Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells

Abstract

Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, β2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones.

Figure 1.. Super-resolution CLEM of core CME components and cargo on platinum replica clathrin lattices.

(a) Platinum replicas of clathrin in WT HeLa cells (scale bar, 100 nm). (b-c) Correlative images with GFP fusions to the core clathrin components, clathrin light chain and AP2 (beta subunit), shown in magenta and EM shown in grayscale. (d-e) Correlative images with GFP fusions to the CME cargo proteins, Transferrin Receptor and VAMP2, shown in magenta and EM shown in grayscale. For b-e, a zoomed-out view is shown on the left (scale bar, 500 nm) with two structures shown at larger magnification on the right (scale bar,100 nm). Larger source images can be found in Supp. Fig. 9. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1 and discussed in the Methods “Statistics and Reproducibility” section.

Figure 2.. Super-resolution CLEM of CME initiation proteins on platinum replica clathrin lattices.

(a-d) Correlative images with GFP fusions to the CME initiation proteins, EPS15, intersectin1s, FCHO2, and NECAP, shown in magenta and EM shown in grayscale. For each, a zoomed-out view is shown on the left (scale bar, 500 nm) with two structures shown at larger magnification on the right (scale bar,100 nm). Larger source images can be found in Supp. Fig. 9. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1 and discussed in the Methods “Statistics and Reproducibility” section.

Figure 3.. Super-resolution CLEM of clathrin adaptors on platinum replica clathrin lattices.

(a-f) Correlative images with GFP fusions to the clathrin adaptors, CALM, epsin1, epsin2, HIP1R, DAB2, β2-arrestin, and stonin2, shown in magenta and EM shown in grayscale. For each, a zoomed-out view is shown on the left (scale bar, 500 nm) with two structures shown at larger magnification on the right (scale bar,100 nm). Larger source images can be found in Supp. Fig. 9. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1 and discussed in the Methods “Statistics and Reproducibility” section.

Figure 4.. Super-resolution CLEM of CME scission-associated proteins on platinum replica clathrin lattices.

(a-f) Correlative images with GFP fusions to the scission-associated proteins, dynamin2, syndapin2, amphiphysin1, and SNX9, shown in magenta and EM shown in grayscale. For each, a zoomed-out view is shown on the left (scale bar, 500 nm) with two structures shown at larger magnification on the right (scale bar,100 nm). Larger source images can be found in Supp. Fig. 9. Number of cells, structures, membrane area imaged, related protein controls, and independent coverslips imaged are listed in Supplementary Table 1 and discussed in the Methods “Statistics and Reproducibility” section.

Figure 5.. CLEM image analysis.

(a-d) Profiles were generated based on mean fluorescence pixel intensity binned by distance from the edge in 12 nm increments. Protein shown is GFPFCHO2. (a) EM image of single clathrin structure. (b) Example edge (tan) outline is shown and bins toward (green) or away (blue) from the center. (c) Binning is then applied to fluorescence data to create a 1DFLIP as in (d). (e) Single 1DFLIPs are normalized by cell intensity (see methods) and averaged to obtain profiles such as these for clathrin light chain fluorescence on domed and flat structures. The 1DFLIPs for clathrin fluorescence on (f) flat and (g) domed CCSs is shown again with the Y-axis being depicted with gray-scale pixel intensity and displayed scaled to a cartoon image of a CCS. The displayed N values are number of clathrin structures analyzed. Standard error is shown in e. Error is also shown as cell-to-cell standard deviation in Supp. Fig. 2. Scale=200 nm in a-c. Scale=24 nm in f-g.

Figure 6.. One-dimensional Fluorescence Intensity Profiles (1DFLIPs) for GFP fusions.

All 1DFLIPs are shown separately with clathrin light chain (black, from Fig. 5e) for (a) flat and (b) domed CCSs. Axes match the axes shown in Fig. 5e. Asterisks denote the side of the protein containing the GFP. Only structures that contained fluorescence were included in the 1DFLIPs and the number of structures included are listed as N. All the 1DFLIPs from a and b are combined with controls from Supp. Fig. 2e (antibody=Ig) and projected with their fluorescence axis depicted with pixel intensity for (c) flat and (d) domed CCSs as was done in Fig. 5f–g. The names of the proteins are given in order according to their colored groupings. Standard error is shown in a-b. Error is also shown as cell-to-cell standard deviation in Supp. Fig. 2. Scale=24 nm in c,d.

Figure 7.. Fluorescence Density Ratios.

FDR_cd vs FDR_fd are calculated as ratios of total intensity per area as described in the methods. Dashes at FDR_cd=2, FDR_fd=0.5 indicate the expected density ratios for proteins statistically distributed throughout the surface of a spherical, hemispherical, or planar object as discussed in the text. Standard Error is shown. These data include structures lacking fluorescence and the number of structures used (N) are shown in Supp. Table 1. Box plots of these data and cell-to-cell variance is shown in Supp. Fig. 8a–c. Total fluorescence ratios, which do not account for structure size, are shown in Supp. Fig. 8e.

Figure 8.. Architectural Model of CME.

(a-f) Six key observed structural phenomena of clathrin-associated proteins are hypothesized to be occurring during different stages of pit development. See last paragraph of text for full description.