FSK7 is a mouse MEC strain isolated from luminal epithelial cells and was used at a low passage number (Kittrell et al., 1992
). FSK7 cells were cultured in DF12 medium (BioWhittaker; Lonza) supplemented with 5 ng/ml EGF, 880 nM insulin, and 2% FCS at 37°C in a humidified atmosphere of 5% CO2
. Primary MECs were extracted from 19-d pregnant mice and cultured in growth medium containing 5 µg/ml insulin, 1 µg/ml hydrocortisone, 3 ng/ml EGF, 10% FCS, 50 U/ml penicillin/streptomycin, 0.25 µg/ml fungizone, and 50 µg/ml gentamycin in F12 medium (Pullan and Streuli, 1996
). For growing lentivirus, 293T was cultured in DME supplemented with 10% FCS.
shRNA for talin1 was designed with siRNA Target Finder (Invitrogen), and the sequences used were shTlnA, 5′-AAGAAGCACAGAGCCGATTGA-3′, and shTlnB, 5′-AAGAACAAGATGGATGAATCA-3′. The scrambled shRNA sequence for talin1 contained the same nucleotides as those for shTlnA, 5′-GACAGATGAGGAGCAATCACA-3′, and this sequence did not reveal any known targets by a BLAST (basic local alignment search tool) search. The shRNA sequence for p21 was 5′-TTAGGACTCAACCGTAATA-3′. The shRNAmir for β1 integrin was 5′-GGCTCTCAAACTATAAAGAAA-3′ (provided by P. Stern, Massachusetts Institute of Technology, Cambridge, MA).
Doubled-stranded oligonucleotides were cloned into the shRNA transfer vectors pSilencer 3.1 (Invitrogen) or pLVTHM (Tronolab). pLVTHM was used in most of the experiments and was used to generate lentiviruses. In a few experiments, the same sequences in pSilencer, which does not also express GFP, were used in transfection experiments, and the empty vector was used as a control (; and , pSi). The extent of knockdown with pSilencer is the same as for pLVTHM. In the case of sh–β1 integrin-mir, the transfer vector was pLB2Cap2Gm (pLB2).
For rescue vectors, human full-length wild-type talin1 cDNA was obtained from Source BioScience LifeSciences. To avoid deletion by shTln1, specific mutations were created by using site-directed PCR and mutagenic oligonucleotides (QuikChange; Agilent Technologies). The shRNA target sequence for mouse talin1 is nucleotides 3,707–3,728, which contains two variations from the corresponding human talin1 sequence (A → T and A → G), and additional G → A and C → T mutations were placed in the human talin1 sequence (no amino acid change). The plasmids containing shTln1-resistant human talin1 or FAK constructs were cotransfected with pLVTHM-shTln1 using Lipofectamine plus (Invitrogen).
Full-length talin (aa 1–7,623), head domain (aa 1–1,305), and rod domain (aa 1,305–7,623) as well as HDVBS (aa 1–5,925) and talinC (aa 5,922–7,623) were cloned into pcDNA6/V5-His(A) (Invitrogen). Additional talinC mutants described in this paper were created by using site-directed PCR and mutagenic oligonucleotides.
wt-myrFAK or an autophosphorylation-site Y397F mutant form of FAK (mu-myrFAK) was generated by cloning the viral Src myristoylation sequence N terminal to the FAK coding sequence in pcDNA6/V5-His(A) and provided by A. Gilmore (University of Manchester, Manchester, England, UK). Full-length p21 cDNA (Thermo Fisher Scientific) was generated in pCMV-SPORT6.
The lentiviral shRNA system was provided by D. Trono (University of Geneva, Geneva, Switzerland). For lentivirus production, the transfer vectors were cotransfected with the envelope plasmid pMD2G and the packaging plasmid psPAX2 by the calcium phosphate method into 293T cells that had been precultured for 8–16 h. Media were replaced after 8–10 h. 10 ml viral supernatants was harvested 48–60 h after transfection, passed through a 0.45-µm filter, and further concentrated by centrifugation at 25,000 rpm at 4°C for 2.5 h. Viral pellets were resuspended in 0.1 ml fresh DF12 medium.
Lentiviral infection was performed by adding the appropriate amount of lentiviral particles directly to precultured 75% confluent cells. Dishes were spun at 3,000 rpm at 37°C for 20 min to increase the infection rate followed by incubation for 3 h. The infected cells were cultured for 48–72 h before being replated for further experiments. This protocol was necessary to ensure turnover of preexisting talin.
Depending on the batch of lentivirus, we normally obtained a 70–100% efficiency of infection in MECs. For some experiments, we FACS sorted the GFP+ cells to enrich to ~100% infected cells for the endpoint assays.
Cells were fixed with 4% formaldehyde for 10 min, permeabilized for 5 min with 0.5% Triton X-100 (Sigma-Aldrich), and subsequently incubated for 1 h with primary antibodies directed against the proteins indicated in this section followed by the appropriate conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). For actin fluorescence staining, Alexa Fluor 647–phalloidin (Invitrogen) was added to cells together with the secondary antibody. For integrin staining, cells were briefly treated with 0.5% CHAPS before fixing.
In the case of costaining for talin1 with the other monoclonal antibodies for vinculin, paxillin, ILK, and Py20, the antitalin antibody was preconjugated to Alexa Fluor 548 by using a monoclonal antibody-labeling kit (Invitrogen). Cells were incubated with monoclonal antibodies followed by a Cy5-conjugated donkey anti–mouse fragment antigen-binding region (Jackson ImmunoResearch Laboratories, Inc.), fully washed with PBS, and then the Alexa Fluor 548–antitalin antibody was added to stain talin1. Antibodies used for immunostaining were as follows: β1 integrin (Klinowska et al., 1999
); FAK (a gift from L. Romer, Johns Hopkins University School of Medicine, Baltimore, MD); 9EG7, paxillin, and py20 (BD); pFAK-Y397 (Invitrogen); V5 and GFP (Invitrogen); secondary mouse Cy2 and Rhodamine Red-X (Rhrx), rabbit Cy2 and Rhrx, and rat Rhrx (Jackson ImmunoResearch Laboratories, Inc.); talin and vinculin (Sigma-Aldrich); and ILK (Millipore).
After staining, coverslips were mounted with antifade reagent (ProLong; Invitrogen). Images were acquired on a restoration microscope (DeltaVision RT; Applied Precision) using either a 100×/1.40 Uplan S Apochromat or a 60×/1.42 Plan Apochromat objective and the Sedat filter set (89000; Chroma Technology Corp.). The images were collected using a camera (CoolSNAP HQ; Photometrics) with a z optical spacing of 0.2 µm. Raw images were then deconvolved using the softWoRx software (Applied Precision), and maximum intensity projections of these deconvolved images are shown in the Results and followed by analysis with ImageJ 3.4 (National Institutes of Health) or Openlab 4 software (PerkinElmer).
Equivalent amounts of protein were separated by SDS-PAGE and immunoblotted for proteins. Detection was achieved with peroxidase-conjugated anti–rabbit, anti–goat, and anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) followed by enhanced chemiluminescence (GE Healthcare). Antibodies different to those used in immunostaining were as follows: talin1- and talin2-specific antibody (a gift from D.R. Critchley, University of Leicester, Leicester, England, UK); FAK (a gift from A. Ziemiecki, University of Bern, Bern, Switzerland); p21 (BD); pY31-paxillin (Invitrogen); pT202/Y204-p42/44-MAPK, Akt, pS473-Akt, pY416-Src, and pY527-Src (Cell Signaling Technology); c-myc (Roche); cyclin A, cyclin D1, Cdk2, Erk2, Src, p16, and p27 (Santa Cruz Biotechnology, Inc.); and actin and p57 (Sigma-Aldrich).
Cell adhesion experiments were performed as previously described (Edwards and Streuli, 1999
). In brief, wells of 96-well plates were precoated with ECM proteins as indicated 4°C overnight. After blocking with 0.5% bovine serum albumin, 105
cells were added into wells in triplicate and incubated for 2 h at 37°C. Wells were washed with PBS, and the adherent cells were quantified by labeling with 5 mg/ml Crystal violet (Sigma-Aldrich) and reading the absorbance at 595 nm.
Replated cells were cultured for 16 h and then labeled with either 10 µM BrdU or EdU for 2 h before fixation. Cells were stained with either anti-BrdU antibodies or with the EdU Alexa Fluor 647 imaging kit (Click-iT; Invitrogen). BrdU/EdU staining was quantified within the GFP-positive, infected/transfected cells. 100–200 cells were counted per experimental point. Error bars are SEM. Statistical data were obtained using Student’s t test.
For the spreading on micropatterned substrata, coverslips were provided by C. Chen, M. Wozniak, and G. Lin (University of Pennsylvania, Philadelphia, PA). The pattern was etched into a polydimethylsiloxane surface, and the 25 × 25–µm adhesive areas were coated with FN. 105 lentiviral infected cells were plated into 35-mm dishes containing micropatterned coverslips for 16 h to allow cell spreading and then fixed after EdU staining as in the previous paragraph.
Online supplemental material
Fig. S1 shows control experiments. Fig. S2 shows that talin1 depletion causes a severe attachment deficiency in fibroblasts. Fig. S3 shows that tensin may provide a link between adhesion complexes and F-actin in talin1-deficient cells. Fig S4 shows staining for total FAK. Fig S5 shows that Src remains phosphorylated in talin-depleted MECs. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201104128/DC1