Clathrin-independent pathways do not contribute significantly to endocytic flux
- Medical Research Council Laboratory of Molecular Biology, United Kingdom
- New England Biolabs, Inc., United States
Figures
Figure 1 with 1 supplement
Experimental strategy and assay validation.
(A) Following surface biotinylation with sulfo-NHS-SS-biotin, HeLa cell lysate was serially diluted with non-biotinylated control cell lysate, and the amount of labelling was detected using …
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Figure 1—source data 1
Plasma membrane proteins identified by mass spectrometry.
Biotinylated proteins identified in this study are listed by accession number, together with the estimated abundance takenfrom Kulak et al., 2014. A zero in the biotinylated proteins column indicates that we did not identify that protein. Calculation of PM abundance is described in the methods section.
- https://doi.org/10.7554/eLife.03970.004
Figure 1—figure supplement 1
Removal of extracellular fluorophore from BG-SS-fluorophore labelled SNAP-tag by reduction with MESNa is highly efficient.
(A) Cells expressing SNAP-GPI were simultaneously labeled at 4°C with BG-SS-488 and SNAPsurface549. The latter is not reducible. They were then treated with PI-PLC or PI-PLC and then MESNa. Both …
Figure 2 with 7 supplements
Over 95% of total endocytosed protein co-localises with markers for clathrin-mediated endocytosis.
(A) Confocal images of co-internalisation of all membrane proteins, labelled at 4°C with sulfo-NHS-SS-biotin, and transferrin-546. Internalisation was for 20 s at 37°C. Biotin was detected with …
Figure 2—figure supplement 1
Macropinosomes are readily identified by labelling with sulfo-NHS-SS-biotin.
Cells were labeled with sulfo-NHS-SS-biotin and transferrin-546, allowed to endocytose at 37°C for the times indicated, MESNA-treated, fixed, and stained with streptavidin-488. Occasionally, cells …
Figure 2—figure supplement 2
Surface-bound transferrin is highly concentrated within clathrin-coated pits.
Cells were transfected with clathrin light chain-GFP, cooled to 4°C, labelled with transferrin-546, fixed, and imaged. Bar is 20 μm. The boxed region is shown in the lower panels.
Figure 2—figure supplement 3
Quantification of percent co-localisation.
All processing was carried out in Image J. Two channel raw images were acquired by confocal microscopy. The channels were separated, subjected to Gaussian blur with σ = 0.7, and then contrast …
Figure 2—figure supplement 4
Co-localisation between internalised sulfo-NHS-SS-biotin and transferrin after labelling at 4°C and 90 s of internalisation at 37°C.
Confocal images of co-internalisation of total membrane protein, labeled at 4°C with sulfo-NHS-SS-biotin, and transferrin-546. Internalisation was for 90 s at 37°C. Biotin was detected with …
Figure 2—figure supplement 5
Co-localisation between internalised sulfo-NHS-SS-biotin and clathrin after 20 s and 60 s of internalisation at 37°C.
HeLa cells labeled at 4°C with sulfo-NHS-SS-biotin, were moved to 37°C for the indicated time-points. Surface biotin was removed by MESNA treatment, the cells were fixed and permeabilised, and then …
Figure 2—figure supplement 6
Total endocytosed protein and transferrin co-localise after 90 s uptake in Cos7 and RPE cells.
Confocal images of co-internalisation of total membrane protein, labeled at 4°C with sulfo-NHS-SS-biotin, and transferrin-546. Internalisation was for 90 s at 37°C. Biotin was detected with …
Figure 2—figure supplement 7
Absence of membrane-positive, transferrin-negative vesicles.
(A) Cells were labelled with the membrane dye FM1-43FX and transferrin at 4°C, warmed to 37°C for 90 s, plasma membrane dye was removed by washes with ice-cold PBS, and were imaged at 4°C without …
Figure 3 with 2 supplements
Over 95% of total endocytosed protein enters the cell via clathrin-coated pits.
(A) 3D projection of cell volumes following interalisation of sulfo-NHS-SS-biotin for 90 s at 37°C. Streptavidin-488 fluorescence is shown in the left panel, vesicle objects recognised with Imaris …
Figure 3—figure supplement 1
Correlation between streptavidin (total endocytosed protein) and transferrin intensities in endocytic vesicles.
(A) Projections of confocal z-stacks showing raw fluorescence images of internalised biotin, labelled with streptavidin, and transferrin, after 90 s internalisation. The streptavidin image was used …
Figure 3—figure supplement 2
Effect of CTB-binding on transferrin intensities in endocytic vesicles.
Analysis of the transferrin cargo load of endocytic vesicle objects identified as in Figure 3A after 90 s of uptake in control and CTB-labeled cells as shown. Frequency distribution of transferrin …
Figure 4 with 1 supplement
Changes in plasma membrane protein composition in cells depleted of AP2.
(A) Western blot for the AP2 alpha subunit following non-targeting or alpha adaptin (AP2) siRNA treatment. (B) Internalisation of sulfo-NHS-SS-biotin and transferrin-647, for 15 min, in cells …
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Figure 4—source data 1
SILAC ratios for biotinylated and non-biotinylated proteins.
Proteins shaded green were classified as depleted from the the plasma membrane upon AP2 siRNA treatment, those shaded redwe classified as accumulated.
- https://doi.org/10.7554/eLife.03970.019
Figure 4—figure supplement 1
Verification of changes in plasma membrane protein levels detected by SILAC.
Flow cytometry was used to analyse plasma membrane abundance of transferrin receptor and CD59. Cells were transfected with control siRNA or siRNA to knock down expression of the alpha adaptin …
Figure 5 with 1 supplement
GPI-anchored proteins co-localise with transferrin in primary endocytic vesicles.
(A) Confocal images of cells transfected with the SNAP-tagged GPI-anchored proteins indicated. Labelling with BG-SS-488 and transferrin-546 at 37°C for 90 s. External 488 fluorophore was removed by …
Figure 5—figure supplement 1
Effect of CTB-binding on transferrin intensities in endocytic vesicles defined by uptake of GPI-linked protein.
Analysis of the transferrin cargo load of endocytic vesicle objects identified as in Figure 3A after 90 s of uptake in control and CTB-labeled cells as shown. Cells were stably expressing SNAP-CD59, …
Figure 6 with 1 supplement
Clathrin-dependent endocytosis of GPI-anchored proteins.
(A) Doubly labelled anti-CD59-546-SS-488 allows comparison of MESNa reduction and PI-PLC treatment as methods for detecting internalised GPI-anchored protein. Cells were labelled at 4°C, warmed to …
Figure 6—figure supplement 1
Endocytic structures induced by high dynamin-2-K44A expression.
(A) Hela cells stably expressing SNAP-CD59 and transiently transfected with dynamin-2-K44A-dsRed were labeled with BG-SS-488 and transferrin-647 for 15 min at 37°C. Note that the cell shown has a …
Figure 7 with 1 supplement
Labelling of the total population of endocytosed proteins does not provide evidence for significant protein flux through clathrin-independent pathways.
(A, C, E, G) Confocal images showing distribution of the marker indicated (caveolin 1, flotillin 1, GRAF1, ARF6), together with total internalised protein after 90 s of endocytosis, revealed as in Fi…
Figure 7—figure supplement 1
Labelling of the total population of endocytosed proteins does not provide evidence for significant protein flux through clathrin-independent endocytic pathways.
(A, B, C, D) Confocal images showing distribution of the marker indicated (caveolin 1, flotillin 1, GRAF1, ARF6), together with internalised biotinylated protein after 15 min of endocytosis. …
Figure 8 with 2 supplements
Differential effects on uptake of transferrin receptor and GPI-anchored proteins via coated pits.
(A) Frequency distribution of the ratio between internalised transferrin and internalised SNAP-CD59 in individual HeLa cells, determined by flow cytometry as in Figure 8—figure supplement 1. (B) …
Figure 8—figure supplement 1
Reduction of AP-2 (alpha adaptin) levels affects the amount of uptake of both transferrin and SNAP-CD59, 50 hr after siRNA transfection.
Cells stably expressing SNAP-CD59 were incubated at 37°C for 15 min with BG-SS-549 and transferrin-647. Surface label was removed with MESNa and acid wash and then the cells were analysed by flow …
Figure 8—figure supplement 2
Incorporation of mutant μ2 subunits into AP2 complexes.
Cells were transfected with plasmids expressing the μ2 YXXΦ-binding mutant-myc as shown. Cell lysates were subjected to immunoprecipitation with anti-alpha adaptin, or as a negative control anti-GFP …
Videos
Video 1
Object recognition for quantification of cargo load in individual endocytic vesicles.
Cells were labelled for 90 s at 37°C with sulfo-NHS-SS-biotin and transferrin-546. After MESNA treatment internalised proteins were labelled with streptavidin-488. 3D reconstructions were obtained …
