Plasmids, molecular cloning and site directed mutagenesis.
For the construction of prey plasmids containing a set of different CD63 domains (the amino acid [AA] sequence length is given in the Supplementary Table 1), we used the vectors pPR3-N and pPR3-SUC (Dualsystems Biotech AG, Schlieren, Switzerland). The indicated primers (Supplementary Table 2) with variable Sfil restriction sites29 at the 5´ and 3´ regions for directional cloning were used to amplify CD63 sequence from the template vector pPR3-N-CD6329 by using 1 unit of PfuUltra HF polymerase (Agilent Technologies, Böblingen, Germany). The amplicons were digested with the SfiI restriction enzyme (New England Biolabs, Frankfurt, Germany) overnight at 50 °C. The digested and purified fragments were ligated with T4 DNA ligase (New England Biolabs, Frankfurt, Germany) into the SfiI digested vector pPR3-N or pPR3-SUC (Dualsystems Biotech AG). To generate bait plasmids pPR3-SUC-TM4LEL containing different C◊A point mutation sites within the LEL of CD63, we used site directed mutagenesis. PCR reaction of a total volume of 50 µl contained 5–50 ng of the plasmid pPR3-SUC-TM4LEL, 50–125 µM of each mutation introducing primer (Supplementary Table 2), 0.25 mM dNTP mix and 2.5 units of PfuUltra HF polymerase (Agilent Technologies, Böblingen, Germany). The PCR cycles were initiated at 94 °C for 2 min, followed by 18 amplification cycles. Each PCR cycle was performed at 94 °C for 0.5 min, and the annealing temperature was set 5 °C below the Tm of the primers for 0.5 min, 72 °C for 1 min/kb. The PCR cycles were finished with an extension step at 72 °C for 4 min. The PCR products were treated with 20 units of DpnI at 37 °C for 3 h. PCR reaction was then transformed into chemocompetent E. coli strain XL1-Blue (Stratagene, Heidelberg, Germany). The expression construct pCMV-CD63-mCherry was generated by using PfuUltra HF polymerase and the primers mCherry_for/mCherry_rev for amplification of the mCherry sequence from the vector template pmCherry-N1 (Clontech, Heidelberg, Deutschland). The amplified fragment and vector pCMV2B-CD63-FLAG40 were digested with XhoI and ApaI enzymes (New England Biolabs, Frankfurt, Germany), then purified and ligated with T4 DNA ligase (New England Biolabs). In the same way as described above, we introduced C◊A point mutation sites in the vector pCMV-CD63-mCherry (pCMV-CD63-mCherry) and generated the vector pCMV-CD63C145A,C146A-mCherry. The control vector pCMV-CD63ΔLEL-mCherry was generated by cloning parts of the CD63 sequence into the vector pCMVtag2B in two steps. First, the transmembrane domain (TM) 1–3 of CD63 was amplified from the template vector pPR3-N-CD6340 using the primers CD63_TM1-3_for/CD63_TM1-3_for (aa 1–110 GenBank accession no. KF998086) and introduced into the vector pCMV-Tag2B through the restriction sites BamHI/PstI; the sequence TM4 of CD63 (aa 201–238, GenBank accession no. KF998086) was amplified with the primers CD63_TM4_for/CD63_TM4_rev and introduced using the restriction sites EcoRV/HindIII. The mCherry reporter sequence was subcloned from the vector pCMV-CD63-mCherry and ligated using the restriction sites XhoI/ApaI in the vector pCMV-CD63ΔLEL. In the constructed vector pCMV-CD63ΔLEL, the mCherry tag was introduced by ligation of XhoI and ApaI digested mCherry fragment from the donor vector pCMV-CD63-mCherry. The vector pCMV-CD63C145A,C146A-YFP used for FRET experiments was generated by exchange of the mCherry sequence with the YFP sequence from the vector pCMV-CD63-YFP-FLAG40 using XhoI and ApaI restriction sites, all other vectors used were previously described40,56. All sequences of cloning sites were verified by Sanger sequencing.
Yeast-based split ubiquitin interaction assay.
All yeast experiments (yeast transformation, bait functionality control assay and yeast growth assay) were performed as previously described29. The strength of PPI was determined by yeast growth and quantification of β-galactosidase. For this, several yeast colonies transformed with bait and prey were collected and grown to an OD600 of 0.8–1, and all samples were diluted to an OD600 of 0.6; 10 µl were dropped on selective plates and incubated overnight at 30 °C. The chlorophenol-red β-d-galactopyranoside (CPRG) assay, in order to quantify the β-galactosidase gene activity, was done according the yeast protocols handbook published by Clontech.
Cells culture and cell transfections.
HEK293T cells were maintained in DMEM medium and Jurkat cells in RPMI medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in 5% CO2 humidified atmosphere. HEK293T cells were transfected with indicated plasmids using METAFECTENE Pro (Biontex, Munich, Germany) according to the manufacturer's instructions. Jurkat cells were transfected with the constructs HIV-1JR − FLGag-iGFP and pCMV-CD63-mCherry or pCMV-CD63C145A,C146A-mCherry using the Amaxa nucleofection system (Amaxa Biosystems) as previously described57.
Cell preparation for indirect immunofluorescence.
Transfected HEK293T cells were grown in 6 wells, the cell culture was removed, and cells were fixed in 2% paraformaldehyde (PFA), diluted in PBS for 20 min at room temperature, washed 2 times with PBS and detached by pipetting. The cell suspension was dropped on poly-L–lysine treated slides within a hydrophobic marked circle area created by a pap pen (Sigma Aldrich), dried and permeabilized with 0.2% saponin (Carl Roth, Karlsruhe, Germany) in PBS for 30 min and blocked in 3% BSA (Carl Roth) in PBS for 2 h. Cells were then incubated with primary goat anti-FLAG (NB600–344, NovusBio, Littleton, USA) and mouse anti-V5 (MCA1360, Serotec, Düsseldorf, Germany) antibodies. Bound primary antibodies were visualized by incubation with fluorescently conjugated secondary antibodies for green channel anti-goat Dylight®488 (Abcam, Cambridge, UK) or red channel anti-mouse Dylight®594 (Abcam). Transfected Jurkat cells were transferred from 6 wells into a reaction tube, washed one time and fixed in 2% PFA for 20 min. Further steps were proceeded, as for the HEK293T cells. Endogenous CD63 expression was detected by incubation with mouse anti-CD63 primary antibodies (1:1 mixture of MEM259, ab8219 and NK1/C3, ab1318, Abcam), and bound primary antibodies were visualized by incubation with anti-mouse Dylight®594 (Abcam). All immunostaining procedures were performed by using 5 µg/ml of primary and 1 µg/ml of secondary antibodies diluted in 5% BSA.
Proximity ligation assay (PLA).
Transfected HEK293T or Jurkat cells were prepared for PLA analysis as previously described40,56. We used the following antibodies for PLA experiments: mouse anti-V5 (MCA1360, Serotec), anti-FLAG (NB600–344, NovusBio) and anti-Na+, K+-ATPase (ab98787, Abcam). For direct PLA experiments on cells, we used the Duolink In Situ Probemaker PLUS and MINUS (Sigma Aldrich) to conjugate the antibodies, anti-2F5 (AIDS Reference and Reagent Program, National Institutes of Health, Bethesda, MD) and anti-CD63 (1:1 mixture of MEM259, ab8219 and NK1/C3, ab1318, Abcam). Jurkat cells were blocked with 5% BSA and 2% chicken albumin (Carl Roth) in PBS. The conjugated antibodies, anti-CD63 (PLA-PLUS) and anti-2F5 (PLA-MINUS), were incubated on cells at a 1:100 dilution in 5% BSA. Duolink in situ detection reagent red was used for all PLA reactions, except for Jurkat cells where detection reagent orange (Sigma Aldrich) was used. PLA samples were mounted with Duolink mounting medium (Eurogentec, Angers, France).
Confocal laser scanning microscopy (cLSM).
All images were acquired using a Zeiss LSM 780 confocal laser scanning inverted microscope (Carl Zeiss, Oberkochen, Germany) and a 63 × oil immersion objective. Fluorescence signals were detected by Zeiss ZEN smart setup instrument settings for 488 nm and 594 nm dyes. PLA images were obtained and quantification of PLA signals was performed as previously described40,56.
FRET experiments and FRET efficiency calculation.
HEK293T cells (1 × 105) were seeded in an IBIDI µ-slide 8 well glass bottom. After 24 h, cells were transfected with 1 µg plasmid DNA of each indicated FRET vector using MetafectenePro (Biontex, Munich, Germany) according to the manufacturer’s instructions, and cells were incubated 24 h before FRET images were obtained. ZEN smart setup instrument settings were done for 405 nm and 514 nm dyes (multitrack channel mode, linewise switch), and the ZEN bleaching option was configured. We defined bleached ROI (red frames), unbleached ROI (green frames) and bleached background ROI (blue frames). Before starting the bleaching procedure, three pre-bleaching and three post-bleaching images were recorded. The 514 nm laser line was set at 100% laser power intensity at the bleaching time point. We calculated FRET efficiency with Zeiss ZEN FRET implemented software to obtain values for each ROI.
Calculation of the recruiting factor ω.
The values of the recruiting factor ω were calculated from fluorescence intensity data by using the following Eq. (1):
Where I is the fluorescence intensity, r1 and r2 are intervals without cell-cell contact and r3 and r4 are intervals at the VS. FindGraph (UNIPHIZ Lab) software was used for calculation of the definite integrals.
Western blot analysis.
HEK293T cells or Jurkat cells were lysed in 100 µl 2 × Laemmli buffer, chromosomal cell DNA was degraded by addition of 25 U of benzonase (Novagen, San Diego, CA); 5 µL β-mercaptoethanol was added, boiled at 95 °C for 5 min and separated by SDS-PAGE using TGX gels (BioRad, Munich, Germany); and then transferred onto an Immobilon-P PVDF membrane (Millipore, Schwalbach, Germany). Expressed proteins were probed with 0.5 µg/ml mouse anti-CD63, 1 µg/mL goat anti-FLAG, mouse anti-V5, 2 µg/mL human anti-2F5, 1 µg/mL mouse anti-α1 Na+,K+-ATPase, and 1 µg/mL of mouse anti-β actin (AM00194PU-N, Acris, Herford, Germany). Primary antibodies were detected with 0.125 µg/mL HRP-conjugated anti-mouse IgG, anti-goat IgG or anti-human IgG (DAKO, Hamburg, Germany) and visualized with Pierce ECL Western blotting substrate (Thermo Scientific, Bonn, Germany). Western blot images were acquired using a Chemocam device (Intas, Göttingen, Germany). PVDF membranes were stripped for reprobing with Roti®-Free Stripping-Puffer 2.2 plus (Carl Roth).
Statistical analysis.
Results are expressed as mean ± standard deviation. A two-tailed, paired Student′s t-test was used to determine statistical significance (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001. A p-value < 0.05 was considered as significant between two data groups.