 6A used for the quantification of data shown in Figure 6B.

https://cdn.elifesciences.org/articles/85601/elife-85601-fig6-data1-v2.zip
Figure 6—source data 2

Original pMLC western blots shown in Figure 6A used for the quantification of pMLC levels shown in Figure 6B.

https://cdn.elifesciences.org/articles/85601/elife-85601-fig6-data2-v2.zip
Figure 6—figure supplement 1
Knockdown of Cav1 in RPE1 cells does not affect the transcription of YAP or YAP target genes.

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.

Figure 6—figure supplement 2
Overexpression and siRNA-mediated knockdown of ARHGAP29.

(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 …

Figure 6—video 1
Time-lapse microscopy of RPE1 cells transfected with an NES-APEX2-EGFP plasmid.
Figure 6—video 2
Time-lapse microscopy of RPE1 cells transfected with an APEX2-EGFP-ARHGAP29 plasmid.
Figure 6—video 3
Time-lapse microscopy of RPE1 cells transfected with an APEX2-EGFP-ARHGAP29 plasmid.
Model of caveolae dynamics and function at the rear of migrating cells.

Low membrane tension promotes caveolae formation at the cell rear, whilst high membrane tension causes caveolae to flatten out, which is accompanied by the dissociation of cavin1/PTRF, EHD2, and …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (human)hTERT RPE-1ATCCATCC CRL-4000
RRID:CVCL_4388
Human retinal pigment epithelium
Cell line (human)hTERT RPE-1 Cavin1-APEX2-EGFPLudwig, 2020NAStable transfection
Cell line (human)hTERT RPE-1 Cavin3-miniSOG-mCherryLudwig et al., 2013NAStable transfection
Cell line (human)hTERT RPE-1
NES-A2E
This studyNAStable transfection
Cell line (human)hTERT RPE-1
Cav1-A2E
This studyNAStable transfection
Transfected construct (human)NES-A2ETan et al., 2020bNARecombinant DNA
Transfected construct (human)Cav1-A2ELudwig et al., 2016NARecombinant DNA
Chemical compound, drugDMEM:F12
1:1 Mixture
WestburgCat# LO BE04-687F/U1NA
Chemical compound, drugGeneticin
Selective Antibiotic (G418 Sulfate)
Fisher ScientificCat# 10131027500 µM
Peptide, recombinant proteinGibco
Fibronectin Bovine Protein, Plasma
Fisher ScientificCat# 330100181:200
OtherLipofectamine
3000 Reagent
Thermo Fisher ScientificCat# L3000015transfection reagent
OtherDharmafect 1Thermo Fisher ScientificCat# T-2001transfection reagent
Peptide, recombinant proteinStreptavidin
Alexa Fluor 568
Thermo Fisher ScientificCat# S11226
RRID:AB_2315774
IF: 1:1000
Peptide, recombinant proteinStreptavidin-HRPThermo Fisher ScientificCat# 434323
RRID:AB_2619743
WB: 1:10,000
Commercial assay or kitDuolink In Situ Orange Starter Kit Mouse/RabbitSigma-AldrichCat# DUO92102-1KTNA
Chemical compound, drugBiotin phenolIris BiotechCat# LS-35000.5 mM
Chemical compound, drugTroloxSigma-AldrichCat# 2388135 mM
Chemical compound, drugGlutaraldehyde (32%)Electron Microscopy Sciences (EMS)Cat# 162202%
Chemical compound, drugParaformaldehyde (32%)Electron Microscopy Sciences (EMS)Cat# 100504–8581–4%
Chemical compound, drugDiaminobenzidine (DAB) (Free-Base)Sigma-AldrichCat# D80010.5 mg/ml
Chemical compound, drugDurcupan
ACM resin
Electron Microscopy Sciences (EMS)Cat# 44610NA
Chemical compound, drugOsmium
tetroxide (2%)
Electron Microscopy Sciences (EMS)Cat# 191521–2%
Chemical compound, drugPotassium ferricyanideSigma-AldrichCat# 7025871–2% (w/v)
Peptide, recombinant proteinSequencing
Grade Modified Trypsin
Promega CorporationCat# V5111
Peptide, recombinant proteinLysyl
Endopeptidase, MS Grade
FUJIFILM Wako Pure Chemical CorporationCat# 125–05061
Peptide, recombinant proteinComplete,
EDTA-free Protease Inhibitor Cocktail
RocheCat# 11873580001
Peptide, recombinant proteinThermo Scientific Pierce
Streptavidin Magnetic Beads
Fisher ScientificCat# 10615204
Commercial assay or kitEZQ Protein Quantitation KitFisher ScientificCat# R33201
AntibodyRabbit monoclonal anti-PARG1 (ARHGAP29)InvitrogenCat# PA5-55336
RRID:AB_2645210
WB: 1:1000
AntibodyRabbit polyclonal anti-Caveolin-1 (CAV1)BD Bioscience/
Transduction labs
Cat# 610060
RRID:AB_397472
WB: 1:10,000
IF/PLA: 1:500
AntibodyMouse monoclonal anti-Caveolin-1 pY14BD Bioscience/
Transduction labs
Cat# 611339
RRID:AB_398863
WB: 1:1000
AntibodyRabbit polyclonal anti-Cavin-1 (PTRF)AbcamCat# ab48824
RRID:AB_882224
WB: 1:5000
IF/PLA: 1:500
AntibodyGoat polyclonal anti-EHD2AbcamCat# ab23935
RRID:AB_2097328
WB: 1:2000
AntibodyMouse monoclonal anti-Filamin 1 (E-3) (FLNA)Santa Cruz BiotechnologyCat# sc-17749
RRID:AB_627606
IF/PLA: 1:100
AntibodyMouse monoclonal anti-Cortactin (clone 4F11)Merck MilliporeCat# 05–180
RRID:AB_309647
IF/PLA: 1:100
AntibodyMouse monoclonal anti-Rock-1 (G-6)Santa Cruz BiotechnologyCat# sc-17794
RRID:AB_628223
IF/PLA: 1:100
AntibodyMouse monoclonal anti-HSP27 (F-4) (HSPB1)Santa Cruz BiotechnologyCat# sc-13132
RRID:AB_627755
IF/PLA: 1:100
AntibodyMouse monoclonal anti-GFPRocheCat# 11814460001
RRID:AB_390913
WB: 1:2000
AntibodyRabbit polyclonal anti-GFPAbcamCat# ab290
RRID:AB_2313768
IF/PLA: 1:100
AntibodyMouse monoclonal anti-YAPCell Signalling TechnologyCat# 14074
RRID:AB_2650491
WB: 1:1000
AntibodyMouse monoclonal anti-YAP pS127Cell Signalling TechnologyCat# 13008
RRID:AB_2650553
WB: 1:5000
AntibodyRabbit polyclonal anti-ppMLC2 (Thr18/Ser19)Cell Signalling TechnologyCat# 3674
RRID:AB_2147464
WB: 1:1000
AntibodyMouse anti-GAPDHSanta Cruz BiotechnologyCat# 47724
RRID:AB_627678
WB: 1:5000
AntibodyDonkey anti-Mouse IgG (H+L) Alexa Fluor 488InvitrogenCat# A-21202
RRID:AB_141607
IF: 1:500
AntibodyDonkey anti-Rabbit IgG (H+L) Alexa Fluor 488InvitrogenCat# A-21206
RRID:AB_2535792
IF: 1:500
AntibodyDonkey anti-Mouse IgG (H+L) Alexa Fluor 555InvitrogenCat# A-31570
RRID:AB_2536180
IF: 1:500
AntibodyDonkey anti-Rabbit IgG (H+L) Alexa Fluor 555InvitrogenCat# A-31572
RRID:AB_162543
IF: 1:500
AntibodyGoat anti-Rabbit IgG (H+L) Alexa Fluor 633InvitrogenCat# A-21071
RRID:AB_2535732
IF: 1:500
AntibodyGoat anti-Mouse IgG (H+L) Alexa Fluor 633InvitrogenCat# A-21052
RRID:AB_2535719
IF: 1:500
AntibodyGoat anti-rabbit IgG (H+L) HRP conjugateInvitrogenCat# A16104
RRID:AB_2534776
WB: 1:5000
AntibodyGoat anti-mouse IgG (H+L) HRP conjugateInvitrogenCat# A16072
RRID:AB_2534745
WB: 1:5000
Recombinant DNA reagentCaveolin-1 siRNA ON-TARGET Plus SMART poolDharmaconCat# L-003467-00-0020100 nM
Recombinant DNA reagentARHGAP29 Mission esiRNASigmaCat# EHU13457100 nM
Recombinant DNA reagentFLUC Control Mission esiRNASigmaCat# EHUFLUC100 nM
Recombinant DNA reagentEHD2 For
(HindIII)
Integrated DNA TechnologiesCGCAAAGCTTCTATGTTCAGCTGGCTGAAGCGGForward primer for cloning of EHD2-A2E
Recombinant DNA reagentEHD2 Rev
(EcoRI)
Integrated DNA TechnologiesATACGAATTCTCTCGGCGGAGCCCTTGTGGCGReverse primer for cloning of EHD2-A2E
Recombinant DNA reagentCCN1
(NM_001554.5)
Integrated DNA TechnologiesFor: TGAAGCGGCTCCCTGTTTTT
Rev: TGAGCACTGGGACCATGAAG
primers for qPCR
Recombinant DNA reagentCCN2
(NM_001901.4)
Integrated DNA TechnologiesFor: CACCCGGGTTACCAATGACA
Rev: GGATGCACTTTTTGCCCTTCTTA
primers for qPCR
Recombinant DNA reagentANKRD1
(NM_014391.3)
Integrated DNA TechnologiesFor: TAGCGCCCGAGATAAGTTGC
Rev: GTCTGCCTCACAGGCGATAA
primers for qPCR
Recombinant DNA reagentYAP
(NM_001130145.3)
Integrated DNA TechnologiesFor: ACTCGGCTTCAGGTCCTCTT
Rev: GGTTCATGGCAAAACGAGGG
primers for qPCR
Recombinant DNA reagentARHGAP29
(NM_001328664.2)
Integrated DNA TechnologiesFor: ACATCTAAAGCGGGTAGTAG
Rev: AAGGGAGGAGATGGTGATAG
primers for qPCR
Recombinant DNA reagentCAV1
(NM_001753.5)
Integrated DNA TechnologiesFor: ACGTAGACTCGGAGGGACA
Rev: TCGTACACTTGCTTCTCGCT
primers for qPCR
Recombinant DNA reagentGAPDH
(NM_002046.7)
Integrated DNA TechnologiesFor: TCGGAGTCAACGGATTTGGT
Rev: TGAAGGGGTCATTGATGGCA
primers for qPCR
Software, algorithmImageJ/FIJINAhttps://Imagej.nih.gov/ij
RRID:SCR_002285
Software, algorithmMaxQuant (version 1.6.7.0)NAhttps://www.maxquant.org/
RRID:SCR_014485
Software, algorithmUniProtNAhttps://www.uniprot.org/
RRID:SCR_002380
Software, algorithmProTIGY (version 1.1.7)Broad Institute, Proteomics Platformhttps://github.com/broadinstitute/protigy
Software, algorithmCytoscapeNAhttps://cytoscape.org
RRID:SCR_003032
Software, algorithmBioGRIDNAhttps://thebiogrid.org
RRID:SCR_007393
Software, algorithmSTRINGNAhttps://string-db.org
RRID:SCR_005223
Software, algorithmInkscapeNAhttps://inkscape.org/
RRID:SCR_014479
Software, algorithmR (version 4.0.3)NAhttps://www.r-project.org/
RRID:SCR_001905

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