NECAP 1 Regulates AP-2 Interactions to Control Vesicle Size, Number, and Cargo During Clathrin-Mediated Endocytosis (original) (raw)
Figure 6
NECAP 1 controls accessory protein levels during vesicle formation, which in turn regulate vesicle number, size, and cargo.
(A) Molecular surface representation of PHear. Amino acids implicated in AP180 binding by NMR are labeled and colored according to the size of the amide chemical shift changes observed upon ligand binding (red, Δδ>0.3; orange, 0.3>Δδ>0.2; yellow, 0.2>Δδ>0.1 ppm). (B) Immunofluorescence analysis of endogenous CALM in control and NECAP 1 KD COS-7 cells. GFP is expressed as part of the viral expression cassette to verify transduction. The large panel is a magnification of the area boxed in the small CALM panel. The bar represents 15 µm in the small and 5 µm in large panels. (C) Quantification of CALM intensity per punctum detected in (B). Repeated measures one-way ANOVA followed by Bonferroni's Multiple Comparison Test revealed a significant difference between control and KD cells, **p<0.01, ***p = 0.0008, N = 3. (D) Quantification of AP-2 intensity per vesicle formation site in control, NECAP 1 KD cells, and NECAP 1 KD cells expressing mCherry-tagged CALM. Repeated measures one-way ANOVA followed by Dunnett's Multiple Comparison Test revealed a significant difference between control and KD cells and between KD cells and KD cells expressing CALM, *p<0.05, ***p = 0.0004, N = 6. (E) Immunofluorescence analysis of Texas Red–labeled EGF endocytosis over 2.5 min in control and NECAP 1 KD COS-7 cells. GFP is expressed as part of the viral expression cassette to verify transduction. The bar represents 15 µm. (F) Quantification of EGF endocytosis in control and NECAP 1 KD COS-7 cells over 2.5 min presented in (E). Repeated measures one-way ANOVA followed by Bonferroni's Multiple Comparison Test revealed significant differences between control and NECAP 1 KD cells, **p = 0.0017, N = 4. (G) Quantification of transferrin endocytosis in control and NECAP 1 KD COS-7 cells over 2.5 min. Repeated measures one-way ANOVA followed by Bonferroni's Multiple Comparison Test revealed no significant differences, N = 5. (H) FACS analysis of transferrin endocytosis and recycling in control and NECAP 1 KD COS-7 cells alongside nontransduced wild-type COS-7 cells. Repeated measures two-way ANOVA followed by Bonferroni posttests revealed no significant differences, N = 4. (I) Quantification of the immunofluorescence analysis of the overlap of endogenous EEA1 and Cy3-labeled transferrin endocytosed in control and NECAP 1 KD COS-7 cells at the time points indicated. Repeated measures two-way ANOVA followed by Bonferroni posttests revealed no significant differences, N = 4. (J) Affinity selection of mCherry-tagged FCHo1 and 2 from HEK-293–T cells using purified GST or GST fused to PHear or PHear R95A. (K) Affinity selection of Flag-tagged FCHo1 variants (aa1–327, F-BAR-x alone; aa1–610, F-BAR-x+linker; aa1–837, full-length; aa, amino acids) from HEK-293–T cells using purified GST or GST fused to PHear. (J and K) Starting material (SM) represents 10% of the lysate used in the binding assays. (L) Immunofluorescence analysis of myc-tagged FCHo2 in control and NECAP 1 KD COS-7 cells. GFP is expressed as part of the viral expression cassette to verify transduction. Endogenous myc is detected in the nuclei. The large panel is a magnification of the area boxed in the small myc panel. The bar represents 15 µm in the small and 5 µm in large panels. (M) Quantification of the number of myc-FCHo2 puncta detected in (L). Repeated measures one-way ANOVA followed by Bonferroni's Multiple Comparison Test revealed a significant difference between control and KD cells, ** p = 0.0014, N = 3. (N) Quantification of number of AP-2 puncta per 500 µm2 in control, NECAP 1 KD cells, and NECAP 1 KD cells expressing mCherry-tagged FCHo1. Repeated measures one-way ANOVA followed by Dunnett's Multiple Comparison Test revealed a significant difference between control and KD cells and between KD cells and KD cells expressing FCHo1, ***p<0.001, N = 6.