Current Biology, Volume 23 Supplemental Information Recruitment of UBPY and ESCRT Exchange Drive HD-PTP-Dependent Sorting of EGFR to the MVB Nazim Ali, Ling Zhang, Sandra Taylor, Alex Mironov, Sylvie Urbé, and Philip Woodman Supplemental information inventory The supplemental information contains: Supplemental Figure 1, related to Figure 1 Supplemental Figure 2, related to Figure 2 Supplemental Figure 3, related to Figure 3 Supplemental Figure 4, related to Figure 4 Supplemental Figure 5, related to Figure 6 Supplemental Figure 6, related to Figure 6 Supplemental Table 1 Supplemental Movie 1, related to Figure 1 (available online) Supplemental Movie 2, related to Figure 1 (available online) Supplemental Movie 3, related to Figure 1 (available online) Supplemental Movie 4, related to Figure 6 (available online) Supplemental Experimental Procedures Supplemental References
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Current Biology, Volume 23
Supplemental Information
Recruitment of UBPY and ESCRT Exchange
Drive HD-PTP-Dependent Sorting
of EGFR to the MVB Nazim Ali, Ling Zhang, Sandra Taylor, Alex Mironov, Sylvie Urbé, and Philip Woodman Supplemental information inventory The supplemental information contains: Supplemental Figure 1, related to Figure 1 Supplemental Figure 2, related to Figure 2 Supplemental Figure 3, related to Figure 3 Supplemental Figure 4, related to Figure 4 Supplemental Figure 5, related to Figure 6 Supplemental Figure 6, related to Figure 6 Supplemental Table 1 Supplemental Movie 1, related to Figure 1 (available online) Supplemental Movie 2, related to Figure 1 (available online) Supplemental Movie 3, related to Figure 1 (available online) Supplemental Movie 4, related to Figure 6 (available online) Supplemental Experimental Procedures Supplemental References
Figure S1, related to Figure 1. A: Tomogram of endosome from control cell; view of first section. See Movie S1 for tomogram and model. B: Western blot of HD-PTP knockdown, showing actin as a loading control. C: Tomogram of vacuolar endosomal region from HD-PTP depleted cell; view of first section. See Movie S2 for tomogram and model. D: Tomogram of tubulovesicular endosomal region from HD-PTP depleted cell; view of first section. See Movie S3 for tomogram and model. Scale bars for all tomograms = 200 nm. E: Western blot of EGFR showing levels are slightly reduced in HD-PTP depleted cells. F: EGFR was localized by immunofluorescence in control and HD-PTP depleted cells. Note the accumulation of EGFR on intracellular membranes upon HD-PTP depletion. Bar = 10 µm.
Figure S2, related to Figure 2. A: Directed Y2H with the indicated HD-PTP or Alix baits. B: Yeast cultures transformed with STAM2 prey and indicated baits were assayed for α-galactosidase. Means of triplicates +/- S.D. C: Yeast cultures transformed for STAM2 and as indicated were assayed for α-galactosidase. Means of triplicates +/- S.D. D: GST or GST-CHMP4B was incubated with or without WT or L/I-DD mutant His6-HD-PTP Bro1-V. Samples were IP’ed with anti-His and Coomassie stained. E: Y2H with the indicated baits and FL UBAP1 as prey. F: Lysates from HeLaM cells expressing HA-STAM2 and HD-PTP-myc were immunoprecipitated with anti-HA and probed for HA and myc. G: Top panel: the indicated point mutants of full-length HD-PTP were analysed by Y2H against STAM2 (note that the L/I-DD mutant is from a separate experiment). Bottom panel: the indicated HD-PTP mutants were analyzed against STAM2, or against TSG101 as a control.
Figure S3, related to Figure 3. Colour images and merge versions of Figure 3D. RPE cells were immunostained for endogenous Hrs (green) and HD-PTP (magenta). Bar = 10 µm.
Figure S4, related to Figure 4. A: Left; HeLaM cells were treated with the indicated HD-PTP siRNA oligos, and cell lysates blotted for Hrs or for tubulin as a control. Right; RPE cells treated with or without HD-PTP siRNA were blotted for Hrs or for tubulin as a control. B: Control or HD-PTP depleted cells were incubated for 8 h with or without 1 µM Iressa, then lyzed and analyzed by WB. Top: typical blot. Bottom: blots from 3 experiments were quantified, +/- SD, and normalised to control values. C: Control or HD-PTP kd HeLaM cells were incubated in serum-free medium for 8 h with or without Iressa. After washing out the drug, cells were pulse-chased with fluorescent EGF for 3 h. Cells were scored for the appearance of EGF in clusters, indicative of defective EGF trafficking. Values are from a single experiment [n = 400]). D: Membrane and cytosol fractions from control or HD-PTP depleted cells were analyzed by Western blot. TfR; transferrin receptor, used as a membrane marker. E: EGFR IPs from control or siRNA-treated cells were analyzed by Western blotting.
Figure S5, related to Figure 5. A: Myc-CHMPs were translated in wheat germ lysates, then blotted for anti-myc or anti-CHMP4B. B: Untreated HD-PTP-myc cells, or cells transfected with either GFP alone or with GFP and HA-STAM2 were subjected to a PLA analysis using a combination of anti-myc and anti-CHMP4B, and PLA product was counted. For transfected samples, PLA product was counted in GFP-expressing cells. Means +/- SD from 3 experiments, 100 cells counted for each, to illustrate inter-experimental variation. Mann-Whitney test used for statistics. C: Control or HD-PTP depleted cells were pulsed with EGF as indicated and EGFR was IP’ed. IPs were blotted for EGFR (top), or for ubiquitin-protein conjugates using FK2 antibody (bottom). The control IP is from control cells treated with EGF for 15 min. D: Control HeLaM cells, or cells depleted of HD-PTP or Hrs, were immunoblotted as indicated. E: EGFR was immunoprecipitated from control or Hrs depleted HeLaM cells and analyzed for UBPY. Left; total lysates. Right; IPs. F: The experiment shown in Figure 5D was also blotted for GST. The asterisk points to GST-CHMP4 degradation products. G: HD-PTP provides a platform for ESCRT exchange on EGFR. Recruitment of CHMP4B and UBPY to HD-PTP (solid lines) displaces STAM2 (broken line) from HD-PTP. In conjunction, STAM2 binding facilitates UBPY-dependent deubiquitination of EGFR, ensuring release of ESCRT-0 from EGFR in favour of ESCRT-III.
Figure S6, related to Figure 6. A: WB from control or UBPY depleted HeLaM cells for UBPY or for actin. B: Control or UBPY depleted HeLaM cells were pulse-chased with Alexa488-EGF for 3 h and stained for EGFR. C: As above, but stained with FK2 antibody. D: HD-PTP depleted HeLaM cells were pulse-chased with Alexa488-EGF for 3 h and stained with FK2 antibody. Bars = 10 µm. E: Stills from Movie S4, showing cross sections through a complex endosome consisting of a large vacuole (V) and connected early MVBs (large arrow). The large vacuole contains a few large inclusions, as well as many ILVs, the majority of which are closely associated with the limiting membrane. The linked endosomes have abundant ILVs. Most anti-EGFR-gold is clustered close to the limiting membranes, though some ILVs are also gold-labelled. Note the presence of fiducial (marker) gold particles on the top section. Bar = 200 nm.
Table S1. Anti-EGFR-gold particle distribution after UBPY or HD-PTP
depletion.
Control, HD-PTP or UBPY depleted cells were incubated with α-EGFR-gold and stimulated with EGF for 30 or 180 minutes, then chemically fixed and processed for thin-section EM. Compartments were classified by morphology, and gold particles were scored over each of these as a percentage of total labelling over the cell (SD in brackets). The indicated numbers of cells (shown in bold, brackets) were from a single experiment (control and HD-PTP knock-down) or from 2 experiments (UBPY knock-down). EE = early endosome; vacuolar, electron translucent endosome with internal vesicles; LE = late endosome; electron dense vacuole with internal vesicles. LM; limiting membrane, ves; vesicle, tub; tubule. Statistical significance was determined for the presence of anti-EGFR-gold in compartment categories: *; p < 0.01, ✝; p < 0.02 vs control sample at same time-point.
glutaraldehyde into culture medium with a 1:1 ratio. Fixed cells were treated with
reduced OsO4 for 1 hour and then embedded in resin. Polymerised resin blocks were
cut as described and sections were examined using FEI BioTWIN. Cells were
randomly picked from the sections to be imaged for further analysis. For control and
HD-PTP depleted samples, 5 cells were selected from a single experiment. For UBPY
depleted samples, cells were selected from two experiments.
Electron Tomography
Section thickness was approximately 300 nm for all tomograms. Grids with thick
sections were coated with 10 nm gold particles (fiducial gold) on both sides before
imaging to provide reference points for generating tomograms. Coated grids were
dried and then imaged using a Tecnai G2 Polara TEM. The specimen holder was tilted
using 1° or 1.5° steps, and images of the sample area at tilt angles from between -60°
to -65° and +60° to +65° were taken. Image data from a second tilting axis was
collected after rotating the sample for 90°. Collected images were subsequently
processed using IMOD software package (Boulder Laboratory for 3-D Electron
Microscopy of Cells, University of Colorado, US) to generate final tomogram(s).
Statistical analysis
For quantitative analysis of Duolink experiments, data from 3 independent replicates
were obtained. For each experiment, the number of reaction product foci was counted
over each of 100 cells for each sample, using a x20 objective on a BX-60 microscope
(Olympus). Product from a blank sample, lacking one of the primary antibody pairs,
was subtracted from all values. Mean values for each 100-cell sample were
calculated, and these values from the 3 experiments were combined to generate a
mean and SD, which was used to show inter-experimental errors on figure panels. The
Mann-Whitney test was applied to values obtained from all 300 cells. Student’s T-test
was used to provide statistics for His6-HD-PTP(Bro1-V) binding experiments. For
comparative pulse-chase experiments (Figure 6B and Table S1) a one way Anova was
applied after testing for normality of data.
Supplemental References
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