Effect of WIPI2 knockdown on EGFR degradation.

A, B) WIPI2CT and WIPI2KD cells were serum-starved for 24 h and then supplemented with EGF (100 ng/ml). After the indicated periods of time, cells were fixed, permeabilized, DAPI-labeled (blue) and decorated with antibodies to EGFR (red), EEA1 or LAMP1 (green). Scale bars: 10 μm. C) Colocalization of EGFR with EEA1 or LAMP1 was quantified over time using the images from A and B and Manders’ correlation coefficients were calculated. Values are the mean ± s.d.; (n = 3 independent experiments). 150 cells were quantified per sample. D) EGFR degradation. WIPI2CT and WIPI2KD cells were stimulated with EGF for different periods of time, lysed and subjected to SDS-PAGE and Western blot analysis for EGFR. α-Tubulin served as a loading control. E) Quantification of EGFR from (E), using the value at time 0 (cells starved for 24 h) as 100% reference. Data are means ±s. d., from three independent experiments.

GLUT1 expression and localization upon WIPI2 knockdown.

A. GLUT1 cell surface exposure. Control and WIPI2KD cells were fixed and stained with antibody against GLUT1 and with DAPI. Where indicated, cells had been permeabilized with 0.05% before staining. Scale bars: 10 μm. B. Quantification of GLUT1-immunofluorescence in cells from A. Regions of interest (ROIs) corresponding to each cell and in some regions outside the cells (background) were manually defined using image J software. Total cell fluorescence was integrated and corrected for background fluorescence. 150 cells per condition, stemming from three independent experiments, were quantified. Red bars show the means. P values were calculated applying an unpaired Student’s t-test with unequal variances. The analysis was performed with 99% confidence. NS = not significant (P > 0.05).

Impact of WIPI2 knockdown on β1-Integrin localization.

A. β1-Integrin cell surface expression. Control and WIPI2KD cells were fixed and stained with antibody to β1- Integrin (green) and with DAPI (blue), without detergent permeabilization. Scale bars: 10 mm. B. Quantification of β1-Integrin immunofluorescence in cells from A. Regions of interest (ROIs) corresponding to each cell and in some regions outside the cells (background) were manually defined using ImageJ software. Total cell fluorescence was integrated and corrected for background fluorescence. 150 cells per condition, stemming from three independent experiments, were quantified. Red bars show the means. P values were calculated applying unpaired Student’s t-test with unequal variances. The analysis was performed with 99% confidence. ***P < 0.001. C. Immunofluorescence staining of intracellular β1-Integrin (green) and EEA1 (red) or LAMP1 (red) in CTRL and WIPI2KD cells. Cells were fixed, permeabilized with 0.05% saponin and stained. Scale bars: 10 mm. D. Colocalization of β1-Integrin with LAMP1 or EEA1 was measured in cells from C, using Manders’ colocalization coefficient M2, calculated in Image J. Colocalization was quantified from three independent experiments with a total of 150 cells. An unpaired Student’s t-test with unequal variances was used to calculate P-values. The analysis was performed with 99% confidence. ***P < 0.001.

Role of the amphipathic α-helix of WIPI2 in β1-Integrin sorting.

A. The amphipathic α-helix. Helical wheel projections showing the CD-loop on blade 6 of the wildtype sequence, WIPI2WT, and WIPI2Sloop. Coloured arrows indicate the two pairs of amino acids that have been swapped in WIPI2Sloop. The magnitude and direction of the hydrophobic moment of the helices was predicted using the online tool Heliquest (Gautier et al., 2008). It is indicated by the vector in the centre of the wheels. Sequences of the hydrophobic loop region of WIPI2WT and WIPI2Sloop are shown, and predicted α-helices are plotted in red. The two pairs of hydrophobic/hydrophilic amino acids that are swapped in WIPI2Sloop are highlighted by rectangles in the sequences. B,C. β1-Integrin localisation. WIPI2KD (B) and WIPI2CT (C) cells were transfected with a plasmid carrying siRNA-resistant EGFPWIPI2WT or EGFPWIPI2Sloop. After 18h of viral transfection, cells were fixed, permeabilized with 0.05% saponin and stained with DAPI and antibodies to β1-Integrin. Scale bars: 10 mm. D. Quantification of β1-Integrin immunofluorescence in cells from B and C. Regions of interest (ROIs) corresponding to cells expressing the indicated WIPI2 variants, and some regions outside the cells (background), were manually defined using Image J software. Total cell fluorescence was integrated and corrected for background fluorescence. 105 cells per condition stemming from three independent experiments were analysed. Red bars indicate the means. P-values were calculated by Welch’s t-test. The analysis was performed with 99% confidence: ****P < 0.00001. NS: not significant (P>0.05)

Effects of the WIPI2 FSSS motif on β1-Integrin recycling.

A,B. Influence of WIPI2 variants on β1-Integrin. WIPI2-knockdown (WIPI2KD, A) and control (WIPI2CT) HK2 cells were transfected with a plasmid carrying siRNA-resistant wildtype or the indicated FSSS variants of EGFPWIPI2. After 18 h of viral transfection, cells were fixed and stained (without detergent permeabilisation) with DAPI and antibodies to β1-Integrin (red). Scale bars: 10 mm. C,D. Quantification of β1-Integrin immunofluorescence in cells from A and B. Regions of interest (ROIs) corresponding to cells expressing the indicated WIPI2 variants, and some regions outside the cells (background), were manually defined using Image J software. Total cell fluorescence was integrated and corrected for background fluorescence. 105 cells per condition stemming from three independent experiments were analysed. Red bars indicate the means. P-values were calculated by Welch’s t-test. The analysis was performed with 99% confidence: ****P < 0.00001.

Integration of WIPI2 with coat subunits.

A. Interaction of WIPI2HA with CCDC93. HK2 cells and HK2 cells stably expressing WIPI2HA were detergent solubilised, the total cell extracts were incubated with anti-HA beads and washed. Adsorbed protein was analysed by SDS-PAGE and Western blotting using the indicated antibodies. The intensity of the interacting CCDC93 was quantified with a LICOR Odyssey fluorescence imager. The background from the corresponding position in the sample from cells without HA-tag was subtracted. The resulting intensity is shown relative to the intensity of WIPI2HA signal on the beads. N = 3 biological replicates. Red bars show the means and error bars represent the SEM. P values were calculated with an unpaired Student’s t-test. *P < 0.01. B. Interaction of WIPI2HA with SNX17. HK2 cells stably expressing WIPI2HA were treated with siRNA to VPS26C (VPS26KD) or with non-specific siRNA (VPS26CT) and lysed. The total cell extracts were incubated with anti-HA beads and adsorbed proteins were analysed using the indicated antibodies as in A. N = 3. C,D. Selectivity for Retriever versus Retromer. The co-immunoadsorption experiments were performed as in A and analysed for co-adsorbed (C) VPS26C or (D) VPS26. N=3. *P < 0.01.**P < 0.001. NS=not significant.

Interaction of WIPI1 with VPS26 and SNX27, but not VPS26C.

A. Interaction of WIPI1HA with VPS26. HK2 cells and HK2 cells stably expressing WIPI1HA were detergent solubilised, the total cell extracts were incubated with anti-HA beads and washed. Adsorbed protein was analysed by SDS-PAGE and Western blotting using the indicated antibodies. The intensity of the interacting VPS26 was quantified with a LICOR Odyssey fluorescence imager. The background from the corresponding position in the sample from cells without HA-tag was subtracted. The resulting intensity is shown relative to the intensity of WIPI1HA signal on the beads. N = 3 biological replicates. Red bars show the means and error bars represent the SEM. P values were calculated with an unpaired Student’s t-test. B. Lack of interaction of WIPI1HA with VPS26C. HK2 cells stably expressing WIPI1HA were used for co-immunoadsorption experiments as in A and decorated with the indicated antibodies. N = 3. C. Interaction of WIPI1HA with SNX27. HK2 cells stably expressing WIPI1HA were used for co-immunoadsorption experiments as in A and decorated with the indicated antibodies. N = 3. **P < 0.001. NS: not significant.

Impact of the FSSS motif on the WIPI2-Retriever interaction.

A. HK2 cells and HK2 cells stably expressing the indicated WIPI2HA variants were detergent solubilised. Anti-HA beads were incubated with the total cell extracts, washed, and adsorbed proteins were analysed by SDS-PAGE and Western blotting using the indicated antibodies. B. Band intensities from the blots in A were quantified with a LICOR Odyssey infrared fluorescence imager and plotted as the ratio of VPS26C over WIPI2HA. N=3 independent biological replicates. Red bars show the means and error bars represent the SEM. P values were calculated with an unpaired Student’s t-test. **P < 0.001. HC: Heavy chain.

Role of the WIPI2 FSSS motif for recruiting WIPI2 and Retriever to Rab11 endosomes.

WIPI2-knockdown HK2 cells were transfected with plasmids expressing the indicated siRNA- resistant EGFPWIPI2 variants and mCherry-RAB5, mCherry-RAB7, or DsRed-Rab11 were fixed, permeabilized, immuno-stained for VPS35L and analyzed by confocal microscopy. Examples of the quantified images are presented in Supplementary Figures S5 to S7. Colocalization with EGFPWIPI2 and VPS35L was assessed for: A. RAB11 B. RAB5 C. RAB7. Colocalization was quantified using Manders’ colocalization coefficient M2, calculated in ImageJ. M2 refers to the fraction of VPS35L colocalising with the different Rab-proteins. For the triple colocalizations WIPI2/VPS35L/RAB M2 indicates the fraction of green pixels (WIPI2) overlapping with the pixels positive for the VPS35L/RAB colocalization. Colocalization was quantified from three independent experiments in a total of 120 cells. P values were calculated applying Welch’s t-test with unequal variances. The analysis was performed with 99% confidence. ***P < 0.0001.

Efficiency of WIPI2 knockdown.

Lysates of HK2 cells (50 μg per sample) were treated with non-target siRNA (WIPI2CT) or siRNA against WIPI2 (WIPI2KD). HK2 cells were analyzed by SDS-PAGE and Western blot against the indicated proteins. a-tubulin was used as a loading control. A representative blot is shown. The signals were quantified on a LICOR Odyssey infrared fluorescence image WIPI2/a-Tubulin ratios were calculated. Red bars indicate the mean and error bars the SEM; n=3 independent experiments. P-values were calculated by Welch’s t-test. ***P < 0.0001.

Surface expression of β1-Integrin in cells depleted of WIPI1.

A WIPI1CT and WIPI1KD cells were fixed and stained with DAPI and with antibody to β1-Integrin. Scale bars: 10 μm. B Quantification of β1-Integrin-immunofluorescence in cells from A. Regions of interest (ROIs) corresponding to each cell and in some regions outside the cells (background) were manually defined using ImageJ software. Total cell fluorescence was integrated and corrected for background fluorescence. 150 cells per condition, stemming from three independent experiments, were analyzed. Red bars show the means. P values were calculated by unpaired Student’s t-test. The analysis was performed with 99% confidence. NS = not significant (P>0.05).

Expression levels of WIPI2 variants.

A. FSSS variants. Cell lysates from wild-type and WIPI2KD cells were transfected for 18 h with siRNA resistant lentiviral expression constructs for EGFPWIPI2WT, EGFPWIPI2S67A and EGFPWIPI2S67E. The cells were analyzed by SDS–PAGE and Western blotting with antibodies to WIPI2 and α-Tubulin. A representative blot is shown. B Quantification of the signals for virally expressed EGFPWIPI2 variants and for endogenous WIPI2. Blots from three independent experiments were quantified on a Licor Odyssey infrared scanner. Graphs on the right side show the mean and SEM. P-values were calculated by Welch’s t-test. NS: not significant. C. SLoop variant. EGFPWIPI2 and EGFPWIPI2SLoop were expressed and analyzed by Western blotting as in described in B. D Blots from C were quantified as in B.

Live cell imaging of cells expressing EGFPWIPI2 variants

WIPI2KD cells were transfected with plasmids carrying the indicated siRNA-resistant EGFPWIPI2 variants. After 18h of viral transfection, cells were analysed by live cell confocal microscopy. Arrows point to tubular structures that are readily detectable in cells expressing the SLoop and S67 variants of WIPI2. Scale bars: 10 mm.

WIPI2 colocalization with RAB5 and Retriever.

HK2 cells expressing mCherry-RAB5 and EGFPWIPI2 wild-type (A) or its indicated variants (B-C), were fixed, permeabilized and stained with antibodies to VPS35L (blue). Scale bars: 10 μm. Insets show enlargements of the outlined areas. Quantifications from multiple experiments are shown in Fig. 9

WIPI2 colocalization with RAB7 and Retriever.

HK2 cells expressing mCherry-RAB7 and wild-type EGFPWIPI2 (A) or its indicated variants (B-C) were fixed, permeabilized and stained with antibodies to VPS35L (blue). Scale bars: 10 μm. Insets show enlargements of the outlined areas. Quantifications from multiple experiments are shown in Fig. 9

WIPI2 colocalization with RAB11 and Retriever.

HK2 cells expressing mCherry-RAB7 and wild-type EGFPWIPI2 (A) or its indicated variants (B-C) were fixed, permeabilized and stained with antibodies to VPS35L (blue). Scale bars: 10 μm. Insets show enlargements of the outlined areas. Quantifications from multiple experiments are shown in Fig. 9