Fig 1.
Interaction between endogenous EHD1 and EHD4.
A, EHD4 co-immunoprecipitates with EHD1. HeLa lysates were incubated at 4°C overnight in the presence or absence of anti-EHD1 antibody. Protein G beads were then added to the lysate-only (beads only) or lysate-antibody (anti-EHD1) mix at 4°C for 3 h. Bound proteins were then eluted by boiling for 10 min at 95°C in β-mercaptoethanol-containing loading buffer, separated by SDS-PAGE, and immunoblotted with anti-EHD4 antibodies (left panel) or stripped and then immunoblotted with anti-EHD1 antibodies (right panel). Input lysates (25%) are depicted (left and right panels, right lane). B, Schematic diagram depicting the domain architecture of C-terminal EHD proteins, indicating residues that were replaced by site-directed mutagenesis and identifying the truncations used in this study. C, Yeast two-hybrid colony growth characterizing the interaction between EHD1 and EHD4. Co-transformation of both pGADT7 and pGBKT7 vectors is required for growth on plates lacking leucine and tryptophan (left panel; +His plates), whereas interaction between the fusion proteins is required for histidine synthesis and growth on -His plates (right panel). + His plates illustrate that both target vectors have been transformed into the yeast.—His plates inform whether the proteins of interest interact. 1: V203P represents an amino acid substitution at residue 203 of EHD1 that is predicted to interfere with coiled-coil formation, whereas 1: 1–439, 1: 1–309 and 1: 1–199 represent various EHD1 truncations. D, Yeast two-hybrid colony growth characterizing the interaction between EHD4 and other EHD proteins. EHD3Δcc represents full-length EHD3 with a valine to proline substitution at residue 203, whereas EH-1 represents the EHD1 EH domain only (residues 436–534). n.s.; non-specific band, ad; activation domain, bd; binding domain.
Fig 2.
EHD4 depletion induces enlarged sorting endosomes.
A-F, Representative micrographs and insets depicting EEA1-labeled endosomes in untreated, mock-treated, and EHD4 knock-down cells. HeLa cells were either untreated (A and inset in D), mock-treated with transfection reagent (B and inset in E), or transfected with an EHD4 shRNA construct (C and inset in F) for 72 h, fixed and immunostained with an EEA1 antibody prior to imaging. G, Validation of EHD4 shRNA efficacy by immunoblot analysis. H, Graph depicting differences in mean EEA1-labeled SE size in untreated, mock-treated and EHD4 knock-down cells. Error bars denote standard deviation and p-values for each experiment were determined by one-way ANOVA for individual experiments using a post-hoc Tukey HSD calculator to determine significance. All 3 experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. Significance between samples for the 3 experiments was calculated by deriving a consensus p-value (see Materials and methods). Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z-stacks collected for each treatment per experiment. Bar, 10 μm. n.s. = not significant (p > 0.5), **p < 0.00001.
Fig 3.
EHD1 and EHD4 coordinately control endosome size.
A-H, Representative micrographs and insets for parental NIH3T3 cells (Parental; A and inset in E), EHD1 knock-out NIH3T3 cells (EHD1 KO; B and inset in F), EHD4 knock-out NIH3T3 cells (EHD4 KO; C and inset in G), and EHD1/EHD4 double knock-out cells (EHD4 DKO; D and inset in H). Parental NIH3T3 and CRISPR/Cas9 gene-edited NIH3T3 cells lacking either EHD1 (EHD1 KO), EHD4 (EHD4 KO), or both EHD1 and EHD4 (EHD1/EHD4 DKO) were fixed and immunostained with antibodies to EEA1, and then imaged by confocal microscopy. I, Immunoblot showing reduced EHD1 expression in EHD1 KO cells, reduced EHD4 expression in EHD4 KO cells and reduced EHD1 and EHD4 expression in EHD1/EHD4 DKO (1/4 DKO) cells. J, Graph depicting mean EEA1-labeled endosome size in parental and KO cells. Individual experiments were performed 3 times. Error bars denote standard deviation and p-values were determined by one-way ANOVA for individual experiments using a post-hoc Tukey HSD calculator to determine significance. A consensus p-value was then derived as described in the Materials and methods to assess significant differences between samples from the 3 experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z-stacks collected for each treatment per experiment. Bar, 10 μm. Consensus p-values from Tukey HSD: *p = 0.003, **p = 0.001.
Fig 4.
Reduced EHD1 recruitment to endosomes upon EHD4 knock-down.
A-F, Representative micrographs and insets depicting EHD1-GFP recruitment to endosomes in untreated (A and inset in D), mock-treated (B and inset in E), and EHD4 knock-down (C and inset in F) cells. CRISPR/Cas9 gene-edited NIH3T3 cells expressing endogenous levels of EHD1 with GFP fused to the C-terminus (EHD1-GFP) were either untreated, mock-treated with transfection reagent, or transfected with EHD4 siRNA for 72 h. Cells were then incubated with anti-LRP1 antibody (30 min on ice, 30 min at 37°C), fixed, and imaged via confocal microscopy. G, Immunoblot showing reduced EHD4 (but not EHD1-GFP) expression in EHD1-GFP cells, with actin used as a loading control. The nitrocellulose filter paper was then stripped and immunoblotted with anti-GFP to show EHD1-GFP expression upon EHD4 loss. H, Graph depicting the mean count of EHD1-labeled endosomes in untreated, mock-treated and EHD4-depleted cells. Individual experiments were performed 3 times. Error bars denote standard deviation and p-values were determined by one-way ANOVA for individual experiments using a post-hoc Tukey HSD calculator to determine significance. A consensus p-value was then derived as described in the Materials and methods to assess significant differences between samples from the 3 experiments. Micrographs are representative orthogonal projects from three independent experiments, with 10 sets of z-stacks collected for each treatment per experiment Bar, 10 μm. n.s., not significant (consensus p > 0.5). Consensus p-values from Tukey HSD: *p < 0.00001.
Fig 5.
EHD4 interacts with sorting endosome resident proteins.
A, Yeast two-hybrid colony growth demonstrating interactions between both EHD1 and EHD4 with Rabenosyn-5, and between EHD1 and MICAL-L1. Two Rabenosyn-5 constructs were utilized: Rabenosyn-5 151–784 contains 5 Asparagine-Proline-Phenylalanine (NPF) motifs, whereas Rabenosyn-5 1–263 is devoid of NPF motifs. B, Schematic illustration depicting residue homology between a region within the EH-domains of EHD1 and EHD4. C, Yeast two-hybrid colony growth assessing the interactions between either EHD1, EHD4, or EHD4 mutants with MICAL-L1. D, Yeast two-hybrid assay depicting an interaction between either EHD1 or EHD4 with Syndapin2. ad; activation domain, bd; binding domain.
Fig 6.
Increased sorting endosome size and decreased EHD1 recruitment upon Rabenosyn-5 knock-down.
A-L, Representative micrographs and insets depicting EEA1-labeled endosomes and EHD1-GFP in mock-treated and Rabenosyn-5 knock-down cells. CRISPR/Cas9 gene-edited NIH3T3 EHD1-GFP cells were either mock-treated with transfection reagent (A-F) or treated with Rabenosyn-5 siRNA (G-L) for 72 h. Cells were then incubated with anti-LRP1 antibody (30 min on ice, 30 min at 37°C), fixed and immunostained using anti-EEA1, and imaged by confocal microscopy. M, Immunoblot showing reduced Rabenosyn-5 expression in EHD1-GFP NIH3T3 cells. N, Graph depicting mean endosome size of mock-treated and Rabenosyn-5 knock-down cells. O, Graph depicting EHD1 recruitment to endosomes in mock-treated and Rabenosyn-5 knock-down cells. Error bars denote standard deviation and p-values were determined by independent two-tailed t-test, with significance derived from consensus p-values from the 3 experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z-stacks collected for each treatment per experiment. Bar, 10 μm. **p < 0.00001.
Fig 7.
Increased sorting endosome size and decreased EHD1 recruitment upon Syndapin2 knock-down.
A-L, Representative micrographs and insets depicting EEA1-labeled endosomes and EHD1-GFP in mock-treated and Syndapin2 knock-down cells. CRISPR/Cas9 gene-edited NIH3T3 EHD1-GFP cells were either mock-treated with transfection reagent (A-F) or treated with Syndapin2 siRNA (G-L) for 72 h. Cells were then incubated with anti-LRP1 antibody (30 min on ice, 30 min at 37°C), fixed and immunostained using anti-EEA1, and imaged by confocal microscopy. M, Immunoblot showing reduced Syndapin2 expression in EHD1-GFP NIH3T3 cells. N, Graph depicting mean endosome size of mock-treated and Syndapin2 knock-down cells. O, Graph depicting EHD1 recruitment to endosomes in mock-treated and Syndapin2 knock-down cells. Error bars denote standard deviation and p-values were determined by independent two-tailed t-test, with significance derived from consensus p-values from the 3 experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z-stacks collected for each treatment per experiment. Bar, 10 μm. **p < 0.00001.
Fig 8.
Increased sorting endosome size and decreased EHD1 recruitment upon MICAL-L1 knock-down.
A-L, Representative micrographs and insets depicting EEA1-labeled endosomes and EHD1-GFP in mock-treated and MICAL-L1 knock-down cells. CRISPR/Cas9 gene-edited NIH3T3 EHD1-GFP cells were either mock-treated with transfection reagent (A-L) or treated with MICAL-L1 siRNA (G-L) for 72 h. Cells were then incubated with anti-LRP1 antibody (30 min on ice, 30 min at 37°C), fixed and immunostained using anti-EEA1, and imaged by confocal microscopy. M, Immunoblot showing reduced MICAL-L1 expression in EHD1-GFP NIH3T3 cells. N, Graph depicting mean endosome size of mock-treated and MICAL-L1 knock-down cells. O, Graph depicting EHD1 recruitment to endosomes in mock-treated and MICAL-L1 knock-down cells. Error bars denote standard deviation and p-values were determined by independent two-tailed t-test, with significance derived from consensus p-values from the 3 experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z-stacks collected for each treatment per experiment. Bar, 10 μm. **p < 0.00001.
Fig 9.
Model depicting potential mechanisms for EHD1 endosomal recruitment.
Syn2, Syndapin2; R5, Rabenosyn-5; EHD1, Eps15 Homology Domain Protein 1; EHD4, Eps15 Homology Domain Protein 4; EH, Eps15 Homology Domain.