Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells (original) (raw)

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Acknowledgements

We thank the US National Heart Lung and Blood Institute (NHLBI) electron microscopy core, the NHLBI flow cytometry core, and the NHLBI light microscopy core for use of equipment; specifically, C. Bleck, E. Stempinski, and C. Keshavarz for help in the EM core, P. Dagur with help doing FACS, and X. Wu for help with TIRF-SIM; D. Drubin (University of California, Berkeley, USA) for the generous gift of the SK-MEL-2 hDNM2EN cell line; K. Neuman, J. Hinshaw and K. Swartz for helpful reading of the manuscript; A. Trexler, A. Somasundaram, T. Davenport and J. Ciemniecki for scientific discussion, H.-J. Yang for help in image processing, and E. Tyler of NIH Medical Arts for creating Fig. 4. J.W.T. is supported by the Intramural Research Program of the National Heart Lung and Blood Institute, National Institutes of Health.

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Authors and Affiliations

  1. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
    Kem A. Sochacki, Andrea M. Dickey, Marie-Paule Strub & Justin W. Taraska

Authors

  1. Kem A. Sochacki
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  2. Andrea M. Dickey
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  3. Marie-Paule Strub
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  4. Justin W. Taraska
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Contributions

K.A.S. designed, performed and analysed experiments. A.M.D. aided in microscopy preparation. M.-P.S. expressed and purified GFP-nanotrap and other plasmid preparations. K.A.S. and J.W.T. processed data and wrote the manuscript.

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Correspondence toJustin W. Taraska.

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Integrated supplementary information

Supplementary Figure 1 Outlining CCSs and assessing correlation error.

(a) Zoomed-out image of the entire imaged portion of a HeLa cell membrane. (b) The mask created after structures have been outlined. Red pixels on domed CCSs, green pixels are on flat CCSs, blue pixels are on highly curved CCSs, white pixels are area that were not analyzed. We did not analyze regions within 500 nm of a gold nanoparticle fiducial marker or the edge of the membrane. (c) zoomed in region from the cell shown in a. (d) Same region with outlining shown. Outlining color scheme is the same as in b. (e–h) Examples of single localization clusters from four different days of imaging. (e) Horizontal linescans through the center of images shown in (e-h). The grey dotted lines are drawn down from the approximate half height to show that the localization precision can be described by the full-width at half max (∼20 nm). (j) To understand how correlation error may affect our analysis, we induced additional correlation error into our existing experiments to see how the 1DFLIPs would change. All data used to study nanotrap labeled GFP-clathrin in HeLa cells were analyzed again with an additional lateral shift of the distance described (0, 24, 48, or 72 nm). This artifically induced a correlation error causing a decrease in 1DFLIP slope. (k) The same induced correlation error analysis was done on nanotrap labeled EPS15-GFP HeLa data. (l) A theoretical ring of fluorescence with 120 nm diameter was convolved with a Gaussian of 20 nm full-width at half maximum. The Gaussian width was chosen based off the localization microscopy precision measured in (e–i). This modeled ring was then shifted with respect to the analyzed circle as was done in (b) for real data. According to this analysis, the slope of the data is largely dependent on the correlation error or outlining precision. The slope in our data is regularly 40 nm over half height indicating that correlation error and outlining precision are similar throughout different images. (a,b) Scale, 4 μm. (c,d) Scale, 300 nm. (e,h) Scale, 50 nm. N shown in j,k are number of separate structures analyzed. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1.

Supplementary Figure 2 1D fluorescence intensity profiles (1DFLIPs) statistical and experimental controls and hierarchical clustering.

(a) Data of nanotrap-labeled GFP-clathrin light chain on CCSs from Fig. 5e are shown with cell-to-cell averaging and standard deviation. 1DFLIPs from flat (b) and domed (c) CCSs are shown for the 18 clathrin-associated proteins studied also shown with cell-to-cell averaging and standard deviation. The N value listed is number of cells. Clathrin data from (a) is referenced in black. (d) Dendrogram of 1DFLIPs hierarchical clustering. For each protein that was studied, the flat 1DFLIP was appended to the domed 1DFLIP making a single linescan. The clustering was based on the correlation distance (one minus the correlation value) between points. This clustering was done using the Matlab function ‘linkage.m’. This is a way of grouping the 1DFLIP pairs from each data set with others of a similar shape. The results from the automated clustering are similar to how one would cluster these linescans by eye. (e–i) Control protein 1DFLIPs. Clathrin Heavy Chain Antibody is compared to clathrin light chain GFP labeling for domed and flat CCSs. (b) Epsin1 antibody is shown in comparison to epsin1-GFP data. In a,b, the antibody data shifts slightly outward which is likely due to the 25 nm antibody tether. (c) EPS15-GFP (AF647 GFP-nanotrap) is compared with EPS15-flag (direct primary antibody AF647 labeling) and EPS15 primary and secondary labeling using anti-EPS15. Low-expression in the EPS15-flag data caused increased sampling noise. (d) GFP-FCHO2 (AF647 GFP-nanotrap) is compared with flag-FCHO2 (direct primary antibody AF647 labeling) and FCHO2 primary and secondary labeling using anti-FCHO2. Less than 25% of clathrin structures were labeled (Table S1) with anti-FCHO2 antibody and those that were labeled had only one label causing increased error. (e) GFP-β-adaptin is compared with _σ_-adaptin-GFP. These are both components of AP2 and exhibit the same trend in domed and flat CCSs. Additional antibody data was used for in depth dynamin analysis (Supplementary Fig. 4), or visual comparison of SK-MEL-2 versus HeLa cells (Supplementary Fig. 3). Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1.

Supplementary Figure 3 Super-resolution CLEM of protein-specific antibodies on platinum replica clathrin lattices compared between HeLa and SK-MEL-2 cells.

Primary antibodies specific for dynamin2, AP2, epsin1, EPS15, and DAB2 were used in conjunction with AF647 secondary antibodies to label either HeLa or SK-MEL-2 hDNM2EN cells. In the case of dynamin2, antibody labeling is also shown in wild type SK-MEL-2 cells. Specific antibodies used are listed in methods. Quantity of labeling appears different for EPS15 and DAB2 but average localization does not differ. Scale, 100 nm.

Supplementary Figure 4 Dynamin 2 quantitative comparison between overexpressed, endogenous, and genome-edited cell lines in HeLa and SK-MEL-2 cell lines.

(a) A histogram of the area of domed (red), flat (green), and highly rounded (blue) clathrin structures in HeLa cells are compared to those in SK-MEL-2 cells showing the sizes of flat structures in SK-MEL-2 and HeLa cells are different. SK-MEL-2 cells have fewer flat CCSs and do not exhibit large flat ‘plaque’ CCSs like those seen in HeLa cells. (b) Dynamin2 data is summarized with cell type, labeling method, number of cells, number of all domed, number of domed containing fluorescence, number of all flat, and number of flat CCSs containing fluorescence. Number of independent coverslips imaged is listed in Supplementary Table 1. (c) 1DFLIPS of domed CCSs from all the data in (b) are plotted together. (d) 1DFLIPS of flat CCSs from all of data in (b) are plotted together. N includes only structures containing fluorescence. Error is standard error as in Fig. 6. Shifts toward the center of the flat CCSs are due to labeling in the center of flat structures that have broken lattices.

Supplementary Figure 5 Overexpression of some proteins cause clathrin structure size and density changes in HeLa cells.

(a) A histogram of the two-dimensional area of domed, flat, and highly curved CCSs in WT HeLa cells measured from platinum-replica EM images. There is a broad range of sizes. This highlights the need to look at a large amount of data before making statements about sizes changing due to overexpression or knockout experiments. Domed CCSs occupy on average a larger area than highly curved CCSs consistent with pits needing to increase their curvature to become a vesicle. Some flat CCSs are quite large and likely could yield multiple domed CCSs. (b) The density of flat CCSs is plotted versus the median area of flat CCSs for all the data discussed in this paper. Most notably, cells overexpressing EPS15-gfp, amphiphysin1-gfp, and gfp-SNX9 all exhibited small flat CCSs at a higher density than in wild type. Cells overexpressing dynamin2-gfp exhibited abnormally large flat CCSs. (c) The density of domed CCSs is plotted versus the median area of domed CCSs for all the data. (d) The density of highly curved CCSs versus the median area of highly curved CCSs for all data discussed in this paper. For (b–d) the median areas from each cell were combined to obtain an average and standard deviation amongst the cells. The average and standard deviation of structure density amongst the cells is also shown. (e–g) All area data shown in b–d are shown as box plots (25–75% boxes, whiskers set with outlier coefficient of 1.5, central line is median). The median areas from each cell are also shown as grey diamonds. WT HeLa cells with are highlighted in red. Each plot is organized in the order of decreasing median area. Little change is observed in domed or highly rounded CCSs but there is a notable change in the area of flat CCSs for some proteins. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1.

Supplementary Figure 6 Two-color localization microscopy with protein-specific antibodies and skylan-clathrin (light chain) in intact HeLa cells.

A gallery of example images for clathrin, dynamin2, AP2, epsin1, EPS15, and DAB2 are shown. Skylan-clathrin is shown in green, and antibody staining is shown in magenta (specific antibodies are described in methods, primary antibodies were supplemented with AF647-labeled secondary antibodies). Note the increased background fluorescence in intact cells. Scale bar, 400 nm. Two independent images were processed for each except for clathrin (N = 3) and DAB2 (N = 1).

Supplementary Figure 7 Two-color TIRF-SIM with GFP-labeled proteins of interest and mCherry-Clathrin (light chain) in living HeLa cells at 37C.

Proteins of interest imaged were GFP-clathrin light chain (N = 6 cells), GFP-β-adaptin (N = 9 cells), GFP-DAB2 (N = 7 cells), EPS15-GFP (N = 7 cells), GFP-epsin2 (N = 6 cells), and GFP-FCHO2 (N = 8 cells). For each, representative zoomed out images of two-color TIRF-SIM image are shown at the top (a,d,g,j,m,p, scale = 5 μm) The yellow box is shown in higher magnification below (b,e,h,k,n,q, scale = 500 nm). Fluorescence linescans along the yellow lines are shown below both sets of images (c,f,i,l,o,r). One coverslip was imaged for each protein pair.

Supplementary Figure 8 Fluorescence Density Ratio Box Plots, edge proteins at broken lattice, and absolute fluorescence ratios.

(a–c) Fluorescence density ratios from Fig. 7 are shown are shown as box plots. The box plots are 25–75 percentiles and the whiskers are set with an outlier coefficient of 1.5. All proteins are listed in order of decreasing mean FDRrds. Gray diamonds are means from all separate cells imaged for that protein. The cell averages in b middle plot are all one because all FDRs are ratios with respect to the mean domed fluorescence density per cell. (d) Edge proteins found within flat CCSs are found where there is a broken lattice. Images from the three edge proteins, EPS15, FCHO2, and dynamin are shown with correlative images on the left (fluorescence in magenta) and EM only images on the right. At the locations where it looks like the edge protein is located in the middle of the CCS, the EM images show that the clathrin lattice is broken. Scale bar, 400 nm. (e) Absolute fluorescence ratios. The absolute highly curved to domed CCS fluorescence ratio (mean) is plotted against the absolute flat to domed CCS fluorescence ratio (mean). This plot is similar to Fig. 3 except the sizes of the structures are not taken into account. Therefore, this plot shows that on average, a domed CCS turning into a highly-curved CCS gains 25–50% more clathrin. Similarly, it shows that over 50% of the edge proteins, EPS15 and FCHO2 decrease in concentration as the average domed CCS turns into the average highly curved CCS. The flat-to-domed ratios are more difficult to interpret because some flat CCSs in HeLa cells are quite large and most likely turn into several pits. Standard error is shown. N values are based off separate CCSs as given in Supplementary Table 1. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1.

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Sochacki, K., Dickey, A., Strub, MP. et al. Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells.Nat Cell Biol 19, 352–361 (2017). https://doi.org/10.1038/ncb3498

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