Supplementary Fig.1 Luton a 175 Brain Thymus Spleen Small Intestine Kidney Testis HeLa b 250 Lung Kidney MDCK c EFA6B si Control si Mismatch #637 #1564 #1770 83 62 47.5 175 IB: anti-efa6b #B1 130 66 Lysates IP: anti-efa6b SB Occludin Claudin 1 E-Cadherin ß-catenin 83 IB: anti-efa6b #B1 Arf6 62 47.5 IB: anti-efa6b #B2 Actin
1 1 Supplementary Fig.2 Luton a TER (ohm.cm 2 ) 50 30 10 0 EFA6A-E242K 1 2 3 4 +Dox - Dox sicontrol +Dox - Dox % Total TRITC-Dextran 50 30 10 Dox EFA6A-E242K Time (h) sicontrol TER (ohm.cm 2 ) 50 30 10 0 EFA6A- Cter 1 2 3 4 +Dox - Dox sicontrol +Dox - Dox % Total TRITC-Dextran 50 30 10 Dox EFA6A- Cter Time (h) sicontrol b A. U. Densitometry 1 1 80 0 EFA6B Occludin Tfn-R 0 2 4 6 8 10 Time (min) % of Total 100 80 0 0 80 100 1 Time (min) EFA6B Occludin Tfn-R c MG-132 IP vsvg GST GST-S5a GST-Eps15 0 116 IB: vsvg
Supplementary Fig.3 Luton a MDCK GFP-EFA6A GFP-PH ZO-1 b sicontrol siusp7 #1118 siusp9x #182 siusp9x #3432 0.5 hr USP9x GFP USP7 Actin ZO-1 1 hr GFP ZO-1 2 hr GFP c siusp7 NC LC 30 90 1 siusp9x NC LC 30 90 1 Afadin E-cadherin ß-catenin Occludin Claudin 1
Supplementary Fig.4 Luton USP9x a Phalloidin Merge USP9x GFP-EFA6A Merge h anti-n1 i b j anti-n1 + peptide c anti-n1 + sirna E-cadherin k d Abcam l e Abcam + sirna f anti-n1 + muusp9x g Abcam + muusp9x USP9x Merge
Supplementary Legends Figure 1 (a) Two independent anti-sera (#B1 and #B2) raised in guinea-pig and immunopurified with the antigenic N-terminal domain (1-308 amino-acids) of human EFA6B were analyzed by immunoblot against lysates from the indicated murine tissues. A doublet at about 175kD was detected in the thymus and spleen while a single band or doublet at about 65kD was detected in all tissues. In the brain in addition to the band at 65kD, two bands were detected at about 110kD and kd. (b) The rabbit antiserum anti-efa6b raised against the Sec7 domain of EFA6B was used to immunoprecipitate EFA6B from MDCK lysate. The IP was resolved on SDS-PAGE along with murine lung and kidney lysates. After transfer the membrane was immuno-blotted with the anti-efa6b #B1 antiserum which recognized a band at 66kD in the IP and the lysates. In addition the doublet at about 175kD was detected in the lung. (c) Specificity of EFA6B RNA interference. 24 h after nucleofection of the following sirnas: sirna control, sirna mismatch or EFA6B targeted sirnas #637, #1564 and #1770, the expression of the indicated proteins was analyzed by immunoblotting. All three EFA6B-targeted sirna decreased EFA6B levels, whereas the control sirnas had no effect. In addition, neither Arf6 nor any of the AJ and TJ protein levels were affected by the EFA6Btargeted sirnas. Figure 2 (a) Both its catalytic activity and its C-terminal remodeling actin domain are required for EFA6 to stimulate TJ formation. Two mutants of EFA6A expressed under the control of the tetracycline-repressible promoter were analyzed for their capacity to rescue the formation of functional TJ in EFA6B-knockdown cells (sirna #637). EFA6A-E242K contains a point mutation of the conserved glutamic acid within the Sec7 domain essential for the nucleotide exchange activity and EFA6A- C lacks the C-terminal actin remodeling domain. The gain of TJ barrier function was analyzed in a calcium switch assay by measuring the TER (n=4) over time and the paracellular diffusion of TRITC-dextran (n=3) at 2 h after calcium repletion. For TER measurement with EFA6A-E2K2K: sicontrol cells +/- Dox p>0.35 at all times, for cells +/- Dox p>0.34 at all times, for +Dox cells +/- p<0.003 after 30 min, for Dox cells +/- p<0.0004 after 30 min. For the paracellular diffusion of the TRITC-Dextran with EFA6A-E242K: sicontrol cells +/- Dox p=0.35, for cells +/- Dox p=0.30, for +Dox cells +/- p=0.0027, for Dox cells +/- p=0.0097. For TER measurement with EFA6A- C: sicontrol cells +/- Dox p>0.15 at all times, for cells +/- Dox p>0.42 at all times, for +Dox cells +/-
p<0.001 at all times, for Dox cells +/- p<0.004 at all times. For the paracellular diffusion of the TRITC-Dextran with EFA6A- C: sicontrol cells +/- Dox p=0.48, for cells +/- Dox p=0.44, for +Dox cells +/- p=0.0066, for Dox cells +/- p=0.0089. Error bars represent the SEM. (b) EFA6B has a rapid rate of protein synthesis and a short half-life. The rates of synthesis (right panel) and the half-life (left panel) of EFA6B, occludin and transferrin receptor (Tfn-R) were determined after incorporation of 35 S-methionine and 35 S-cysteine. To measure the rate of protein synthesis MDCK cells were subjected to radioactive pulses of different durations and to measure the half-life MDCK cells were subjected to a 10 min pulse followed by a chase over a 2 h period (See Supplementary Methods). In both cases, the numbers represent the amount of radioactivity incorporated in each protein measured after immunoprecipitation, SDS-PAGE, fluorography and corrected for the number of cysteine and methionine residues in each protein. (c) BHK cells transfected with vsvg-efa6a (97kD) were treated or not with MG-132 (50 µm) for 4 h and lysed in SDS lysis buffer. The lysate was subjected to immunoprecipitation with an anti-vsvg antibody or incubated with GST, GST-S5a or GST-Eps15 (UIM1/2) beads for 2 h. The precipitates were resolved by SDS-PAGE and the immunoblot probed with an anti-vsvg antibody. Figure 3 (a) GFP-PH from EFA6A hampers TJ assembly. MDCK GFP-EFA6A and GFP-PH #1 cells grown on 12-mm filters were subjected to a calcium switch and fixed 0.5, 1 or 2 h after calcium repletion. The samples were prepared for immunofluorescence analysis and costained for ZO-1. (b) Specificity of USP9x RNA interference. The following sirnas: sirna control, USP7-targeted sirna #1118 or USP9x-targeted sirnas #182 and #3432 were transfected by nucleofection. 24 h post-transfection USP7 and USP9x levels were analyzed by immunoblotting. Actin was used as a loading control. Both USP9x sirnas decreased USP9x levels without affecting USP7. Conversely the USP7 sirna knocked down USP7 without affecting USP9x. (c) The lysates described in Fig. 4b were immunoblotted for the indicated proteins. Knocking down USP9x decreased afadin levels but did not affect any of the other AJ and TJ proteins. Figure 4 Characterization of the rabbit polyclonal (anti-n1) and mouse monoclonal (Abcam) USP9x antibodies. USP9x localization (green) was visualized in cells labeled for filamentous actin with fluorescent phalloidin (red). (a) The anti-n1 gave a strong intracellular staining as described earlier (Murray et al., 04). However, no staining was detected at cell-cell contacts. The anti-n1 staining was decreased by pre-incubation with the immunizing peptide
(b) or by the USP9x-specific sirna #182 (c). The same staining was observed with the Abcam antibody (d) and similarly repressed after USP9x knockdown (e). Finally, the exogenously expressed murine USP9x was recognized by the anti-n1 (f) and Abcam antibodies (g). Staining for murine USP9x detected by both antibodies was cytoplasmic and absent from cell-cell contacts as seen for endogenous USP9x, that is intracellular and absent from cell-cell contacts. In (h) and (i), are shown the co-localization of GFP-EFA6A and USP9x at the periphery of a membrane ruffle or the extremity of a long plasma membrane extension, respectively. In (j) is shown a more mature contact where GFP-EFA6A is still present but from which USP9x is already excluded. The panels in (k) show the localization of USP9x in mature contacts established within a cluster of MDCK cells. USP9x is totally excluded from the cell-cell adhesion marked by E-cadherin. In (l), USP9x co-localizes with E- cadherin in a newly-formed contact. Scale bars, 25 µm.
Supplementary Methods Measure of protein synthesis and half-life. MDCK cells grown on 12 mm filters were rinsed and starved in MEM, 5% dialyzed FCS, without cysteine or methionine (MEM-Cys- Met) for min at 37 C. The cells were then labeled on a 30 µl drop of MEM-Cys-Met containing a mix of 35 S-Cys/Met (66 µci / filter at 1175 Ci / mmol). To measure the rate of protein synthesis the cells were pulsed for 2 min, 5 min and 10 min. At the end of the pulse, the cells were quickly rinsed in ice-cold PBS and solubilized in Triton X-100 lysis buffer (1% Triton-X100, mm Hepes ph 7.4, EDTA 2 mm, NaCl 125 mm, 0.2 mm PMSF and protease inhibitors) for min at 4 C. To measure half-life, the cells were pulsed for 15 min, quickly washed 4 times in MEM-BSA (MEM, 0.6% BSA, mm Hepes ph 7.4, penicilline/streptomycine), and further incubated in MEM-BSA containing an excess of nonradioactive cysteine (2 mm) and methionine (1 mm) for the indicated times before solubilization in Tx-100 lysis buffer. For both experiments, the lysates were spun 30 min at 16,000 g in a microfuge and the supernatants pre-cleared. The indicated proteins were immunoprecipitated using specific antibodies coupled to protein A sepharose beads, the immunoprecipitates washed 5 times in lysis buffer, resolved on SDS-PAGE, then revealed and analyzed using a PhosphorImager (Fujifilm BAS-1500, Fuji Film, France).