Endothelial CD2AP Binds the Receptor ICAM-1 To Control Mechanosignaling, Leukocyte Adhesion, and the Route of Leukocyte Diapedesis In Vitro This information is current as of September 30, 2021. Antje Schaefer, Trynette J. van Duijn, Jisca Majolee, Keith Burridge and Peter L. Hordijk J Immunol published online 8 May 2017 http://www.jimmunol.org/content/early/2017/05/06/jimmun ol.1601987 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 8, 2017, doi:10.4049/jimmunol.1601987 The Journal of Immunology

Endothelial CD2AP Binds the Receptor ICAM-1 To Control Mechanosignaling, Leukocyte Adhesion, and the Route of Leukocyte Diapedesis In Vitro

Antje Schaefer,*,†,1 Trynette J. van Duijn,* Jisca Majolee,*,2 Keith Burridge,†,‡ and Peter L. Hordijk*,x,1,2

Inflammation is driven by excessive transmigration (diapedesis) of leukocytes from the blood to the tissue across the endothelial cell monolayer that lines blood vessels. Leukocyte adhesion, crawling, and transmigration are regulated by clustering of the endothelial mechanosensitive receptor intercellular adhesion molecule-1 (ICAM-1). Whereas several proteins are known to promote ICAM-1 function, the molecular mechanisms that limit ICAM-1–mediated adhesion to prevent excessive leukocyte transmigration remain

unknown. We identify the endothelial actin-binding protein CD2-associated protein (CD2AP) as a novel interaction partner of Downloaded from ICAM-1. Loss of CD2AP stimulates the dynamics of ICAM-1 clustering, which facilitates the formation of ICAM-1 complexes on the endothelial cell surface. Consequently, neutrophil adhesion is increased, but crawling is decreased. In turn, this promotes the neutrophil preference for the transcellular over the paracellular transmigration route. Mechanistically, CD2AP is required for mechanosensitive ICAM-1 downstream signaling toward activation of the PI3K, and recruitment of F-actin and of the actin- branching protein cortactin. Moreover, CD2AP is necessary for ICAM-1–induced Rac1 recruitment and activation. Mechanical

force applied on ICAM-1 impairs CD2AP binding to ICAM-1, suggesting that a tension-induced negative feedback loop promotes http://www.jimmunol.org/ ICAM-1–mediated neutrophil crawling and paracellular transmigration. To our knowledge, these data show for the first time that the mechanoreceptor ICAM-1 is negatively regulated by an actin-binding adaptor protein, i.e., CD2AP, to allow a balanced and spatiotemporal control of its adhesive function. CD2AP is important in kidney dysfunction that is accompanied by inflammation. Our findings provide a mechanistic basis for the role of CD2AP in inflamed vessels, identifying this adaptor protein as a potential therapeutic target. The Journal of Immunology, 2017, 198: 000–000.

idney disease is often linked to vascular inflammation, TCR CD2 (3), E-cadherin–based cell contacts (4), and the endo- which results in major complications for patients. cytosis of the EGF and VEGF receptors (5, 6). Finally, CD2AP K However, the molecular mechanisms that connect both binds F-actin and actin-regulating proteins such as cortactin to by guest on September 30, 2021 diseases are largely unexplored. Focal segmental glomerulo- stimulate lamellipodia formation and cell migration (7, 8). sclerosis (FSGS) results in kidney failure and is characterized by A hallmark of inflammation is leukocyte transendothelial mi- impaired protein filtration at the podocyte cell-cell junction, the slit gration, also known as transmigration or diapedesis, from the diaphragm, located between foot processes in the kidney glo- circulation across endothelial cells lining the blood vessels (9–12). merulus (1). CD2-associated protein (CD2AP) is required for Upon capture, rolling, and arrest, leukocytes such as neutrophils formation of this junction as it connects the adhesion receptor adhere, spread, and crawl on the endothelial cell surface, in a Nephrin with the F-actin cytoskeleton. Mutations in the human process mediated by binding of the b2 integrins Mac1 (macro- CD2AP gene are associated with FSGS (2). Similar to FSGS phage 1 Ag; aMb2 integrin) and LFA1 (leukocyte function as- patients, heterozygous CD2AP mice develop glomerular lesions. sociated Ag 1; aLb2 integrin) to the endothelial receptor Intriguingly, increased leukocyte infiltration was also observed in intercellular adhesion molecule-1 (ICAM-1). Crawling leukocytes these lesions, but the molecular basis for this finding is unknown. probe the surface for permissive transmigration sites, guided by In addition to this function, CD2AP controls the clustering of the gradients in adhesion receptors, chemokines, and endothelial cell

*Department of Molecular Cell Biology, Sanquin Research and Landsteiner Labora- 026; to A.S. and P.L.H.), Sanquin Research Grant PPO-C 6-001 (to P.L.H. and T.J.v.D.), tory, Academic Medical Center, University of Amsterdam, Amsterdam 1066CX, the and National Institutes of Health Grants GM029860 and HL114388 (to K.B.). Netherlands; †Lineberger Comprehensive Cancer Center, University of North Caro- A.S. and P.L.H. conceived and designed the study; A.S., T.J.v.D., and J.M. performed lina at Chapel Hill, Chapel Hill, NC 27599; ‡Department of Cell Biology and Phys- experiments; A.S. and P.L.H. analyzed data; K.B. provided conceptual input and iology and McAllister Heart Institute, University of North Carolina at Chapel Hill, x analytic tools; and A.S. and P.L.H. wrote the paper. Chapel Hill, NC 27599; and Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam 1098XH, the Netherlands Address correspondence and reprint requests to Dr. Antje Schaefer, Lineberger Com- prehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, 1A.S. and P.L.H. have shared senior authorship. NC 27599. E-mail address: [email protected] 2Current address: Department of Physiology, VU University Medical Center, Univer- The online version of this article contains supplemental material. sity of Amsterdam, Amsterdam, the Netherlands. Abbreviations used in this article: CD2AP, CD2-associated protein; DIC, differential ORCIDs: 0000-0003-4305-4001 (T.J.v.D.); 0000-0003-4450-3948 (J.M.); 0000- interference contrast; FRAP, fluorescent recovery after photobleaching; FSGS, focal 0002-3348-078X (P.L.H.). segmental glomerulosclerosis; ICAM-1, intercellular adhesion molecule-1; LARG, Received for publication November 22, 2016. Accepted for publication April 12, leukemia-associated RhoGEF; PECAM-1, platelet endothelial cell adhesion molecule-1; 2017. Rac1, ras-related C3 botulinum toxin substrate 1; RT, room temperature; siRNA, small interfering RNA. This work was supported by a research fellowship from Sanquin Research and the Dutch Blood Supply Foundation (to A.S.), the Netherlands Organisation for Scien- Ó tific Research (ZonMW More Knowledge with Less Animals Project 40-42600-98- Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601987 2 CD2AP CONTROLS ICAM-1 FUNCTION stiffness. Finally, leukocytes transmigrate through endothelial cell- Expression vectors cell junctions (paracellular) or through the endothelial cell body Human CD2AP-GFP was a kind gift from Andrey Shaw (Washington (transcellular) toward the underlying tissue. University School of Medicine, St. Louis). N-CD2AP-GFP (aa 1–329) and ICAM-1 is a single-span transmembrane, mechanosensitive C-CD2AP-GFP (330–640) (4), ICAM-1-mCherry (23), and Lifeact-GFP receptor that responds to the pulling forces of adherent leukocytes (24) were previously described. Human ICAM-1-GFP was a kind gift from (13, 14). The adhesive function of ICAM-1 is strongly promoted Francisco Sanchez-Madrid, University of Madrid (Spain). GFP-PH (GFP- Akt-PH) was a gift from Tamas Balla [#51465; Addgene plasmid (25)]. by its clustering, induced by leukocyte adhesion. This clustering leads to ICAM-1 binding to various actin-binding proteins such as Abs a filamin B, -actinin-4, and cortactin. These link ICAM-1 to the The mouse monoclonal Abs directed against actin and a-Tubulin were from cortical F-actin cytoskeleton (15) forming functionally distinct Sigma, against cortactin (clone 4F11) from Millipore, against Rac1 from adhesive complexes (16). Deletion of the ICAM-1 cytoplasmic BD Transduction Laboratories, against CD2AP (B4) from Santa Cruz domain inhibits this ICAM-1–F-actin connection, the consequent Biotechnology, against FITC-labeled ICAM-1 (IF, FACS) from R&D downstream signaling, and leukocyte transmigration (17). In ad- Systems, and against GFP (JL8) from Clontech. The rabbit polyclonal Ab directed against filamin B was from Bethyl Laboratories, against ICAM-1 dition, ICAM-1 is regulated through inside-out signaling. Several (WB) from Santa Cruz Biotechnology, and against GAPDH (D16H11) endothelial proteins are required for efficient ICAM-1 clustering from Cell Signaling Technology. Hoechst-33258, phalloidin-Alexa-488, and function including filamin B and a-actinin-4 (16, 18), in ad- -Texas-Red, and secondary Alexa-labeled Abs were from Invitrogen. dition to RhoGTPase signaling driven by the GEFs Trio and Secondary HRP-conjugated Abs for Western blotting were from Dako. leukemia-associated RhoGEF (LARG) (13, 14, 19, 20). However, Western blot analysis much less is known about negative regulators in endothelial cells Downloaded from Samples were analyzed by SDS-PAGE and transferred to nitrocellulose that limit ICAM-1 clustering to prevent uncontrolled leukocyte membranes (Whatman) (16). After blotting, membranes were blocked in transmigration, in particular in the context of the force-dependent 10% low-fat milk in TBST for 30 min at room temperature (RT) and in- regulation of ICAM-1 function. cubated with primary Abs in 1% milk-TBST overnight at 4˚C. Next, Using biochemical binding assays, live-cell confocal micros- membranes were blocked with 10% milk-TBST for 20 min at RT, incu- copy, and mechanobiological studies, we identify endothelial bated with the secondary Abs in 1% milk-TBST for 1 h at RT, and washed in TBST for 1 h at RT. Blots were developed by ECL (Thermo Fisher CD2AP as a novel interaction partner of clustered ICAM-1. This Scientific). For quantification, integrated intensity was calculated and http://www.jimmunol.org/ interaction mediates ICAM-1 mechanosignaling toward, and drives corrected for background using ImageJ. recruitment of cortactin and F-actin to the adhesion complex. Pull-down assays Moreover, CD2AP binding to clustered ICAM-1 is required for efficient ras-related C3 botulinum toxin substrate 1 (Rac1) re- N-terminal biotinylated peptides encode the intracellular domain of human cruitment and activation. Conversely, Rac1 activity limits the ICAM-1 or VCAM-1 were synthesized using Fmoc-solid–phase chemistry (Netherlands Cancer Institute, Amsterdam). To analyze binding to these CD2AP-ICAM-1 interaction. Our data suggest that CD2AP acts as peptides, a TNF-a–stimulated HUVEC monolayer in a 10 cm dish was a negative regulator of ICAM-1 clustering, which limits the for- washed with PBS containing 1 mM CaCl2 and 0.5 mM MgCl2, and lysed mation of ICAM-1 adhesion complexes to prevent uncontrolled for 10 min on ice in NP40-buffer (25 mM Tris, 100 mM NaCl, 10 mM neutrophil adhesion and transcellular transmigration. Mechanical MgCl2, 10% glycerol, 1% NP40, pH 7.4), supplemented with a phospha- by guest on September 30, 2021 tension applied on clustered ICAM-1 impairs CD2AP binding, tase inhibitor mixture (Sigma) and protease inhibitor mixture tablets (Roche). After cell lysis and centrifugation (10,000 3 g, 10 min, 4˚C), indicating a force-dependent regulation of this adhesive complex. supernatant was incubated with Streptavidin agarose beads (Sigma) and 5 CD2AP may represent a therapeutically relevant, molecular link mg biotinylated peptide for 3 h at 4˚C under continuous mixing (16). between inflammation and kidney dysfunction. Empty beads were used as control. Beads were washed five times with NP40-buffer, resuspended in SDS-sample buffer. Proteins were detected using Western blotting. To analyze the binding of the CD2AP constructs to Materials and Methods the cytoplasmic ICAM-1 domain, HeLa cells were transfected with Cell culture, treatments, and transfections CD2AP-GFP, N-CD2AP-GFP, C-CD2AP-GFP, or GFP and pull-down studies with the N-terminal biotinylated peptide encode the intracellular Primary HUVECs (pooled from three donors) were purchased from Lonza domain of human ICAM-1 were performed as described above. and cultured on 10 mg/ml fibronectin (Sanquin)-coated dishes in EGM-2 medium, supplemented with SingleQuots (Lonza) at 37˚C and 5% CO2 Pull-out clustering experiments until passage 8. The HUVEC monolayer was stimulated with 10 ng/ml a Pull-out clustering experiments were performed using beads coated with TNF- (PeproTech EC) for 18 h prior to each experiment. If indicated, m TNF-a–stimulated HUVECs were treated with 5 mg/ml Cytochalasin B anti-ICAM-1 or IgG1 control (16). The 10 m magnetic goat anti-mouse (Sigma) for 30 min (DMSO as solvent) or with indicated concentrations of IgG1 Ab-coated Dynabeads (Invitrogen) were coated with mouse mAb NSC23766 (Calbiochem) for 30 min (H O as control) or EHT1846 anti-ICAM-1 (BBIG-l1; R&D Systems) or IgG1 control (Sanquin) 2 according to the manufacturers’ protocol. To induce clustering, 45 ml Ab- (Sigma) for 1 h (H2O as control). HUVECs were transfected with a vali- 3 5 m a dated pool of small interfering RNAs (siRNAs) against CD2AP [Santa coated beads (4 10 beads/ l) were incubated with TNF- –stimulated Cruz Biotechnology (4, 21)] and, as control, with siRNA against luciferase HUVEC monolayers (10 cm dish) for 25 min at 37˚C and 5% CO2. Cells (59-CGUACGCGGAAUACUUCGA-39; Eurogentec). Cells were trans- were washed with PBS containing 1 mM CaCl2 and 0.5 mM MgCl2, lysed fected with 1.3 nM siRNA, INTERFERin (Polyplus-transfection), and for 5 min on ice with radioimmunoprecipitation assay buffer (50 mM Tris OptiMEM (Invitrogen) according to the manufacturers’ recommendations, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 0.1% SDS, and and used for assays after 72 h. For overexpression of CD2AP-GFP, ICAM- 0.25% deoxycholic acid), and incubated for 1 h at 4˚C under continuous 1-GFP, ICAM-1-mCherry, Lifeact-GFP, or GFP, HUVECs were trans- mixing. Beads were isolated using a magnetic holder, washed twice with fected by electroporation (Neon Transfection System; Life Technologies) radioimmunoprecipitation assay buffer, three times with NP40 buffer, and according to the manufacturers’ recommendations. After electroporation, resuspended in SDS sample buffer. For the non-clustered condition, beads the viability of the cells was 50–80%. Cells were used for assays after 48 h. were added to the cells upon cell lysis, washed as described above, and resuspended in SDS sample buffer. Protein levels were analyzed using HL60 cells and HeLa cells were cultured at 37˚C and 5% CO2 in IMDM Medium (Sigma) containing 10% heat-inactivated FCS (Life Technologies), Western blotting. Quantified signals were normalized to the clustered 300 mg/ml glutamine, and 100 U/ml penicillin/streptomycin (PAA Cell ICAM-1 condition. Culture Company). Differentiation of the HL60 cells to a monocyte-like Rac1 activation assay upon ICAM-1 clustering phenotype was achieved by adding 1.3% DMSO in the medium for 3–5 d (22). HeLa cells were transfected with CD2AP-GFP, N-CD2AP-GFP, TNF-a–stimulated HUVEC monolayers (10 cm dish), transfected with si- C-CD2AP-GFP, or GFP using Trans IT-LTI (Mirus) to the manufacturer’s Control or si-CD2AP, were incubated with 45 ml beads coated (4 3 105 protocol and used for assays after 24 h. beads/ml) with anti-ICAM-1 Ab (see above) for the indicated times at 37˚C The Journal of Immunology 3

and 5% CO2 to induce ICAM-1 clustering. Cells were washed with PBS versus CD2AP-GFP) or 20 s (ICAM-1-GFP in knockdown studies) or 10.3 s containing 1 mM CaCl2 and 0.5 mM MgCl2 and lysed in 50 mM Tris pH (Lifeact-GFP) and with z-sections at three positions (2.5 mmintervals). 7.4, 500 mM NaCl, 0.5 mM MgCl2, 1% Triton X-100, 0.5% deoxycholic Fluorescence intensity was quantified in a donut-shaped area positioned at acid, 0.1% SDS supplemented with protease inhibitor mixture tablets the center of the bead and corrected for background, bleaching, and trans- (Roche). To measure Rac1-GTP levels, lysates were centrifuged (10,000 3 g, fection efficiency for each cell, time point, and confocal plane using 10 min, 4˚C) and supernatant was incubated with Streptavidin agarose Zen2009 and Zen2 software, ImageJ, andPrism5(GraphPad)(16).For beads (Sigma) and 30 mg biotinylated peptide encoding the CRIB domain normalization, the average of the saturated signal was calculated as 100% of the effector PAK1 (synthesized using Fmoc-solid–phase chemistry, and the time point of adding the beads as 0 s. Netherlands Cancer Institute, Amsterdam) for 30 min at 4˚C under con- tinuous mixing (19). Beads were washed four times in 50 mM Tris, Fluorescent recovery after photobleaching studies 150 mM NaCl, 0.5 mM MgCl , 1% Triton X-100, (pH 7.4) supplemented 2 Upon recruitment to anti-ICAM-1-Ab-coated beads, fluorescent recovery with protease inhibitor mixture tablets (Roche), resuspended in SDS after photobleaching (FRAP) assays were performed in the area of accu- sample buffer and analyzed using Western blotting. mulated fusion protein directly next to the bead. Experiments were per- Application of continuous force in pull-out clustering formed using 50 iterations with 488-nm laser illumination, at maximum power (25 mW) and a Zeiss LSM-510-META confocal laser scanning experiments microscope. Fluorescence recovery was measured by time lapse imaging for Tosyl-activated 4.5 mm Dynabeads (Thermo Fisher Scientific) were coated 80 s with intervals of 0.784 s. The signal was corrected for background and with mouse mAb anti-ICAM-1 (BBIG-l1; R&D Systems) in 0.1 M borate bleaching for each cell and time point. For normalization, the average of the buffer pH 9.5, according to the manufacturer’s protocol, overnight at RT saturated signal was calculated as 100%. under continuous mixing (13, 26). Next, beads were incubated with 0.1% Application of continuous force and PI3K activation fatty acid-free BSA (Sigma) for 2 h at RT under continuous mixing, and washed three times with PBS. To prepare BSA-coated beads, beads were HUVECs were seeded on a fibronectin-coated glass slides and transfected Downloaded from incubated with 0.1% fatty acid-free BSA instead of anti-ICAM-1 Ab. For with si-Control or si-CD2AP (see above). After 24 h, cells were transfected biochemical experiments, 55 ml of the coated beads (∼3 beads per cell) were with 3 mg of GFP-PH, which contains the PH domain of the Akt kinase and incubated with a TNF-a–stimulated HUVEC monolayer (10 cm dish) for serves as sensor for PI3 lipid binding and PI3K activation (27, 28) or the 15 min at 37˚C and 5% CO2 to induce ICAM-1 clustering. For the force GFP vector, using Lipofectamine2000 (Invitrogen) according the manu- condition, a continuous force (∼10 pN) was applied for 1 min to the beads facturer’s protocol. Cells were used for assays 48 h after transfection with using a permanent ceramic magnet (K&J Magnetics) placed 1 cm above and GFP constructs (72 h after adding siRNA) and stimulated with TNF-a 18 h parallel to the cells (13, 26). No significant changes in cell morphology, prior to experiments. Tosyl-activated 4.5 mm anti-ICAM-1-Ab-coated http://www.jimmunol.org/ movement of the nucleus, cell edges, and organelles were observed. Sub- beads (∼3 beads per cell) were incubated with TNF-a–stimulated sequently, cells were lysed and the pull-out experiment was carried out as HUVEC monolayers for 15 min at 37˚C and 5% CO2 to induce ICAM-1 described above. The clustered (no force) and the non-clustered conditions clustering. For the force condition, a continuous force (∼10 pN) was ap- (no force) using the same beads were also performed as described above. plied for 1 min to the beads using a permanent ceramic magnet (K&J Magnetics) placed 1 cm above and parallel to the cells. No significant Confocal laser scanning microscopy changes in cell morphology, movement of the nucleus, cell edges, and organelles were observed. Cells were fixed and images in seven random HUVECs were cultured on fibronectin-coated glass coverslips, washed, fields were captured on a Zeiss Axiovert2000M widefield microscope fixed with 3.7% formaldehyde (Sigma), immunostained, and mounted with (633/1.4 oil objective) and processed with ImageJ. Recruitment of GFP-PH Mowiol (Sigma) as described (16). or GFP was counted as positive if at least half of the bead was surrounded

Quantification of fluorescence intensities (F-actin). To determine F-actin by the GFP construct, based on the GFP fluorescence. The percentage of by guest on September 30, 2021 levels, TNF-a–treated HUVEC monolayers were fixed and stained with beads with a ring formation was calculated as (number of beads with ring phalloidin. Alternatively, HUVECs were transfected with Lifeact-GFP and formation)/total number of adherent beads 3 100. GFP levels were measured in live cells. Images were recorded in five random fields with a Zeiss LSM510-META confocal laser scanning mi- FACS analysis croscope (633/NA 1.4 oil objective). Using ImageJ software, mean fluo- rescence intensity of each cell was corrected for background and TNF-a–stimulated HUVEC monolayers were detached with Accutase transfection efficiency. (Sigma), stained with mouse mAb anti-ICAM-1-FITC (R&D Systems) or isotype IgG1 control (Sanquin), and analyzed by flow cytometry (FACS Leukocytes. Freshly isolated human neutrophils or differentiated HL60 cells CantoII; BD Biosciences) and FlowJo software (Treestar). were incubated for 10 min at 37˚C and 5% CO2 with TNF-a–stimulated HUVEC monolayers, transfected as indicated. Cells were washed, fixed, Neutrophil isolation and immunostained as indicated. Anti-ICAM-1-Ab-coated beads. To analyze protein recruitment to anti- Polymorphonuclear neutrophils were isolated from whole blood derived ICAM-1-Ab-coated beads, 10 mm polystyrene beads (Polysciences) were from healthy donors by 1:1 dilution with 10% trisodium citrate/PBS coated with mouse mAb anti-ICAM-1 (BBIG-l1; R&D Systems) using glu- (Sigma) and a Ficoll-Paque plus density gradient (GE Healthcare) (16). taraldehyde according to the manufacturer’s protocol (16). Beads were in- After erythrocyte lysis in cold lysis buffer (155 mM NH4Cl, 10 mM cubated with a TNF-a–stimulated HUVEC monolayer for 15 min at 37˚C and KHCO3, 0.1 mM EDTA, pH 7.4), neutrophils were washed once with lysis 5% CO2. Cells were washed twice with PBS containing 1 mM CaCl2 and buffer, once with 10% trisodium citrate/PBS, and resuspended in HEPES 0.5 mM MgCl2 to remove unbound beads, fixed, and immunostained. Images buffer [20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM were recorded in six random fields with a Zeiss LSM510-META confocal K2HPO4, 1 mM CaCl2, 5 mM Glucose (Sigma) and 0.4% human serum laser scanning microscope (633/NA 1.4 oil objective) with z-sections at two albumin (Sanquin), pH 7.4]. Neutrophils were stored at RT for a maximal positions (1.5 mm interval). Protein recruitment was counted as positive if at 5 h upon isolation. For experiments, neutrophils were activated by incu- least half of the adherent bead at the apical plane was surrounded by the bation at 37˚C for 15 min. fluorescence signal of the immunostained protein. The percentage of beads with a ring formation was calculated as (number of beads with ring Neutrophil transmigration studies formation)/total number of adherent beads 3 100. Numbers were then nor- HUVECs cultured in a fibronectin-coated 10 cm dish were transfected with malized to the si-Control condition, which was set to 1. indicated siRNAs. Cells were trypsinized after 32 h and half of them seeded on a fibronectin-coated 30 mm glass coverslip (for studies under static Live-cell confocal imaging conditions) and the other half in a fibronectin-coated m-slide VI 0.4 (Ibidi, To study recruitment to anti-ICAM-1-Ab-coated beads, TNF-a–treated for flow studies). Cells were cultured for 2 d until confluency and stimu- HUVECs were transfected as indicated, cultured on fibronectin-coated lated with TNF-a 18 h prior to experiments. 30 mm glass coverslips (Thermo Fisher Scientific), and placed in a heat- Under static conditions. A total of 500,000 freshly isolated neutrophils were ing chamber at 37˚C and 5% CO2. GFP- and differential interference contrast added to TNF-a–treated HUVEC monolayers cultured on the glass cov- (DIC)–signals were monitored using a Zeiss LSM-510-META confocal laser erslip and placed in a heating chamber at 37˚C and 5% CO2. Live-cell scanning microscope (633/NA 1.4 oil objective). After 2 min 10 mm imaging under static conditions was performed for 25 min with intervals of polystyrene beads coated with anti-ICAM-1 Ab (see above) were added, and 10 s using DIC on a Zeiss LSM510-META confocal microscope (633/NA imaging was performed for up to 23 min with intervals of 24 s (ICAM1-GFP 1.4 oil objective). Image acquisition was performed with the Zen2009 4 CD2AP CONTROLS ICAM-1 FUNCTION software. Neutrophil spreading was determined after 7 min by calculating intracellular ICAM-1 domain, independent from its clustering, is the length:width ratio using ImageJ. To measure neutrophil adhesion, non- sufficient for CD2AP binding, we analyzed the association of adherent neutrophils were washed away with EGM-2 medium after 25 min CD2AP to a biotin-tagged peptide encoding the intracellular and cells were fixed. Adherent neutrophils were counted based on DIC images in five random fields. Numbers were normalized to the control ICAM-1 region. This largely unstructured region mediates down- condition, which was set to 1. stream signaling and contains only 28 residues, but no described Under physiological flow. In an Ibidi m-slide, 1 3 106 freshly isolated protein-binding motif (Fig. 1C) (14, 17). Confirming our results of neutrophils in HEPES medium were perfused over TNF-a–treated the clustering binding experiments, CD2AP bound to a peptide 2 HUVEC monolayers at 0.8 dyne/cm at 37˚C and 5% CO2 (16). After 3 encoding the intracellular ICAM-1 region (Supplemental Fig. 1A). min, the solution was replaced by HEPES medium. Transmigrated neu- trophils were distinguished from adherent neutrophils by their bright to CD2AP interacted, to a lesser extent, with the intracellular region of phase-dark transition in the phase contrast. Absolute numbers of trans- VCAM-1 (Supplemental Fig. 1A), whereas filamin B, as we showed migrated neutrophils were counted after 25 min upon neutrophil addition. previously, bound specifically to ICAM-1 (16, 18). Transmigration was counted as a transcellular event if opening and closing To determine which part of CD2AP binds to the cytoplasmic of the cell-cell junctions through which the full body of the neutrophil ICAM-1 region, in HeLa cells we expressed the GFP-fusion protein transmigrated was observed (403 objective and zoom). The time required for the transmigration step was defined as the time-point at which the of either full-length CD2AP, the N-terminal CD2AP region crawling neutrophil localizes at the position where it will transmigrate and (N-CD2AP), which contains the three SH3 domains, or the C-terminal thus rounds off again [reverse spreading (14)] until the time-point at which CD2AP region (C-CD2AP), which is required for F-actin binding (8) the entire body of the neutrophil is below the endothelial cell. To determine (Fig. 1D). Subsequently, we lysed the cells and analyzed the neutrophil adhesion, neutrophils were counted after 25 min upon addition, independent of their transmigration behavior. Numbers were normalized to binding of these proteins to the biotin-tagged peptide encoding the si-Control, which was set to 1. Neutrophil crawling time, speed, and dis- intracellular ICAM-1 region. Similar to full-length CD2AP-GFP, Downloaded from tance were quantified after 90 s upon neutrophil addition until they C-CD2AP-GFP, but not N-CD2AP-GFP or GFP, bound to ICAM-1 transmigrated, using the ImageJ plug-in manual tracking. The crawling (Fig. 1E). Of note, we observed in all conditions that filamin B time was defined as the time from adhesion until neutrophils round off and binding to ICAM-1 was not altered. localize at the position where they will transmigrate. Using confocal immunofluorescence microscopy, we found that Neutrophil adhesion at endothelial cell-cell borders in TNF-a–stimulated endothelial cells, CD2AP is partly localized

A total of 500,000 freshly isolated neutrophils were added to TNF-a–treated at clustered ICAM-1–positive adhesion sites, induced by adherent http://www.jimmunol.org/ HUVEC monolayers cultured on a 30 mm glass coverslip and placed in a human neutrophils (Fig. 1F) and monocytic HL60 cells heating chamber at 37˚C and 5% CO2. After 10 min incubation, non- (Supplemental Fig. 1B). Of note, we transfected HUVECs with adherent neutrophils were washed away with EGM-2 medium, cells CD2AP-GFP in these experiments as CD2AP is also expressed were fixed and stained with phalloidin (F-actin). Images with three z-stacks m endogenously in leukocytes. Furthermore, live-cell confocal im- (every 2.3 m) in 14 random fields were captured on a Zeiss LSM510- a META confocal microscope (633/NA 1.4 oil objective) and processed aging with anti-ICAM-1-Ab-coated beads and TNF- –stimulated with Zen2009 software and ImageJ. Adherent neutrophils were counted endothelial cells expressing CD2AP-GFP, ICAM-1-GFP, or GFP based on DIC images, endothelial cell-cell borders were defined by en- revealed that CD2AP-GFP, but not GFP, is recruited to anti- dogenous F-actin staining (phalloidin). An adherent neutrophil was defined ICAM-1-Ab-coated beads with similar kinetics as ICAM-1-GFP

as being located at the cell-cell border when at least half of the neutrophil by guest on September 30, 2021 was adherent above the cell-cell contacts. The percentage of adherent (Fig. 1G). Using this approach, we previously showed that neutrophils above endothelial cell-cell contacts was calculated by the a-actinin-4 is recruited as fast as ICAM-1 itself (16), whereas number of bound neutrophils above cell-cell border/number of all adherent recruitment of cortactin and filamin B requires significantly more neutrophils 3 100. time. These differences in recruitment kinetics underscore the adaptor-specific formation of ICAM-1–based adhesion complexes. Statistical analysis Moreover, these data show that CD2AP, like a-actinin-4, directly Mean values and SEs (SEM) of the indicated number of independent ex- coclusters with ICAM-1. Importantly, anti-ICAM-1-Ab-coated periments (n) were calculated. The p values were determined using an beads are an established in vitro mimicry of adherent leukocytes unpaired two-tailed Student t test (Prism5). A p value ,0.05 was con- sidered statistically significant. (16, 18, 30, 31), but the process of leukocyte diapedesis in which leukocytes arrest, spread, crawl, and transmigrate within 1–4 min is more complex and faster (9–12). Results In summary, these results show that CD2AP binds through its CD2AP interacts preferentially with clustered ICAM-1 C-terminal region to the intracellular domain of ICAM-1. ICAM-1 Previous studies showed that CD2AP regulates adhesion receptors clustering is necessary for efficient CD2AP association, but the in different cell types (3–6, 29). In this study, we tested whether intracellular ICAM-1 domain is sufficient for this interaction. CD2AP binds to the endothelial adhesion receptor ICAM-1. Furthermore, CD2AP localizes to ICAM-1 positive adhesion sites, ICAM-1 clustering on the cell surface is induced by adherent induced by adherent leukocytes or anti-ICAM-1-Ab-coated beads, leukocytes, but also by anti-ICAM-1-Ab-coated beads bound to in primary human TNF-a–stimulated endothelial cells. live endothelial cells (16, 18, 30, 31). We isolated the clustered ICAM-1 adhesion complex by adding anti-ICAM-1-Ab-coated Depletion of endothelial CD2AP increases neutrophil adhesion beads to activated primary HUVECs, which were stimulated and transcellular transmigration with the inflammatory cytokine TNF-a. Binding of beads to intact To determine the role of endothelial CD2AP in ICAM-1–mediated cells prior to cell lysis allows subsequent extraction (pull-out) of transmigration of neutrophils, we reduced the expression of clustered ICAM-1. Non-clustered ICAM-1 was isolated by adding CD2AP in TNF-a–stimulated human endothelial cells (Fig. 2A). the beads post cell lysis. Endogenous CD2AP, similar to actin, We used a validated pool of CD2AP-specific siRNAs (4, 21); bound preferentially to clustered ICAM-1 and to a lesser extent to however, we cannot completely exclude some off-target effects. non-clustered ICAM-1 (Fig. 1A). Of note, equal ICAM-1 quan- We next performed transmigration assays with primary human tities were isolated in the clustered and non-clustered condition neutrophils under physiological flow conditions. Through their b2 (Fig. 1A, 1B). CD2AP may constitutively associate with non- integrins, neutrophils adhere to the activated endothelial mono- clustered ICAM-1 as the CD2AP levels were low or below de- layer, which allows us to specifically study the signaling of the tection under these experimental conditions. To study whether the endothelial ligand ICAM-1. Unexpectedly, our data indicate that The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. CD2AP binds preferentially to clustered ICAM-1 induced by anti-ICAM-1-Ab-coated beads or adherent leukocytes. (A) ICAM-1 clustering was induced by adding anti-ICAM-1-Ab-coated beads to TNF-a–treated HUVECs. Non-clustered (nc) and clustered (c) ICAM-1 was extracted from cells, and binding of CD2AP and actin was assessed by Western blotting. Beads coated with irrelevant IgG1 Abs were included as control. Total (TCL) and bound proteins (pull-out) are shown in the left panels; quantification in the bar graphs (n = 3 independent experiments). (B) Efficiency of ICAM-1 extraction is not affected by Ab-mediated clustering. n = 3, Western blot in (A). (C) 3D model (16) of the human intracellular ICAM-1 region (28 residues) illustrates an a-helix close to the membrane followed by a large unstructured region. (D) Schematic overview of full-length CD2AP and its N- and C-terminal fragments. (E) GFP fusion proteins of CD2AP full-length and its N- and C-terminal constructs were transfected into HeLa cells and tested for their binding to the biotinylated peptide encoding the cytoplasmic region of ICAM-1 (ICAM-1 C-term). Representative Western blot is shown for cell lysates (CL) and ICAM-1 binding (ICAM-1 PD, pull-down; n = 3). GFP and filamin B were included as controls. (F) Representative confocal images of neutrophils (DIC) adhering to TNF-a–stimulated, CD2AP-GFP transfected HUVECs. Cells were fixed 10 min after neutrophil addition and immunostained for endogenous ICAM-1. CD2AP-GFP localizes at endothelial ICAM-1 positive adhesion sites (arrows), induced by adherent neutrophils (asterisks). Images represent one outof three experiments. (G) After adding anti-ICAM-1-Ab-coated beads (DIC) to TNF-a–stimulated HUVECs transfected with CD2AP-GFP, recruitment of CD2AP-GFP was observed by confocal live-cell imaging. Still images from representative time-lapse recordings are shown. Quantification (left panel) is shown for recruitment of CD2AP-GFP, ICAM-1-GFP, and GFP (beads added at t = 0 s). Bar graphs represent recruitment after 340 s (right panel). n =3, each independent experiment is an average of at least two cells with one or two GFP rings per cell. Scale bars, 10 mm. Data are mean 6 SEM. ***p , 0.001, Student t test. CC, coiled coil; ns, not significant; PRR, prorich region; SH3, SH3 domain; TM, transmembrane. 6 CD2AP CONTROLS ICAM-1 FUNCTION depletion of endothelial CD2AP significantly increased the num- neutrophils bound very close to or on endothelial cell-cell contacts bers of transmigrating neutrophils (Fig. 2B, Supplemental Video (Fig. 2G), as previously reported (34, 35). CD2AP silencing sig- 1) with around 70% of the neutrophils transmigrating via the nificantly reduced the number of adherent neutrophils at the en- transcellular pathway (through the endothelial cell body) and 30% dothelial cell-cell borders (Fig. 2G), which may contribute to their using the paracellular route (through cell-cell junctions) (Fig. 2C). preference in transcellular diapedesis. In line with these results, In contrast, neutrophils preferred to transmigrate using the para- CD2AP depletion reduced the crawling speed, the lateral migra- cellular pathway under control conditions (Fig. 2C) (14, 32, 33). tion distance, and the time crawling neutrophils take prior to Moreover, depletion of CD2AP reduced the time required for the transmigration (Fig. 2H–K, Supplemental Video 1). Taken to- transmigration step itself (Fig. 2D). We next analyzed neutrophil gether, these data show that endothelial CD2AP is a key regulator spreading, adhesion, and crawling, all of which precede the actual of neutrophil diapedesis. The results suggest that the increased step of diapedesis. Reduction of CD2AP expression impaired number of transmigrating neutrophils (Fig. 2B) is likely caused by neutrophil spreading (Fig. 2E), but promoted neutrophil adhesion the increase in their overall adhesion. To our knowledge, CD2AP as shown in live-cell imaging and fixed-cell studies (Fig. 2F, is the first ICAM-1– and actin-binding adaptor protein, the loss of Supplemental Fig. 1C). Under control conditions, the majority of which likely promotes transcellular diapedesis. Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. CD2AP depletion promotes neutrophil adhesion and transcellular diapedesis, but impairs neutrophil spreading and crawling. (A) Repre- sentative Western blot for siRNA-mediated CD2AP reduction in TNF-a–treated HUVECs (TCL, total cell lysate). Tubulin is shown as loading control. Bar graph shows quantification of the knockdown efficiency (n = 9). (B) Quantification of all transmigration events and (C) of paracellular and transcellular transmigration events of adherent neutrophils across TNF-a–stimulated HUVEC monolayers, transfected with control siRNA (si-Ctrl) or si-CD2AP, under physiological flow (n = 6). Absolute numbers of transmigrated neutrophils were counted 25 min after neutrophil addition. (D) Quantification of the time required for the transmigration step of neutrophils across TNF-a–stimulated HUVECs, transfected with indicated siRNA, under physiological flow (n =3, 20 neutrophils per group). (E) Neutrophil spreading across TNF-a–treated HUVECs, transfected with indicated siRNA, was observed by DIC. Stills are from a representative live-cell imaging and show magnifications of a typical neutrophil phenotype. Bar graph shows quantification of the spreading index calculated at 7 min after neutrophil addition (n = 3, 52–79 neutrophils per group). (F) Quantification of adherent neutrophils on TNF-a–stimulated HUVECs, transfected with indicated siRNA, under physiological flow, measured after 25 min as described in (B). (G) Quantification of neutrophils adherent at borders of single endothelial cells in a TNF-a–stimulated HUVEC monolayer, transfected with the indicated siRNAs (n = 5, 31–52 neutrophils for si- Ctrl, 49–152 neutrophils for si-CD2AP). After 10 min incubation, non-adherent neutrophils were washed away and cells were fixed. Adherent neutrophils were counted based on DIC, endothelial cell-cell borders were defined by endogenous F-actin staining (phalloidin, see figures below). (H) Quantification of crawling speed, (I) crawling distance, and (J) crawling time until transmigration of neutrophils across TNF-a–stimulated HUVECs, transfected with in- dicated siRNA, under physiological flow (n = 3, 20 neutrophils per group). (K) Graphical representation of neutrophil crawling tracks from (H)to(K). Scale bar, 10 mm. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. The Journal of Immunology 7

CD2AP is a negative regulator of ICAM-1 clustering dynamics clustering prior to fixation of the cells. Using 3D confocal micros- To study the molecular basis for increased neutrophil adhesion and copy, upon CD2AP depletion we observed a significant increase of transmigration upon CD2AP depletion, we examined ICAM-1 adherent beads that showed local accumulation of endogenous expression and dynamics in TNF-a–stimulated primary human ICAM-1 (Fig. 3C, 3D), suggesting increased numbers of ICAM-1 endothelial cells. SiRNA-mediated CD2AP depletion did not alter adhesion clusters. In contrast, the recruitment of F-actin to anti- total expression and surface expression of ICAM-1 (Fig. 3A, 3B). ICAM-1-Ab-coated beads was impaired (Fig. 3D, also see below). To investigate the dynamics of ICAM-1 clustering, CD2AP was In line with these results, live-cell confocal imaging with TNF-a– depleted in TNF-a–stimulated endothelial cells and anti-ICAM-1- stimulated endothelial cells demonstrated that ICAM-1-GFP is Ab-coated beads were added to intact cells to induce ICAM-1 recruited significantly faster to anti-ICAM-1-Ab-coated beads upon

FIGURE 3. CD2AP depletion in- creases ICAM-1 dynamics during clus- tering and in the clustered complex. (A) SiRNA-mediated CD2AP depletion in TNF-a–treated HUVECs did not change total expression of ICAM-1, assessed by

Western blotting (tubulin as loading Downloaded from control, representative blot from n =3) or (B) ICAM-1 surface expression, ana- lyzed by FACS (IgG1 control as isotype control, n = 2). (C and D) Anti-ICAM-1- Ab-coatedbeadswereaddedtoTNF- a–stimulated, si-CD2AP, or si-Control transfected HUVECs. Cells were fixed http://www.jimmunol.org/ after 15 min and immunostained for ICAM-1 and F-actin. Bar graphs (C) illustrate fraction of anti-ICAM-1- Ab-coated beads (-fold of control), which showed local ICAM-1 accumu- lation (n = 4, at least 50 beads per condition). (D) Representative 3D con- focal images show that ICAM-1, but not

F-actin, is recruited to the bead (DIC) by guest on September 30, 2021 after CD2AP depletion (F-actin quanti- fication is shown in Fig. 5A, 5B). (E) After adding anti-ICAM-1-Ab-coated beads (DIC) to TNF-a–stimulated HUVECs transfected with ICAM-1-GFP and indicated siRNA, ICAM-1-GFP re- cruitment was observed with confocal live-cell imaging. Images are stills of representative movies (Supplemental Videos 2, 3). Quantification (left panel) shows that ICAM-1-GFP is recruited more rapidly to anti-ICAM-1-Ab-coated beads upon CD2AP depletion (beads added at t = 0 s). Bar graphs (right panel) illustrate recruitment after 130 s; n =4, each experiment is an average of at least two cells with one or two GFP rings per cell. (F) Mobility of ICAM-1-GFP was analyzed by FRAP following recruit- ment to anti-ICAM-1 beads (see E). Still images show bleaching and recovery of ICAM-1-GFP after CD2AP depletion. Images for control condition in Supplemental Fig. 1D. Recovery curves (left panel) and bar graph (right) of the mobile fraction, based on the average GFP signal from 50 to 80 s following bleaching (n = 5) demonstrate higher ICAM-1 mobility upon CD2AP deple- tion. Scale bars, 10 mm. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. 8 CD2AP CONTROLS ICAM-1 FUNCTION

CD2AP depletion, indicating that CD2AP limits ICAM-1 dynamics skeleton in TNF-a–stimulated human endothelial cells using (Fig. 3E, Supplemental Videos 2, 3). Finally, using FRAP, we found Cytochalasin B inhibited the binding of CD2AP and actin to that the reduction of CD2AP expression significantly increased the clustered ICAM-1 (Supplemental Fig. 2A, 2B). The interaction of mobile fraction of clustered ICAM-1-GFP around anti-ICAM-1- filamin B was also reduced, as we described previously (16). Ab-coated beads (Fig. 3F, Supplemental Fig. 1D). Conversely, loss of CD2AP affected the overall organization of the These findings suggest that CD2AP is a negative regulator of F-actin cytoskeleton as deduced from the prominent reduction of ICAM-1 clustering, which limits ICAM-1 mobility in the adhesion F-actin cables in TNF-a–stimulated endothelial cells (Fig. 4A, complex. This, in turn, may prevent uncontrolled formation of 4B). Consequently, F-actin recruitment to clustered ICAM-1 was adhesion nanoclusters on the endothelial surface to balance ex- significantly impaired upon CD2AP depletion as observed in live- cessive leukocyte adhesion. cell confocal imaging with TNF-a–stimulated endothelial cells transfected with Lifeact-GFP and anti-ICAM-1-Ab-coated beads CD2AP is required for proper F-actin organization and (Fig. 4C, Supplemental Video 4). CD2AP depletion also impaired recruitment to ICAM-1 the recruitment of endogenous F-actin to clustered ICAM-1 as The endothelial F-actin cytoskeleton regulates ICAM-1 function indicated by the reduced number of anti-ICAM-1-Ab-coated beads and leukocyte adhesion (14, 15). Conversely, ICAM-1 clustering with local F-actin accumulation in confocal microscopy studies controls F-actin dynamics in endothelial cells, underscoring the (Figs. 5A, 5B, 3D) and by biochemical binding assays (Fig. 5C, bidirectionality of signaling in this adhesion complex. As CD2AP 5D, Supplemental Fig. 2C). binds to F-actin (8), we analyzed the role of the F-actin network in Taken together, these data show that endothelial F-actin dy- the ICAM-1-CD2AP-complex. Destabilizing the F-actin cyto- namics regulate CD2AP binding to clustered ICAM-1. Conversely, Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. CD2AP depletion reduces F-actin network formation and delays F-actin recruitment to ICAM-1. (A) Confocal images illustrate the dis- tribution of F-actin in TNF-a–treated HUVECs, transfected with si-Control or si-CD2AP. Cells were fixed and stained with phalloidin. Bar graphs show quantification of the fluorescence intensity based on phalloidin staining per cell (n = 5, 10 cells per group). (B) Live-cell confocal images of F-actin (Lifeact-GFP) distribution in TNF-a–stimulated HUVECs, transfected with si-Control or si-CD2AP. A HUVEC monolayer is shown in which most, but not all cells are transfected with Lifeact-GFP and thus not visible. Bar graphs represent Lifeact-GFP fluorescence intensity per cell (n = 4, 7 cells per group). (C) After adding anti-ICAM-1-Ab-coated beads (DIC) to TNF-a–stimulated HUVECs transfected with Lifeact-GFP and indicated siRNA, Lifeact-GFP re- cruitment was observed in confocal live-cell imaging studies. Images are stills from representative movies (Supplemental Video 4). Quantification (left panel) shows that CD2AP depletion impairs F-actin (Lifeact-GFP) recruitment to anti-ICAM-1-Ab-coated beads (beads added at t = 0 s). Bar graphs (right panel) illustrate recruitment after 600 s; n = 4, each experiment is an average of at least two cells with one or two GFP rings per cell. Scale bars, 10 mm. Data are mean 6 SEM. **p , 0.01, ***p , 0.001, Student t test. The Journal of Immunology 9

CD2AP is required for proper F-actin organization and recruitment Consistent with this result, in biochemical binding studies we to clustered ICAM-1 in TNF-a–stimulated human endothelial observed a reduced cortactin interaction with clustered ICAM-1 cells. upon CD2AP silencing (Fig. 5C, 5D, Supplemental Fig. 2C). Of note, binding of filamin B was not altered. These findings indicate CD2AP is necessary for cortactin binding to clustered ICAM-1 that cortactin binding to clustered ICAM-1 is, in part, dependent Cortactin interacts with CD2AP in podocytes, fibroblasts, and on CD2AP. cancer cells (6, 7), and with clustered ICAM-1 in endothelial cells (16, 36, 37). Therefore, we were interested in the role of cortactin CD2AP is required for Rac1 activation upon ICAM-1 in the ICAM-1-CD2AP pathway. Upon depletion of CD2AP in clustering TNF-a–stimulated endothelial cells, in confocal microscopy Upon leukocyte adhesion, ICAM-1 clustering induces activation of studies we found fewer anti-ICAM-1-Ab-coated beads that the RhoGTPase Rac1 (9, 38). Previously, we showed that in epi- showed local recruitment of endogenous cortactin (Fig. 5A, 5E), thelial cells Rac1 binds to the N-terminal part of CD2AP (4). suggesting impaired recruitment of cortactin to clustered ICAM-1. Whether CD2AP is involved in Rac1 signaling upon ICAM-1 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. CD2AP depletion impairs recruitment and binding of F-actin, cortactin, and Rac1 to ICAM-1. (A) Anti-ICAM-1-Ab-coated beads (blue) were added for 15 min to a monolayer of TNF-a–stimulated HUVECs transfected with indicated siRNA. Cells were fixed and immunostained as indicated for confocal imaging. (B) Bar graphs illustrate that CD2AP depletion reduces the number of anti-ICAM-1-Ab-coated beads (-fold of control) with local accumulation of F-actin (n = 5, at least 50 beads per group). (C) Western blots show that siRNA-mediated CD2AP depletion in TNF-a–treated HUVECs reduces binding (pull-out) of CD2AP, actin, cortactin, and Rac1, but not of filamin B to clustered ICAM-1, induced by anti-ICAM-1-Ab-coated beads. TCL, total cell lysate. (D) Quantification of the ICAM-1 binding (pull-out) for the indicated proteins from (C)(n = 3). Quantification of the extracted ICAM-1 levels in Supplemental Fig. 2C. (E) Quantification (bar graphs) of confocal imaging as explained in (A) illustrate that CD2AP depletion reduces the number of anti-ICAM-1-Ab-coated beads with local cortactin accumulation (n = 3). (F) Anti-ICAM-1-Ab-coated beads (DIC) were incubated for 15 min with a TNF-a–treated HUVEC monolayer transfected with indicated siRNA. Cells were fixed and immunostained for Rac1 and studied with 3D confocal imaging. Bar graphs (G) show that CD2AP depletion reduces the number of anti-ICAM-1-Ab-coated beads (-fold of control) with local Rac1 accumulation (n =4,at least 50 beads per group). Scale bars, 10 mm. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. ns, not significant. 10 CD2AP CONTROLS ICAM-1 FUNCTION clustering in endothelial cells is not yet known. In confocal analyzed. Treatment of NSC23766 significantly stimulated microscopy studies we observed that CD2AP depletion in TNF-a– CD2AP binding to ICAM-1 (Fig. 6B). In line with these data, stimulated endothelial cells reduced the number of anti-ICAM-1- increasing concentrations of EHT1846 also promoted, although to Ab-coated beads that showed local accumulation of endogenous a lesser extent, CD2AP association to ICAM-1 (Supplemental Rac1, indicative of impaired Rac1 recruitment to clustered ICAM-1 Fig. 2D). These results suggest that the loss of Rac1 activity (Fig. 5F, 5G). Similarly, biochemical interaction assays demon- triggers a compensatory pathway, increasing CD2AP binding to strate that silencing of CD2AP reduced Rac1 binding to ICAM-1, which in turn promotes Rac1 activation. Of note, F-actin clustered ICAM-1 (Fig. 5C, 5D, Supplemental Fig. 2C). dynamics as observed by the formation of F-actin stress fibers was To analyze whether CD2AP is important for efficient Rac1 impaired under these conditions, whereas the integrity of the en- activation induced by ICAM-1 clustering, we depleted CD2AP dothelial monolayer was not significantly affected (Fig. 6C). in TNF-a–stimulated endothelial cells, added anti-ICAM-1- In summary, our findings show that CD2AP is required for ef- Ab-coated beads to trigger ICAM-1 clustering, and isolated active ficient Rac1 recruitment to the clustered ICAM-1 complex and for Rac1 (Rac1-GTP). Under control conditions, ICAM-1 clustering Rac1 activation induced by clustered ICAM-1. Conversely, the led to Rac1 activation after 10 and 30 min following clustering CD2AP-ICAM-1 interaction is negatively regulated by Rac1 (Fig. 6A), in line with previous results (19). Importantly, depletion through a feedback mechanism that limits excessive local ICAM-1- of CD2AP significantly impaired ICAM-1–induced Rac1 activa- CD2AP-Rac1 signaling. tion (Fig. 6A). We next investigated if Rac1 may be part of a feedback loop, affecting CD2AP binding to ICAM-1. TNF-a– CD2AP is a force-sensitive protein that is required for ICAM- stimulated endothelial cells were treated with low concentrations 1–dependent mechanotransduction Downloaded from of the pharmacological inhibitors NSC23766 or EHT1846 to Recently, we showed that applying mechanical force on clustered block Rac1 activation. Subsequently, CD2AP binding to a biotin- ICAM-1 promotes the recruitment of the RhoGEF LARG, RhoA sig- tagged peptide encoding the intracellular ICAM-1 region was naling, and cellular stiffening to enhance neutrophil transmigration http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. CD2AP is involved in clustered ICAM-1–induced Rac1 signaling. (A) ICAM-1 clustering was induced by anti-ICAM-1-Ab-coated beads, which were added for the indicated times to TNF-a–treated HUVECs transfected with si-Control or si-CD2AP. Levels of active Rac1 (Rac1-GTP) were precipitated in pull-down assays with a biotinylated peptide encoding the GTPase binding domain of the CRIB effector protein Pak1 and assessed by Western blotting. Tubulin as loading control. Bar graphs (lower panel) show quantification of Rac1 binding (Rac1-GTP) to the CRIB peptide (n = 2). (B) TNF-a–stimulated HUVECs were treated with indicated concentrations of the Rac1 inhibitor NSC23766 and CD2AP binding to the biotinylated peptide encoding the intracellular ICAM-1 region was studied using Western blotting (CL, cell lysate; PD, pull-down). Bar graphs (lower panel) show quantification of CD2AP binding to ICAM-1 (pull-down, n = 3). (C) Confocal images of TNF-a–stimulated HUVECs treated with solvent (Ctrl) or 100 mM NSC23766 (NSC) or 12.5 mM EHT1846 (EHT) as Rac1 inhibitors and stained for F-actin (phalloidin). Scale bar, 10 mm. Data are mean 6 SEM. **p , 0.01, Student t test. CL, cell lysate; PD, pull-down. The Journal of Immunology 11

(13). To study whether CD2AP binding to ICAM-1 is dependent In addition to CD2AP, ICAM-1 clustering induces the recruit- on tension-induced force, we incubated TNF-a–stimulated endo- ment of several proteins including the actin-binding proteins fil- thelial cells with magnetic anti-ICAM-1-Ab-coated beads to in- amin B and a-actinin-4, the Src kinase, and the RhoGEF Trio (16, duce ICAM-1 clustering and applied a continuous force for 1 min 18, 19, 44). In contrast to CD2AP, these proteins are required for using a permanent magnet. The applied forces of 10 pN on ICAM-1 ICAM-1 clustering, indicating that clustering-induced protein re- are in the range of traction forces exerted by migrating leukocytes cruitment represents the first step in the formation of the ICAM-1 (39, 40). CD2AP bound preferentially to clustered ICAM-1 and, to complexes bringing proteins in close proximity, followed by a lesser extent, to the non-clustered form (Figs. 7A, 1A). Intrigu- maturation and positive or negative regulation of the adhesion ingly, applying mechanical force on clustered ICAM-1 reduced complexes. This way of regulation has also been described for significantly the binding of CD2AP to clustered ICAM-1 (Fig. 7A, integrin-based focal adhesions (45, 46), which are regulated by Supplemental Fig. 2E). We observed no significant CD2AP similar signaling pathways as ICAM-1 clusters (47). Consistent binding to beads that were coated with BSA, underlining the with our current findings, our previous work and that from other binding specificity of this assay. groups suggests that separate ICAM-1 complexes, characterized We next questioned whether CD2AP is required for mechano- by distinct interaction partners, spatial organization, and/or sub- sensitive downstream signaling of clustered ICAM-1. We focused cellular localization, exist to control leukocyte behavior during the on PI3K signaling because PI3K activation is a common mecha- different stages of transmigration (16, 30, 48). nosensitive response in endothelial cells that is induced by force on Similar to the mechanoreceptors PECAM-1 and JAM-A as well as adhesion receptors such as platelet endothelial cell adhesion integrins (26–28, 49, 50), ICAM-1 senses and transduces mechanical molecule-1 (PECAM-1), JAM-A, and integrins (27, 28). Previous forces into downstream signaling [this study and (13, 16)]. In line Downloaded from studies indicated a role of the PI3K pathway in leukocyte trans- with our observation that CD2AP limits ICAM-1 clustering, to our migration as well as in ICAM-1 and CD2AP signaling (41–43). knowledge we provide the first evidence that CD2AP binding to We transfected TNF-a–stimulated endothelial cells with Control- ICAM-1 is reduced by force applied to clustered ICAM-1, indicative siRNA or siRNA against CD2AP and a GFP-PH fusion protein, of mechanosensitive feedback. The application of force through which was previously established to serve as sensor for PI-3– magnetic Ab-coated beads mimics the pulling forces of adherent modified lipids and thus PI3K activation (27, 28). Subsequently, leukocytes, which promotes the formation of higher-order ICAM-1 http://www.jimmunol.org/ magnetic anti-ICAM-1-Ab-coated beads were added to live cells nanoclusters, increasing avidity and firm adhesion (13, 51). Mecha- to trigger ICAM-1 clustering and a constant force was applied for notransduction often implicates tension-dependent conformational 1 min. GFP-PH recruitment to anti-ICAM-1-Ab-coated beads was changes to induce force-sensitive signaling cascades, as described for a b analyzed using widefield microscopy. Under control conditions, 5 1 integrin and filamin A (52, 53). This is of particular interest for tension applied on anti-ICAM-1-Ab-coated beads significantly ICAM-1 as its short cytoplasmic region lacks any known protein- increased the number of anti-ICAM-1-Ab-coated beads that binding motif, but is required for downstream signaling (14, 17). showed local accumulation of the GFP-PH construct (Fig. 7B), Although many interaction partners are known, their binding inter- indicating that force imposed on clustered ICAM-1 induces PI3K faces in the intracellular ICAM-1 region are not well described. by guest on September 30, 2021 activation. We observed similar levels of force-induced PI3K Tension-induced force reduced CD2AP binding to the cytoplasmic activation as previously reported for PECAM-1, integrins, and ICAM-1 domain, which implies that under such conditions the JAM-A (27, 28). GFP-PH recruitment was specific for clustered CD2AP binding site in ICAM-1 will become accessible for other ICAM-1 and not due to perturbation of the membrane because proteins. Indeed, force on ICAM-1 also induces recruitment of pro- teins such as the RhoGEF LARG (13). This force-dependent binding GFP was not recruited (Fig. 7B). Depletion of CD2AP inhibited of interaction partners ensures a spatiotemporally separated forma- the force-induced GFP-PH recruitment, and thus PI3K activation tion of ICAM-1 complexes to precisely regulate leukocyte adhesion upon ICAM-1 clustering because only 15% of anti-ICAM-1- and transmigration, representing another regulatory mechanism for Ab-coated beads showed local accumulation of GFP-PH, similar protein-protein association during leukocyte-endothelial interactions. to the observation for the non-force control conditions. Our data suggest that adherent and crawling neutrophils exert control In summary, these findings show that CD2AP is a mechano- on the composition of the ICAM-1–based adhesion complexes sensitive transducer and that its binding to clustered ICAM-1 is through local, traction-mediated force on ICAM-1. negatively regulated by mechanical force. Tension on clustered Similar to its stimulation, limiting the ICAM-1 function needs ICAM-1 activates PI3K, and CD2AP is required for this force- to be regulated by specific proteins and their associated signal induced PI3K response, underscoring the key role of CD2AP in transduction pathways. Knowledge of negative regulation of such the mechanosensitive ICAM-1 downstream signaling. adhesion complexes is limited. Even for the well-studied integrins, inactivation mechanisms are only just beginning to emerge (54). Discussion CD2AP is required for the recruitment of F-actin, cortactin, and Combining high-resolution live-cell confocal imaging with bio- active Rac1 to the clustered ICAM-1 complex (Fig. 7C). Cortactin chemical and mechanobiological assays, we identify the actin- and Rac1 control efficient Arp2/3-dependent F-actin branching binding protein CD2AP as a novel interaction partner of the and polymerization (55, 56), indicating that CD2AP is required for endothelial mechanoreceptor ICAM-1. Our data indicate that cytoskeletal remodeling downstream of the ICAM-1 adhesion CD2AP performs at least three functions in the ICAM-1–based complex. Our data suggest that the F-actin network in the CD2AP- adhesion complex (Fig. 7C): 1) mediating recruitment of F-actin ICAM-1 complex may act as a brake, limiting ICAM-1 recruit- and cortactin as well as activation of Rac1; 2) limiting ICAM-1– ment to sites of leukocyte adhesion. This will need to be carefully mediated signaling in a force-dependent fashion and being a key balanced with the stabilizing function of the F-actin network for player in ICAM-1 mechanotransduction; and 3) determining the ICAM-1 clusters (14, 15, 47). In agreement with the model choice of transmigration route of human neutrophils across TNF- depicting functionally distinct ICAM-1 complexes (see above), a–stimulated primary human endothelial cells. To our knowledge, the scaffolding role of the F-actin cytoskeleton with its multiple CD2AP is the first ICAM-1–binding adaptor protein that combines binding partners may allow fine-tuning of the assembly and dis- these different functions to control leukocyte diapedesis. assembly of differential ICAM-1 complexes, similar to what has 12 CD2AP CONTROLS ICAM-1 FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 7. CD2AP is a force-sensitive protein and required for ICAM-1–dependent mechanotransduction. (A) TNF-a–stimulated HUVECs were in- cubated with magnetic anti-ICAM-1-Ab-coated beads for 15 min to induce clustering and force was applied for 1 min with a permanent magnet, followed by isolation of the clustered ICAM-1 complex. Beads coated with BSA were used as control. Western blots show total cell lysates (TCL) and CD2AP binding to ICAM-1 (pull-out). ICAM-1 levels were used as loading control for the pull-out with anti-ICAM-1-Ab-coated beads. nc, non-clustered; c, clustered condition; c + force, clustered condition with applied force. Bar graphs (lower panel) show quantification of the CD2AP binding (n = 3), demonstrating that force applied on clustered ICAM-1 impairs CD2AP interaction. Quantification of extracted ICAM-1 levels is shown in Supplemental Fig. 2E. (B) TNF-a–stimulated HUVECs, transfected with indicated siRNA and the PI3K activation sensor GFP-PH or the GFP vector, were incubated for 15 min with magnetic anti-ICAM-1-Ab-coated beads prior to being subjected to force (10 pN) for 1 min with a permanent magnet. Cells were fixed and GFP-PH recruitment to the beads was studied. Representative widefield images are shown, beads in phase contrast (PC). Quantification (lower panel) of percentage of beads with local GFP-PH accumulation shows that CD2AP depletion impairs force-induced recruitment of the PI3K activation sensor GFP- PH to anti-ICAM-1-Ab-coated beads (n = 2, 36–53 beads/group). (C) Schematic model depicting how CD2AP limits the adhesive function of the mechanoreceptor ICAM-1. Leukocyte adhesion induces ICAM-1 clustering, which promotes CD2AP binding. Applied mechanical force (e.g., by strong leukocyte binding) on ICAM-1 impairs CD2AP association indicating a tension-dependent negative feedback regulation to ensure a fast adaption of the adhesive ICAM-1 function. CD2AP is a negative modulator of ICAM-1 clustering, which limits the formation of ICAM-1 complexes to likely prevent uncontrolled leukocyte adhesion, reduced crawling and transcellular transmigration. Mechanistically, CD2AP recruits F-actin, cortactin, and active Rac1 to facilitate F-actin branching and polymerization at the adhesion complex. CD2AP binding to ICAM-1 is regulated by Rac1 activity in a negative feedback fashion. The F-actin network may function as a brake for ICAM-1 mobility and may selectively fine-tune the spatiotemporal assembly and disassembly of the adhesion complex. In parallel, CD2AP contributes to mechanosensitive ICAM-1–triggered PI3K activation, which may also signal to regulate cell-cell contacts. Scale bar, 10 mm. Data are mean 6 SEM. **p , 0.01, ***p , 0.001, Student t test. The Journal of Immunology 13 been suggested for focal adhesions (54). In parallel, CD2AP also 1998. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94: 667–677. contributes to mechanosensitive ICAM-1–triggered PI3K activa- 4. van Duijn, T. J., E. C. Anthony, P. J. Hensbergen, A. M. Deelder, and tion, which, through a junctional located pool of Rac1, may signal P. L. Hordijk. 2010. Rac1 recruits the adapter protein CMS/CD2AP to cell-cell to regulate cell-cell contacts (43). contacts. J. Biol. Chem. 285: 20137–20146. 5. Kobayashi, S., A. Sawano, Y. Nojima, M. Shibuya, and Y. Maru. 2004. The Generally, leukocytes prefer to transmigrate through endothelial c-Cbl/CD2AP complex regulates VEGF-induced endocytosis and degradation of cell junctions (paracellular) and only a minority of them cross the Flt-1 (VEGFR-1). 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The regulation of transendothelial migration: new knowl- dition, increased ICAM-1 surface expression, induced by different edge and new questions. Cardiovasc. Res. 107: 310–320. Downloaded from 12. Nourshargh, S., and R. Alon. 2014. Leukocyte migration into inflamed tissues. levels of TNF-a or through overexpression, impairs crawling, re- Immunity 41: 694–707. duces leukocyte adhesion at endothelial cell-cell contacts, and allows 13. Lessey-Morillon, E. C., L. D. Osborne, E. Monaghan-Benson, C. Guilluy, transcellular diapedesis (35, 57). Similarly, depletion of the RacGEF E. T. O’Brien, R. Superfine, and K. Burridge. 2014. The RhoA guanine nucle- otide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction Tiam1 or the small GTPase Rap1b in T lymphocytes, conditions that in endothelial cells to stimulate transendothelial migration. J. Immunol. 192: impair cell spreading, results in a shift of their preference toward the 3390–3398. 14. Schaefer, A., and P. L. Hordijk. 2015. Cell-stiffness-induced mechanosignaling - a transcellular pathway (61, 62). Our data indicate that CD2AP- http://www.jimmunol.org/ key driver of leukocyte transendothelial migration. J. Cell Sci. 128: 2221–2230. controlled clustering of ICAM-1 is also a driving factor as CD2AP 15. Schnoor, M. 2015. Endothelial actin-binding proteins and actin dynamics in depletion leads to faster ICAM-1 clustering, which facilitates for- leukocyte transendothelial migration. J. Immunol. 194: 3535–3541. 16. Schaefer, A., J. Te Riet, K. Ritz, M. Hoogenboezem, E. C. Anthony, F. P. Mul, mation of ICAM-1 complexes. Consequently, neutrophil adhesion is C. J. de Vries, M. J. Daemen, C. G. Figdor, J. D. van Buul, and P. L. Hordijk. increased, which appears to contribute to the increased number of 2014. Actin-binding proteins differentially regulate endothelial cell stiffness, transmigrating neutrophils. In line with previous studies (see above), ICAM-1 function and neutrophil transmigration. J. Cell Sci. 127: 4470–4482. 17. Lyck, R., Y. Reiss, N. Gerwin, J. Greenwood, P. Adamson, and B. Engelhardt. the number of adherent neutrophils at the endothelial cell-cell con- 2003. T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothe- tacts is reduced and neutrophil spreading as well as crawling are lium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for impaired, resulting in enhanced transcellular transmigration. These transendothelial migration of T cells. Blood 102: 3675–3683.

18. Kanters, E., J. van Rijssel, P. J. Hensbergen, D. Hondius, F. P. Mul, by guest on September 30, 2021 data suggest that both endothelial cells and leukocytes actively A. M. Deelder, A. Sonnenberg, J. D. van Buul, and P. L. Hordijk. 2008. Filamin control the route of transmigration. B mediates ICAM-1-driven leukocyte transendothelial migration. J. Biol. Chem. Excessive leukocyte transmigration and inflammation are driven 283: 31830–31839. 19. van Rijssel, J., J. Kroon, M. Hoogenboezem, F. P. van Alphen, R. J. de Jong, by aging and are often associated with chronic (inflammatory) E. Kostadinova, D. Geerts, P. L. Hordijk, and J. D. van Buul. 2012. The Rho-guanine disease including kidney dysfunction and arthritis or atheroscle- nucleotide exchange factor Trio controls leukocyte transendothelial migration by rosis, resulting in life-threatening complications. The molecular promoting docking structure formation. Mol. Biol. Cell 23: 2831–2844. 20. Schaefer, A., N. R. Reinhard, and P. L. Hordijk. 2014. Toward understanding mechanisms that link these events are not well understood. In this RhoGTPase specificity: structure, function and local activation. Small GTPases 5: 6. study, we show that the actin-binding protein CD2AP, which is 21. Monzo, P., N. C. Gauthier, F. Keslair, A. Loubat, C. M. Field, Y. Le Marchand- required for kidney function, limits ICAM-1–mediated leukocyte Brustel, and M. Cormont. 2005. Clues to CD2-associated protein involvement in cytokinesis. Mol. Biol. Cell 16: 2891–2902. transmigration. Elucidating the molecular mechanisms underlying 22. Back, A. L., K. A. Gollahon, and D. D. Hickstein. 1992. Regulation of ex- the role of CD2AP and similar key proteins in diapedesis may pression of the leukocyte integrin CD11a (LFA-1) molecule during differentia- open new possibilities for the development of therapeutics to treat tion of HL-60 cells along the monocyte/macrophage pathway. J. Immunol. 148: 710–714. chronic inflammatory disease. 23. van Buul, J. D., J. van Rijssel, F. P. van Alphen, M. Hoogenboezem, S. Tol, K. A. Hoeben, J. van Marle, E. P. Mul, and P. L. Hordijk. 2010. Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium. PLoS Acknowledgments One 5: e11336. We thank Prof. Andrey Shaw (Washington University School of Medicine, St. 24. Riedl, J., A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, Louis, MO) for CD2AP-GFP and Prof. Francisco Sanchez-Madrid (University F. Bradke, D. Jenne, T. A. Holak, Z. Werb, et al. 2008. Lifeact: a versatile marker of Madrid, Madrid, Spain) for ICAM-1-GFP. We also thank Erik Mul and the to visualize F-actin. Nat. Methods 5: 605–607. 25. Va´rnai, P., and T. Balla. 1998. Visualization of phosphoinositides that bind Central Facility of Sanquin Blood Supply (Amsterdam, the Netherlands) for pleckstrin homology domains: calcium- and agonist-induced dynamic changes help with the microscope and the flow cytometry. and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J. Cell Biol. 143: 501–510. 26. Guilluy, C., V. Swaminathan, R. Garcia-Mata, E. T. O’Brien, R. Superfine, and Disclosures K. Burridge. 2011. The Rho GEFs LARG and GEF-H1 regulate the mechanical The authors have no financial conflicts of interest. response to force on integrins. Nat. Cell Biol. 13: 722–727. 27. Scott, D. W., C. E. Tolbert, and K. Burridge. 2016. Tension on JAM-A activates RhoA via GEF-H1 and p115 RhoGEF. Mol. Biol. Cell 27: 1420–1430. 28. Collins, C., C. Guilluy, C. Welch, E. T. O’Brien, K. Hahn, R. Superfine, References K. Burridge, and E. Tzima. 2012. Localized tensional forces on PECAM-1 elicit 1. Scott, R. P., and S. E. Quaggin. 2015. Review series: the cell biology of renal a global mechanotransduction response via the integrin-RhoA pathway. Curr. filtration. J. Cell Biol. 209: 199–210. Biol. 22: 2087–2094. 2. Kim, J. M., H. Wu, G. Green, C. A. Winkler, J. B. Kopp, J. H. Miner, 29. Shih, N. Y., J. Li, V. Karpitskii, A. Nguyen, M. L. Dustin, O. Kanagawa, E. R. Unanue, and A. S. Shaw. 2003. CD2-associated protein haploinsufficiency J. H. Miner, and A. S. Shaw. 1999. Congenital nephrotic syndrome in mice is linked to glomerular disease susceptibility. Science 300: 1298–1300. lacking CD2-associated protein. Science 286: 312–315. 3. Dustin, M. L., M. W. Olszowy, A. D. Holdorf, J. Li, S. Bromley, N. Desai, 30. Barreiro, O., M. Zamai, M. Ya´n˜ez-Mo´, E. Tejera, P. Lo´pez-Romero, P. N. Monk, P. Widder, F. Rosenberger, P. A. van der Merwe, P. M. Allen, and A. S. Shaw. E. Gratton, V. R. Caiolfa, and F. Sa´nchez-Madrid. 2008. Endothelial adhesion 14 CD2AP CONTROLS ICAM-1 FUNCTION

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Endothelial CD2AP binds the receptor ICAM-1 to control mechanosignaling, leukocyte adhesion and the route of leukocyte diapedesis in vitro

Antje Schaefer, Trynette J. van Duijn, Jisca Majolee, Keith Burridge and Peter L. Hordijk

Supplemental Fig. 1. CD2AP is involved in ICAM-1-dependent leukocyte transmigration.

(A) Pull-down using biotinylated peptides encoding the intracellular regions of ICAM-1 and

VCAM-1 with cell lysates of TNF α-treated HUVECs. Binding of CD2AP and FilaminB was assessed by western blotting (left panel, CL, cell lysate; PD, Pull-Down). Empty beads (beads) were used as control. Western blots are in the left panel, quantification in the right panel (n=2).

(B) Representative confocal image of HL60 cells bound to TNF α-stimulated HUVECs transfected with CD2AP-GFP and ICAM-1-mCherry. Cells were fixed after 25 min. CD2AP-

1

GFP localizes at ICAM-1 positive adhesion sites, induced by adherent HL60 cells (asterisks).

Scale bar, 10 µm (n=2). (C) Human neutrophils were added to TNF α-treated HUVECs, transfected with the indicated siRNAs. After 25 min, non-adherent neutrophils were washed away and cells were fixed. Adherent neutrophils were counted based on DIC images, bar graphs shows quantification (n=3, 40-133 neutrophils for si-Ctrl, 101-306 neutrophils for si-CD2AP).

(D) Mobility of ICAM-1-GFP was analyzed by FRAP following the recruitment to anti-ICAM-

1-antibody-coated beads in TNF α-stimulated HUVECs. Still images show bleaching and recovery of ICAM-1-GFP after transfection with si-Control (see Fig. 3F). Scale bars, 10 µm.

Data are mean ± s.e.m., * P < 0.05; *** P < 0.001; Student’s t-test.

2

Supplemental Fig. 2. CD2AP interaction with Rac1 is regulated by F-actin dynamics and

Rac1 activity (A) TNF α-activated HUVECs were treated with CytochalasinB (CytoB), followed by isolation of the clustered ICAM-1 complex using anti-ICAM-1-antibody-coated beads.

Western blots (left panels) show total cell lysate (TCL) and association of indicated proteins

(Pull-out). Bar graphs (right panel) show quantification of CD2AP, actin and FilaminB binding to clustered ICAM-1 (n=3). (B) Bar graphs show equal levels of extracted ICAM-1 under the experimental conditions in A (n=3). (C) Quantification of extracted ICAM-1 upon pull-out with anti-ICAM-1-antibody-coated beads from TNF α-treated HUVECs, transfected with indicated siRNAs (Fig. 5C,D). (D) TNF α-stimulated HUVECs were treated with indicated concentrations

3 of the Rac1 inhibitor EHT1846. CD2AP binding to the biotinylated peptide encoding the intracellular ICAM-1 region was studied using western blotting (left panels, CL, cell lysate; PD,

Pull-down). Bar graphs (right panel) show quantification of CD2AP binding to ICAM-1 (Pull- down, n=3). Increasing EHT1846 concentrations stimulate CD2AP interaction with ICAM-1.

However, the data did not reach statistical significance due to a higher variation, but a similar trend was observed as for the NSC23766 inhibitor (Fig. 6B). Expression levels of CD2AP,

ICAM-1 and total Rac1 in the CL are not altered. (E) Quantification of extracted ICAM-1 levels upon pull-out with anti-ICAM-1-antibody-coated beads and applied force (see also Fig. 7A).

Data are mean ± s.e.m., *** P < 0.001, ns, not significant; Student’s t-test.

4

Endothelial CD2AP binds the receptor ICAM-1 to control mechanosignaling, leukocyte adhesion and the route of leukocyte diapedesis in vitro

Antje Schaefer, Trynette J. van Duijn, Jisca Majolee, Keith Burridge and Peter L. Hordijk

LEGENDS FOR SUPPLEMENTAL VIDEOS

Supplemental Video 1. Transmigration steps of human neutrophils under physiological flow conditions. Human neutrophils adhere, spread, crawl and transmigrate across a monolayer of TNF α-treated HUVECs transfected with si-Control (si-Ctrl, left side), whereas depletion of endothelial CD2AP (si-CD2AP, right side) impairs crawling and increases absolute numbers of adherent and transmigrated neutrophils. Transmigrated neutrophils were distinguished from adherent neutrophils by their bright to phase-dark transition, monitored using the phase-contrast of a widefield microscope. To limit the size of the file, videos show frames every 40 s for 25 min.

Supplemental Video 2. Recruitment of ICAM-1-GFP to anti-ICAM-1-antibody-coated beads under control conditions. TNF α-stimulated HUVEC monolayer was transfected with

ICAM-1-GFP and si-Control (si-Ctrl). Time-lapse video shows recruitment of ICAM-1-GFP to adherent beads coated with anti-ICAM-1 antibody, monitored on a confocal microscope. Still images of selected time points are in Fig. 3E. Numbers of frames were reduced to limit the size of the file.

Supplemental Video 3. Upon depletion of endothelial CD2AP, ICAM-1-GFP is faster recruited to anti-ICAM-1-antibody-coated beads. TNF α-stimulated HUVEC monolayer was

1 transfected with ICAM-1-GFP and si-CD2AP. Time-lapse video shows recruitment of ICAM-1-

GFP to adherent beads coated with anti-ICAM-1 antibody, monitored on a confocal microscope.

Still images of selected time points are in Fig. 3E. Numbers of frames were reduced to limit the size of the file.

Supplemental Video 4. Depletion of endothelial CD2AP impairs recruitment of Lifeact-

GFP to anti-ICAM-1-antibody-coated beads. TNF α-stimulated HUVEC monolayer was transfected with Lifeact-GFP and si-Control (si-Ctrl, left side) or si-CD2AP (right side). Time- lapse videos show recruitment of Lifeact-GFP to adherent beads coated with anti-ICAM-1 antibody, monitored on a confocal microscope. Frames were taken every 10.3 s. Still images of selected time points are in Fig. 4C.

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