Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis

Abstract

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP-binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1-depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP-binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1-depleted cells.

Keywords: ATP-binding; CP110; EHD1; EHD2; EHD3; EHD4; MICAL-L1; SNAP29; ciliary vesicle; ciliogenesis; distal appendage vesicle; mother centriole; primary cilium.

FIGURE 1

EHD4 regulates primary ciliogenesis and its depletion prevents CP110 removal from the m‐centriole. (A, B) Representative fields of cells depicting primary cilia labeled with acetylated tubulin (red) and DAPI (blue) in mock‐treated (A) and EHD4 knock‐down cells (B). (C–J) Representative micrographs depicting primary cilia labeled with acetylated tubulin (red) and marked by CP110 (green) and DAPI (blue) in mock‐treated (C–F) and EHD4 knock‐down cells (G–J). NIH3T3 cells were either mock‐treated with transfection reagent (A; C, inset in D–F), or transfected with EHD4 siRNA oligonucleotides (B; G, inset in H–J) for 48 h, fixed and immunostained with DAPI and antibodies to detect acetylated tubulin and CP110 prior to imaging. Arrowheads denote primary cilia and arrows mark centrosomes/basal bodies in the micrographs. (K) Validation of EHD4 siRNA efficacy by immunoblot analysis, with actin used as a control. (L) Graph depicting the percentage of ciliated cells in mock‐treated and EHD4 knock‐down cells. (M) Graph illustrating the percentage of centrosomes/basal bodies with two CP110 dots in mock‐treated and EHD4 knock‐down cells. Error bars denote standard deviation, and p values for each experiment were determined by an independent two‐tailed t test. Percentage of ciliated cells and percentage of cells with two CP110 dots per centrosome/basal body were calculated from two separate sets of three experiments. All six experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. Significance between samples for each set of three experiments was calculated by deriving a consensus p value based on Folks and Rice and our previous study (see Section 2). Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bars (B and G), 10 μm, Bar for insets, 2.7 μm. i: consensus p < 0.00001

FIGURE 2

EHD2 is not required for normal primary ciliogenesis. (A–H) Representative micrographs of NIH3T3 cells that were engineered by CRISPR/Cas9 to express endogenous levels of EHD2 tagged with GFP (EHD2‐GFP) depicting primary cilia labeled with antibodies against acetylated tubulin (red) and DAPI stain (blue). CRISPR/Cas9 gene‐edited NIH3T3 EHD2‐GFP cells were either mock‐treated with transfection reagent (A, inset in B–D), or transfected with EHD2 siRNA (E, inset in F–H) for 48 h, fixed and immunostained with DAPI and an acetylated tubulin antibody prior to imaging. (I) Validation of EHD2 siRNA efficacy by immunoblot analysis. (J) Graph depicting the percentage of ciliated cells in mock‐treated and EHD2 knock‐down NIH3T3 EHD2‐GFP cells. Error bars denote standard deviation and p values for each experiment were determined by an independent two‐tailed t test. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bars, 10 μm. n.s. = not significant (consensus p > 0.05)

FIGURE 3

The EHD1 G65R mutant does not bind to SNAP29 and MICAL‐L1. (A) Amino acid sequence comparison of the four human EHD orthologs, EHD1‐4, in the region adjacent to glycine 65. Sequences are aligned with residues 56–75 of EHD1. (B) Yeast two‐hybrid colony growth reflecting interactions between either EHD1 WT or EHD1 G65R with SNAP29 and MICAL‐L1. The experiment depicted is representative of three independent experiments.

FIGURE 4

Ciliogenesis in EHD1 knock‐out cells is rescued by WT EHD1 but not the EHD1 G65R mutant. (A–I) Representative micrographs depicting primary cilia labeled by acetylated tubulin (red) and GFP‐EHD1 (green) and DAPI stain (blue) in EHD1 knock‐out (KO) cells that were either untransfected, or transfected with GFP‐EHD1 WT, or GFP‐EHD1 G65R. CRISPR/Cas9 gene‐edited NIH3T3 EHD1‐KO cells were either mock‐treated with transfection reagent (no transfection) (A–C), transfected with GFP‐EHD1 WT (D–F), or transfected with the GFP‐EHD1 G65R mutant (G–I) for 48 h, fixed and immunostained with DAPI, an anti‐GFP antibody and an acetylated tubulin antibody prior to imaging. (J) Validation of GFP‐EHD1 WT and G65R transfection efficacy by immunoblot analysis. (K) Graph illustrating the corrected total cell fluorescence values for each cell transfected with either GFP‐EHD1 WT or GFP‐EHD1 G65R. (L) Graph depicting the percentage of ciliated cells in non‐transfected, GFP‐EHD1 WT transfected and GFP‐EHD1 G65R transfected cells. (M) Graph illustrating the percent of cells where EHD1 is localized to the primary cilium or centrosome in non‐transfected, GFP‐EHD1 WT transfected and GFP‐EHD1 G65R transfected cells. Error bars denote standard deviation, and p values for each experiment were determined by one‐way ANOVA. All six experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the six experiments. Micrographs are representative orthogonal projections from six independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bar, 10 μm. n.s. = not significant (consensus p > 0.05). i: p < 0.001; iii consensus p < 0.00001

FIGURE 5

EHD1 E470W, but not P446S, disrupts MICAL‐L1 binding. (A) Amino acid sequence alignment highlighting residue homology between residues 441 and 475 of EHD1 and its paralogs EHD2, EHD3 and EHD4. Based on these alignments, P446S and E470W substitutions in EHD1 were made to conform with the EHD2 sequences. (B) Yeast two‐hybrid colony growth depicting interactions between either EHD1 WT, EHD1 P446S, EHD1 E470W, or EHD1 P446S/E470W with MICAL‐L1.

FIGURE 6

EHD1 P446S and E470W do not rescue ciliogenesis. (A–L) Representative micrographs depicting primary cilia labeled with acetylated tubulin (red), GFP‐EHD1 (green) and DAPI stain (blue) in NIH3T3 EHD1‐KO cells that were mock‐treated (no transfection), or transfected with GFP‐EHD1 WT, GFP‐EHD1 P446S, or GFP‐EHD1 E470W. CRISPR/Cas9 gene‐edited NIH3T3 EHD1‐KO cells were either mock‐treated with transfection reagent (no transfection) (A–C), transfected with GFP‐EHD1 WT (D–F), transfected with GFP‐EHD1 P446S (G–I), or transfected with GFP‐EHD1 E470W (J–L) for 48 h, fixed and immunostained with DAPI, an anti‐GFP antibody and an acetylated tubulin antibody prior to imaging. (M) Validation of GFP‐EHD1 transfection efficiency by immunoblot analysis. (N) Graph depicting the percentage of ciliated cells in mock‐treated, GFP‐EHD1 WT, GFP‐EHD1 P446S and GFP‐EHD1 E470W cells. (O) Graph illustrating the percent of cells with EHD1 localized to the primary cilium or centrosome in mock‐treated, GFP‐EHD1 WT, GFP‐EHD1 P446S and GFP‐EHD1 E470W cells. Error bars denote standard deviation and p values for each experiment were determined by one‐way ANOVA. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three 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. (i) consensus p < 0.05, (iii) consensus p < 0.00001

FIGURE 7

Proposed mechanism of SNAP29 recruitment for distal appendage vesicle fusion and ciliary vesicle formation. Model depicting a proposed mechanism by which EHD1 mediates primary ciliogenesis. EHD1 dimers are recruited to the centrosome by MICAL‐L1, which in turn recruit SNAP29 to mediate the fusion of the distal appendage vesicles to form the ciliary vesicle. Dimerization of EHD1 may facilitate concomitant interactions of individual EHD1 proteins with the NPF‐containing proteins MICAL‐L1 and SNAP29. The EHD1 ATP‐binding/hydrolysis mutant G65R is unable to dimerize and fails to interact with either MICAL‐L1 or SNAP29, preventing fusion of the distal appendage vesicles and formation of the ciliary vesicle. EHD1 E470W exhibits reduced binding to MICAL‐L1 and its ability to interact with SNAP29 is currently unknown, but it remains incapable of supporting ciliogenesis.