Visualizing the dynamic coupling of claudin strands to the actin cytoskeleton through ZO-1.

Cell culture and transfections Rat 1 Tet-off (R1-R12) cells were obtained from the American Type Culture Collection and maintained at 37°C and 5% CO 2 in high-glucose DMEM (4.5 g/l), supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (pen-strep). Stable cell lines were made by cotransfecting tagged constructs with pSVZeo (Thermo Fisher) using Lipofectamine 2000 according to the manufacturer’s directions; cells were selected with 0.8 mg/ml Zeocin (Invivogen) for 14 d, and clonal lines were isolated and analyzed by immunoblot and immunofluorescence. Stable knockout cell lines were made by transfecting pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene 62988; Ran et al. , 2013 ) using Lipofectamine 2000. Individual clones were isolated after initial selection for 48 h in 2 µg/ml puromycin (Life Technologies), followed by dilution cloning to single cells into 96-well plates. Clonal lines were expanded and tested 2–3 wk after transfection by immunoblot.

HEK293 cells

(Tet-off advanced; Takara) were maintained in high-glucose DMEM supplemented with 10% Tet-qualified FBS (Atlanta Biologicals) and pen-strep. FuGENE transfection reagent (Promega) was used for transfection of GFP- and myc-tagged constructs according to the manufacturer’s instructions; cells were collected 48 h after transfection and samples processed for immunoprecipitation experiments as described later. In some experiments, cells were pretreated for 2 h before imaging with the Rho kinase inhibitor Y27632 (30 μM; Sigma-Aldrich), the Arp2/3 inhibitor CK666 (100 μM; Sigma-Aldrich), or S-nitro-blebbistatin (100 μM, Cayman Chemical).

DNA constructs

Sequences for all oligonucleotide primers used for cloning are shown in Supplemental Table S1. GFP cldn2 was constructed by cloning mouse cldn2 ( Colegio et al. , 2003 ) into enhanced GFP (EGFP) C3 vector (Takara) using Eco RI/ Sal I. GFP cldn2(−3) was made from the GFP cldn2 using site-directed mutagenesis (Agilent Technologies) to introduce a stop after amino acid 227. GFP linker cldn2 was generated by site-directed mutagenesis (Agilent Technologies). GFP Linker cldn2 Y224E was made from this plasmid also using site-directed mutagenesis (Agilent Technologies). mCherry cldn2 was generated by excising EGFP from GFP cldn2 and replacing it with mCherry (Takara). SNAP-tagged cldn2 was made by infusion cloning after PCR of cldn2 into the Bam HI/ Xho I sites in pSNAPf (New England Biolabs). GFP ZO-1 ( Fanning et al. , 2012 ), myc-tagged ZNA ( Rodgers et al. , 2013 ), and GFP occludin ( Van Itallie et al. , 2015 ) have been previously described. Halo occludin was made by subcloning full-length occludin into pHalo C1 (kindly provided by John Hammer, National Institutes of Health, Bethesda, MD) into the Nhe I/ Sal I sites. GFP cldn1 and cldn11 were made by direct cloning from previously described pTRE constructs ( Van Itallie et al. , 2003 ) into the Eco RI site of the EGFP N1 vector (Takara). TdTomato F-tractin was kindly provided by Robert Fischer (National Institutes of Health; York et al. , 2012 ). Stable ZO-1, ZO-2, and double-knockout clones were made using the CRISPR/Cas9 system ( Ran et al. , 2013 ); two separate vectors targeting different exons were designed for both ZO-1 and ZO-2. Oligonucleotides were phosphorylated, annealed, and cloned into the Bbs I site of pSpCas9(BB)-2A-Puro vector according to the Zhang laboratory protocols (MIT). All constructs were verified by sequencing.

Antibodies

Mouse anti–ZO-1 (33-9100) and rabbit ZO-2 (38-9100) antibodies were from GE Healthcare, mouse Myc-Tag (9B11) antibody was from Cell Signaling Technology, rabbit anti-GFP (Ab290) and rabbit anti–N-cadherin (Ab76057) antibodies were from Abcam, and rabbit anti–β-catenin (C2206) antibody was from Sigma-Aldrich. Antibodies were validated by recognizing bands of the predicted size on immunoblots and by cellular immunolocalization where previously reported. Species-specific secondary antibodies for immunofluorescence (Cy2, Cy3, and Cy5 conjugated) and immunoblots (IR-labeled 680 and 790/800 antibodies) were from Jackson ImmunoResearch. Rhodamine phalloidin and Alexa Fluor 647 phalloidin were from GE Healthcare. Halo ocln was labeled using Janelia Fluor 646 ( Grimm et al. , 2015 ) at 1:200 in cell culture media plus serum, followed by four 10-min washes to allow free dye to diffuse out. Cells were otherwise fixed and stained normally.

Immunofluorescence microscopy

Rat-1 cells were cultured on uncoated glass coverslips, fixed with 1 or 4% paraformaldehyde in CSK buffer (10 mM 1,4-piperazinediethanesulfonic acid, pH 6.8, 100 mM KCl, 300 mM sucrose, 2 mM MgCl 2 , and 2 mM ethylene glycol tetraacetic acid) at room temperature for 20 min, permeabilized with 0.2% Triton X-100 for 10 min, quenched with 50 mM NH 4 Cl, and incubated in 2% normal goat serum in phosphate-buffered saline (PBS) for 60 min and in primary antibodies for 60 min. After washing, samples were incubated with fluorescent-labeled secondary antibodies; in some cases, rhodamine or Alexa Fluor 647–phalloidin was added with the secondary antibodies. In some cases, cells were fixed with 100% cold ethanol, washed twice with Dulbecco’s PBS, and blocked and incubated in primary and secondary antibodies as described. After washing, samples were mounted with Mowiol containing 1% n -propyl gallate. Fixed samples were imaged on a Zeiss 710 2P Confocal microscope, using either 20×/numerical aperture (NA) 0.8 air or 63×/NA 1.4 oil objectives with 488-, 561-, and 633-nm laser lines or a Zeiss LSM 880 Airyscan in superresolution mode with a 63×/NA 1.4 objective. Raw data were processed using Airyscan processing with “auto strength” (mean strength ± SD = 5.5 ± 1.3) with Zen Black software, version 2.3 Superresolution images were taken using a GE OMX Blaze V4 Ultrafast Structured Illumination Microscope equipped with four scientific complementary metal-oxide semiconductor cameras using a 60×/1.42 NA lens using 488-, 561-, and 647-nm laser lines. Images were acquired using DeltaVision OMX software. For live-cell imaging, normal medium was supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4. Images were taken every 10–15 s for 10 min. Movies are maximum intensity projections of 0.8- to 1.2-μm stacks through the claudin strand patches. FRAP was performed on a Zeiss 780 confocal microscope using a 63x/NA 1.4 oil objective and heated stage in 5% CO 2 . Bleaching of GFP signal was done with the 405-nm laser line (100% power for