JCS062497 Supplementary Material
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- Supplemental Figure S1 -
Fig. S1. Expression and siRNA-mediated gene silencing of TI-VAMP in HeLa cells. (A) TI-VAMP was targeted by two different siRNAs. Immunoblot shows that TI-VAMP migrates at 25 kDa. Silencing is about 95-99% and 80-97% with TI-VAMP siRNA 1 and 2. TI-VAMP expression is not affected by siRNAs targeting other VAMPs or CD82. (B) Immunochemistry using antibodies against TI-VAMP/VAMP7 showing the efficiency after 96 hours of silencing in HeLa cells. TI-VAMP staining is detected in larger perinuclear punctae, which mainly colocalize with the late endosomal markers tetraspanin CD63 (see supplementary material Fig. S3). Scale bar, 20 µm. (C) Comparison of silencing with oligonucleotide and pSuper plasmid. Cells were electroporated with either control pSuper (targeting the canine TI-VAMP sequence: dog TI-VAMP shRNA) or the pSuper plasmid targeting human TI-VAMP. pSuper plasmid encodes short RNA interference sequences (shRNA) against human TI-VAMP or dog TI-VAMP sequences which do not target human mRNA (V. Proux-Gillardeaux, personal communication). Left panel: western blot analysis of TI-VAMP expression in knocked down cells using with anti TI-VAMP antibody. Extinction of the protein expression is observed after 144 hours of incubation, which is a little longer than with oligonucleotides (96 hours). Right panel: immunocytochemistry of TI-VAMP in transfected cells with control and human pSuper plasmid after 144 hours of incubation. Extinction is satisfactory using the plasmid interference technique but less homogenous than using the oligonucleotide method. Whereas most of the cells express very low level of protein, some of them are still expressing TI-VAMP (see arrow). We thus chose the oligonucleotide method for the silencing of the TI-VAMP mRNA. Scale bar, 10 µm.
- Supplemental Figure S2 -
Fig. S2. Specific function of TI-VAMP in EGF endocytosis. (A) HeLa cells were incubated with A488-EGF at 18°C for 1 hour, fixed and processed for immunofluorescence. Note that TI-VAMP depleted cells accumulated more EGF than mock cells (luciferase siRNA) suggesting increased endocytosis. Scale bar, 10 µm. (B) Silenced HeLa cells were incubated with A488-EGF at 18°C for 1 hour, washed and stripped with acidic buffer to remove surface labeling. Endocytosed fluorescent EGF was quantified in various depleted cells by flow cytometry. (C) Top panel: TI-VAMP depleted cells showed significant increased in EGF median fluorescence compared with the mock RNAi (right shift of the fluorescence intensity). No statistical difference is observed using the other v-SNAREs siRNAs. Bottom panel: three independent experiments have been pooled together resulting in 30,000 analyzed cells per condition. TI-VAMP depleted cells showed systematically highly significant increase in EGF endocytosis with both siRNAs (Student�s t-test: siRNA 1: P=0.0004; siRNA 2: P=0.0011). No statistical difference is observed using the other v-SNAREs siRNA (Student�s t-test: VAMP3: P=0.9534; VAMP4: P=0.7308; VAMP8: P=0.0917). (D) Rescue of the increased endocytosis of EGFR by re-expression of rat TI-VAMP. pcDNA3-rat-TI-VAMP was transfected in mock and TI-VAMP depleted cell and incubated for 24 hours. Cells were assayed by western blot (top) and cytometry (bottom). Each time point corresponds to 10,000 cells. Note that the transfection of rat-TI-VAMP was able to reverse the phenotype of increased EGF endocytosis. (E) TI-VAMP KD does not alter EGFR expression. Representative western blot of the total amount of EGFR (25 µg of protein/lane). Quantification done on three independent western blots indicated that the amount of EGFR is not significantly different in mock and TI-VAMP depleted cells (Student�s t-test: siRNA 1: P=0.9342; siRNA 2: P=0.6091). There is no statistical differences between TI-VAMP siRNA 1 and siRNA 2 (P=0.6775). (F) Flow cytometry quantification of the EGFR at the cell surface. Four independent experiments have been pooled together resulting in 40,000 analyzed cells per condition (bottom panel). The amount of surface EGFR is not significantly different in mock and TI-VAMP depleted cells (Student�s t-test: siRNA 1: P=0.1868; siRNA 2: P=0.5170). There is no statistical difference between TI-VAMP siRNA 1 and siRNA 2 (P=0.1978).
- Supplemental Figure S3 -
Fig. S3. Immunocytochemical visualization of EGF endocytosis, TI-VAMP and endosomal markers in HeLa cells. EGF binding to its receptor (the EGFR) at the surface triggers EGFR phosphorylation and endocytosis. The EGF-EGFR can either be recycled back to the membrane via the recycling endosomes, or be targeted to the late endosomes and lysosome to be degraded. The fate of the receptor can be followed using fluorescent EGF because the ligand remains bound to its receptor along its different routes. HeLa cells were incubated with fluorescent EGF (Texas Red) at 18°C for 1 hour, to accumulate EGF-EGFR in the early endosomes. At this temperature, traffic towards degradation compartments is blocked and recycling towards the plasma membrane is strongly impaired (Futter et al., 1996; Sorkin et al., 1991a). Cells were washed at 4°C to remove excess fluorescent EGF. Cells were then incubated at 37°C for 0, 15, 30, 60 minutes and 16 hours and then fixed for immunochemistry. Early endosomes are detected with EEA1 monoclonal antibody, and late endosomes/lysosomal compartments are labeled with CD63 monoclonal antibodies. Triple labeling was carried out to localize EGF in the endocytic pathway. Triple detection of TI-VAMP (green), EGF (red) and endosomes (blue) at 0 minutes (A), 30 minutes (B) and 16 hours (C) at 37°C is shown. EGF colocalizes with EEA1 between 0 minutes and 15 minutes. First colocalization with TI-VAMP and CD63 occurs after 30 minutes at 37°C. EGF labeling decreases after 1 hour of incubation at 37°C and is totally degraded after 16 hours. These results suggest that the EGF-EGFR complex reaches late endosomal compartments about 30 minutes after leaving the early endosomes. Scale bar, 20 µm.
- Supplemental Figure S4 -
Fig. S4. Expression and siRNA-mediated gene silencing of v-SNAREs in HeLa cells. To compare our results with known v-SNARE expressed in mammalian cells we also used siRNA against others endosomal v-SNARE such as VAMP3, VAMP4 and VAMP8. (A) Western blot analysis of knocked down cells with anti-VAMP3, VAMP4 and VAMP8 antibody. Knock down efficiency of endosomal v-SNAREs expression: VAMP3: 91%, VAMP4: 65-70% and VAMP8: 90-95%. (B) Immunochemistry using antibodies against VAMP3, VAMP4, and TI-VAMP/VAMP7 showing the efficiency after 96 hours of silencing in HeLa cells. In control cells, VAMP3 and VAMP4 display a classical punctate pattern evoking endosomal staining with a perinuclear accumulation corresponding to the Golgi apparatus. Each v-SNARE staining is efficiently decreased following specific siRNA. Scale bar, 20 µm. (C) Immunochemistry with a polyclonal antibody against VAMP8 showing that the level of expression of endogenous VAMP8 (see the cell marked �nt�) is too low in HeLa cells to be detectable by immunochemistry with our polyclonal antibody (TG15). However, the specificity of the antibody is confirmed by the fact that HeLa cells transfected with GFP-VAMP8 (marked �t�) can be detected with our antibody. Scale bar, 10 µm.
- Supplemental Figure S5 -
Fig. S5. Distribution of adaptor and Golgi-related protein in TI-VAMP KD cells. Immunochemistry against AP-1, AP-3, M6PR and TGN46 showing that the distribution pattern is not altered in TI-VAMP knocked down cells compared with mock depleted cells. Scale bar, 10 µm.
- Supplemental Figure S6 -
Fig. S6. TI-VAMP KD does not affect EGF-EGFR recycling and degradation. (A) Increased EGF intensity in TI-VAMP depleted cells is not due to a recycling defect. At 18°C, traffic towards the degradative compartments is blocked and recycling towards the plasma membrane is strongly diminished (Futter et al., 1996; Sorkin et al., 1991a) suggesting that the contributions of recycling or degradation to EGF accumulation in TI-VAMP depleted cells would be very minor if they exist. To completely rule out these possibilities, we directly tested the effect of TI-VAMP depletion on recycling. Mock- or TI-VAMP-silenced cells (siRNA 1 or 2) were incubated with 100 ng/ml of A488-EGF in the absence or presence of monensin, an inhibitor of the recycling pathway (Basu et al., 1981; Wiley et al., 1985). The total amount of EGF was quantified by flow cytometry (20,000 cells per condition, in triplicate). The effect of the monensin was equal in both mock and TI-VAMP depleted cells, indicating that the EGF accumulation in TI-VAMP knockdown cells did not result from a recycling defect. (B) Kinetics of EGF disappearance is not altered in TI-VAMP depleted cells. TI-VAMP was previously proposed to mediate the transport from endosomes to lysosomes on the basis of the 25% inhibitory effect of anti-TI-VAMP polyclonal antibodies on EGF degradation (Advani et al., 1999). We further checked if the disappearance of fluorescent EGF, which is the result of both recycling and degradation of the complex, is altered in TI-VAMP depleted cells. Mock and knocked down HeLa cells were incubated for 1 hour at 18°C with A488-EGF. Cells were washed and stripped with acidic buffer to remove any surface labeling, and incubated at 37°C. EGF was quantified by flow cytometry, each time point corresponding to 15,000 cells. As already shown in supplementary material Fig. S2C, fluorescence intensity at t=0 was higher after TI-VAMP knockdown indicating a higher uptake at the initial point. However the kinetics of fluorescence decay was not different between mock and TI-VAMP knocked down cells (n=10,000 cells). (C) Mild effect on degradation of EGFR by TI-VAMP depletion. HeLa cells were incubated with 100 ng/ml EGF in DMEM without serum for 0, 30, 60 or 120 minutes. The amount of EGFR was quantified by western blot analysis (15 µg of protein/lane). As expected we detected a slight delay in EGFR degradation in TI-VAMP knocked down cells but this difference was not statistically significant among four independent experiments (Student�s t-test: TI-VAMP siRNA 1 after 30, 60 and 120 minutes: P=0.09, 0.28; 0.09; siRNA 2: P=0.26; 0.45; 0.26). (D) Degradation and recycling of EGF are not affected in TI-VAMP depleted cells. Cells were incubated with A488-EGF at 18°C for 1 hour, washed, stripped to remove any surface labeling, and incubated at 37°C for 2 hours to allow degradation and recycling. Resting EGF was quantified and normalized to the first time point. The amount of EGF remaining in the mock and TI-VAMP depleted cells are not significantly different (P>0.05). The amount of EGF remaining in the VAMP3 depleted cells is slightly increased (Student�s t-test, mock/VAMP3 siRNA, P=0.0398). (E) Mild effect on degradation of EGFR by CD82 depletion. HeLa cells were incubated with 100 ng/ml of EGF in DMEM without serum for 0, 30, 60 or 120 minutes. The amount of EGFR was quantified by cytometry. We detected a slight delay in EGFR degradation in CD82 knocked down cells in two independent experiments (Student�s t-test: 15 minutes P=0.0497; 30 minutes P=0.0492; 60 minutes P=0.0210; 120 minutes P=0.0075).
- Supplemental Table S1 -
- Movie 1
Movie 1. Confocal video-microscopy of CD82-YFP and TI-VAMP−RFP. HeLa cells were transfected with CD82-YFP and TI-VAMP−RFP and were incubated for 24 hours. Video-microscopy on living cells at 37°C shows that CD82 is transported in TI-VAMP-positive vesicles (movie related to Fig. 3). Scale bar, 10 µm.
- Movie 2
Movie 2. Video-microscopy of EGFR labeled by quantum dots. EGFR was labeled with biotinylated antibody (Ab-3) and streptavidin quantum dots (QDot). EGFR was imaged every 38 mseconds by video-microscopy on living cells at 37°C with an EMCCD camera. Note that the blinking in the fluorescence signal is typical of a single quantum dot molecule switching between bright and dark states (movie related to Fig. 4). Scale bar, 5 µm.