The number of vesicle formation sites and vesicle size are altered upon NECAP 1 KD.

<p>(A) Equal protein amounts of lysates from COS-7 cells transduced with control virus (shRNAmiR-control) or viruses to KD NECAP 1 (NECAP 1 nt162, NECAP 1 nt220) were processed by Western blot for the indicated proteins, CHC, clathrin heavy chain; EEA1, early endosomal antigen 1. (B) Immunofluorescence analysis of endogenous AP-2 in control and NECAP 1 KD COS-7 cells. The large panel is a magnification of the area boxed in the small AP-2 panel. The bar represents 20 µm in the small and 5 µm in large panels. (C) Quantification of (B). Repeated measures one-way ANOVA followed by Bonferroni's Multiple Comparison Test revealed a significant difference between control and KD cells, ***<i>p</i><0.0001, <i>N</i> = 4. (D) NECAP 1 KD COS-7 cells were processed for immunofluorescence to reveal endogenous AP-2 and clathrin light chains (clathrin). The bar represents 20 µm. In (B and D), GFP is expressed as part of the viral expression cassette to verify transduction. (E) Distribution of Cy3-labeled transferrin endocytosed for 1 min in control (shRNAmiR-control) and NECAP 1 KD (NECAP 1 nt162 and NECAP 1 nt220) COS-7 cells. The bar represents 20 µm. (F and G) 3D superresolution analysis of AP-2-labeled vesicle formation sites in control (F) and NECAP 1 KD cells (G). The large panels show a 4 µm² area with biplane signals rendered to a 250 nm particle size (“confocal”) to illustrate a traditional microscope image, and rendered to a 5 nm particle size (superresolution). The small panels show individual structures from the large panels as indicated in x/y and x/z orientation with signals rendered to a 5 nm particle size. The 5 nm spot size exceeds the localization precision of each point but shows the distribution and numbers of localizations. The scale bar represents 1 µm in the large and 100 nm in the small panels. (H) Quantification of the x, y, and z diameters of deeply invaginated CCPs in control and NECAP 1 KD cells. Unpaired two-tailed <i>t</i> tests revealed significant differences in size for all three dimensions, <i>x</i>-axis, **<i>p</i> = 0.0038; <i>y</i>-axis, ***<i>p</i><0.0001; <i>z</i>-axis, ***<i>p</i><0.0001; shRNAmiR-control: four cells, 40 vesicle formation sites; NECAP 1 nt 220: six cells, 60 vesicle formation sites. (I) Quantification of the number of AP-2 signals detected per vesicle formation site. An unpaired two-tailed <i>t</i> test revealed a significant difference, ***<i>p</i> = 0.0002; shRNAmiR-control: four cells, 40 vesicle formation sites; NECAP 1 nt 220: six cells, 60 vesicle formation sites. (J) Quantification of the x, y, and z diameters of deeply invaginated vesicle formation sites at the bottom (cell surface contacting the cover slip) or top (cell surface facing the culture medium) of NECAP 1 KD cells. Unpaired two-tailed <i>t</i> tests revealed no significant differences in size for all three dimensions, each location: three cells, 23 vesicle formation sites. (K and L) Size analysis of CCPs in control and NECAP 1 KD COS-7 cells measuring the width of CCPs detected by EM. (K) The electron micrographs show representative examples of structures found in control and KD cells. The bar equals 100 nm. (L) The bar graph shows the average width of CCPs binned for pits depths of 0–50 nm, 50–100 nm, and 100+ nm in control and NECAP 1 KD cells. Statistical analysis using a two-tailed Mann Whitney test revealed a significant difference in size between the two populations in each bin, 0–50 nm, *<i>p</i> = 0.0286; 50–100 nm, *<i>p</i> = 0.0370; 100+, *<i>p</i> = 0.0280. (M) Size analysis of CCVs in control and NECAP 1 KD COS-7 cells measuring the membrane-to-membrane diameter of CCVs detected by EM. The electron micrographs show representative examples of structures found in control and KD cells (bars equal 100 nm), and the bar graph shows the size distribution within the whole population of vesicles analyzed in each condition. Statistical analysis using a two-tailed unpaired <i>t</i> test revealed a significant difference in size between the two populations, ***<i>p</i><0.0001, <i>N</i> = 3.