Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

Tyler Jones; Naava Naslavsky; Steve Caplan

doi:10.1371/journal.pone.0239657

. 2020 Sep 23;15(9):e0239657. doi: 10.1371/journal.pone.0239657

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

Tyler Jones ¹, Naava Naslavsky ¹, Steve Caplan ^1,^2,^*

Editor: Ruben Claudio Aguilar³

PMCID: PMC7511005 PMID: 32966336

Abstract

Upon internalization, receptors are trafficked to sorting endosomes (SE) where they undergo sorting and are then packaged into budding vesicles that undergo fission and transport within the cell. Eps15 Homology Domain Protein 1 (EHD1), the best-characterized member of the Eps15 Homology Domain Protein (EHD) family, has been implicated in catalyzing the fission process that releases endosome-derived vesicles for recycling to the plasma membrane. Indeed, recent studies suggest that upon receptor-mediated internalization, EHD1 is recruited from the cytoplasm to endosomal membranes where it catalyzes vesicular fission. However, the mechanism by which this recruitment occurs remains unknown. Herein, we demonstrate that the EHD1 paralog, EHD4, is required for the recruitment of EHD1 to SE. We show that EHD4 preferentially dimerizes with EHD1, and knock-down of EHD4 expression by siRNA, shRNA or by CRISPR/Cas9 gene-editing leads to impaired EHD1 SE-recruitment and enlarged SE. Moreover, we demonstrate that at least 3 different asparagine-proline-phenylalanine (NPF) motif-containing EHD binding partners, Rabenosyn-5, Syndapin2 and MICAL-L1, are required for the recruitment of EHD1 to SE. Indeed, knock-down of any of these SE-localized EHD interaction partners leads to enlarged SE, presumably due to impaired endosomal fission. Overall, we identify a novel mechanistic role for EHD4 in recruitment of EHD1 to SE, thus positioning EHD4 as an essential component of the EHD1-fission machinery at SE.

Introduction

Upon internalization, receptors, lipids and extracellular fluid are segregated into budding vesicles that are cleaved from the plasma membrane and trafficked to a key endocytic compartment known as the early or sorting endosome (SE) [1]. The SE is a key sorting organelle, and from this organelle, receptors may be transported to late endosomes and lysosomes for degradation, or alternatively, recycled back to the plasma membrane for additional rounds of internalization [2]. In recent years, significant advances have been made in understanding the complex mechanisms that regulate cargo sorting at the SE. For example, it was demonstrated that the ARF GTPase activating protein (GAP), ARF GAP with coiled-coil ankyrin repeat and PH domain-containing protein 1 (ACAP1), interacts with a variety of receptors to direct them back to the plasma membrane [3]. Moreover, coupled with the retromer complex, which entails a Cargo Selection Complex (CSC) trimer of VPS35, VPS29 and either a VPS26a or VPS26b isoform, along with a dimer of sorting nexins (SNX1 or SNX2, and SNX5, SNX6 or SNX32) [4, 5], two members of the sorting nexin family, SNX17 and SNX27, have recently been implicated in controlling the recycling of multiple receptors via interactions between their FERM domains and the cytoplasmic tails of the receptors [3, 6–9]. In addition, the involvement of the SNX17-associated retriever and CCC complexes [10, 11] have further highlighted the active and complex mechanisms by which proteins are sorted and recycled to the plasma membrane.

Despite the progress in understanding the players and mechanisms of sorting at the SE, how the fission of budding vesicles at the SE occurs remains poorly understood. Upon incorporation of receptors into budding transport vesicles, it is necessary to recruit fission machinery for vesicle release. In addition to the retromer [12], the Wiskott–Aldrich syndrome protein (WASH) complex, comprised of WASH1 (also known as WASHC1), Strumpellin (WASHC5), CCDC53 (WASHC3), KIAA1033/SWIP (WASHC4) and Fam21 (also known as WASHC2) [13, 14] has been implicated in vesicular fission via actin nucleation [15]. A recent study further suggest involvement of a novel complex including Rab11-FIP5, VIPAS39, VPS45, Rabenosyn-5 and the dynamin-like Eps15 Homology Domain protein, EHD1 [16]. However, the potential involvement of dynamin-like proteins such as EHD1 and nucleotide hydrolysis at the SE remains likely.

Over the past two decades, the Eps15 Homology Domain protein 1 (EHD1) has emerged as a major regulator of endocytic recycling [17, 18]. Recent studies have demonstrated that in vitro, EHD1 is capable of membrane fission, whereas in cells it localizes to SE and recycling endosomes, and induces ATP-catalyzed membrane fission [19–26]. We have recently demonstrated that EHD1 undergoes recruitment to endosomal membranes upon induction of receptor-mediated endocytosis, where it interacts with SNX17 and promotes endosomal fission [27]. In addition, a number of key asparagine-proline-phenylalanine motif-containing endosomal proteins have been identified that interact with EHD1 and/or EHD4, and may serve to recruit the latter to endosomes [18, 28], including Rabenosyn-5 [29], Syndapins [30, 31], MICAL-L1 [25], and others. While our data are consistent with a model in which SNX17 and EHD1 couple endosomal sorting and the endosomal fission machinery, the mechanism of EHD1 recruitment to endosomes remains unclear.

Herein, we address the potential role of the EHD1 paralog, EHD4, in the process of EHD1 endosomal recruitment and fission. EHD4 shares ~70% identity with EHD1, and has been characterized as a potential EHD1 interaction partner and regulator of trafficking from SE [32, 33]. We demonstrate that EHD4 hetero-dimerizes with EHD1, apparently displaying higher propensity for hetero-dimerization than homo-dimerization, suggesting that it may contribute to the regulation of EHD1 recruitment to endosomes. Consistent with this notion, we showed that impaired EHD4 expression, via siRNA, shRNA or CRISPR/Cas9 gene-editing all led to decreased EHD1 recruitment to endosomes. Moreover, EHD4-depleted cells displayed enlarged SE, likely resulting from impaired endosomal fission. Finally, we demonstrated that EHD4 shares several key endosomal binding partners with EHD1, including Rabenosyn-5 and Syndapin2, and their depletion similarly leads to reduced EHD1 endosomal recruitment and fission. Our findings recognize EHD4 as an important regulator of EHD1-mediated endosomal recruitment and fission.

Materials and methods

Cell lines

The HeLa cervical cancer cell line was acquired from the American Type Culture Collection (ATCC, CRM-CCL-2). NIH3T3 (ATCC, CRL-1658) parental cells were subjected to CRISPR/Cas9 to generate the NIH3T3 cell line expressing endogenous levels of EHD1 with GFP attached to the C-terminus, as well as the EHD1 knock-out, EHD4 knock-out and EHD1/EHD4 double knock-out cells as described [34, 35]. Both HeLa and NIH3T3 cells were cultured at 37°C in 5% CO2 in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat-inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), 1x Penicillin Streptomycin (Gibco, 15140–122), 50 mg of Normocin (InvivoGen, NOL-40-09), and 2 mM L-Glutamine (Gibco, 25030–081). All cell lines were routinely tested for Mycoplasma infection.

Antibodies

The following antibodies were used: Rabbit anti-EHD1 (Abcam, ab109311), Rabbit anti-EHD4 [32], Rabbit anti-EEA1 (Cell Signaling, #3288), Rabbit anti-HA (SAB, #T501), Mouse anti-GFP (Roche, 11814460001), Mouse anti-LRP1 (Novus, NB100-64808), Donkey anti-mouse-HRP (Jackson, 715-035-151), Mouse anti-rabbit IgG light chain-HRP (Jackson, 211-032-171), Alexa-fluor 568-conjugated goat anti-rabbit (Molecular Probes, A11036).

DNA constructs, cloning, and site-directed mutagenesis

Cloning of PTD1, EHD1, EHD4, MICAL-L1, Rabenosyn-5 1–263, and Rabenosyn-5 151–784 in the yeast two-hybrid vector pGADT7 and cloning of PVA3, EHD1, EHD4, EHD1 V203P, EHD1 aa1-439, EHD1 aa1-309, EHD1 aa1-199, EH-1, EHD2, EHD3, EHD3Δcc, MICAL-L1, and Syndapin2 in the yeast two-hybrid vectors were described previously [31, 32, 36–39]. The following constructs were generated via site-directed mutagenesis using Q5 High-Fidelity 2X Master Mix (New England Biosciences, M0492S) according to the manufacturer’s protocol: pGADT7-EHD4 S522A, pGADT7-EHD4 S523D, and pGADT7-EHD4 SS522AD.

Co-immunoprecipitation

HeLa cells were cultured on 100 mm plates until confluent. Cells were lysed with lysis buffer made from 50 mM Tris, pH 7.4, 100 mM NaCl, 0.5% Triton X-100, and 1 x protease cocktail inhibitor (Millipore, 539131) on ice for 15 min with mixing every 5 min. Lysates were centrifuged to clear insoluble matter and then incubated in the absence of antibody or with rabbit anti-EHD1 (Abcam, ab109311) overnight on a rotator at 4°C. Protein G Sepharose Beads 4 Fast Flow (GE Healthcare, 17-0618-01) were added to both control and antibody-containing lysates and mixed on a rotator at 4°C for 4 h. Samples were then washed with the aforementioned lysis buffer and centrifuged at 22,000 x g at 4°C for 30 s. Washes were performed a total of three times and proteins were eluted from the beads by boiling in 4x loading buffer (250 mM Tris, pH 6.8, 8% SDS, 40% glycerol, 5% β-mercaptoethanol, 0.2% bromophenol blue) for 10 min and detected by immunoblotting.

Yeast two-hybrid assay

AH109 yeast were cultured overnight in YPD media containing 10 g/L Bacto Yeast Extract (BD, Ref. 212750), 20 g/L Peptone (Fisher Scientific, CAS RN: 73049-73-7, BP1420-500), and 20 g/L Dextrose (Fisher Scientific, CAS RN: 50-99-7, BP350-1) at 30°C and 250 RPM. Cultures were then spun down at 975 x g for 5 min and the supernatant was aspirated. Pellets were rinsed with autoclaved MilliQ water and centrifuged for an additional 5 min at 975 x g and the supernatant was aspirated. Pellets were resuspended in a suspension buffer of 80% autoclaved MilliQ water, 10% lithium acetate pH = 7.6, and 10% 10x TE pH = 7.5. 125 μl aliquots of the cell suspension were then incubated each with 600 μl of PEG solution (40% PEG (CAS RN: 25322-68-3, Prod. Num. P0885), 100 mM lithium acetate pH = 7.6 in TE pH = 7.5). 1 μl of Yeastmaker Carrier DNA (TaKaRa Cat# 630440) was added to each aliquot, followed by 1 μg of each respective plasmid, and mixed by inverting twice, then by vortexing twice. Mixtures were then incubated at 30°C and 250 RPM for 30 min. 70 μl of DMSO was added to each tube, followed by inverting/mixing twice, and mixtures were placed at 42°C for 1 h. Samples were then placed on ice for 5 min, followed by centrifugation at 22,000 x g for 30 s. The supernatant was aspirated and the samples were resuspended in 40 μl of autoclaved MilliQ water. 15 μl of each sample was then plated and spread on -2 plates (+His) made using 27 g/L DOB Medium (MP, Cat. No. 4025–032), 20 g/L Bacto Agar (BD, Ref. 214010), and 0.64 g/L CSM-Leu-Trp (MP, Cat. No. 4520012) and incubated at 30°C for 72–96 h. Following the incubation period, three separate colonies from each sample were selected and added to 600 μl of autoclaved MilliQ water. In a clean cuvette, 500 μl of the mixture was added to 500 μl of autoclaved water and measured using a spectrophotometer at 600 nm. Mixtures were then normalized to 0.100 λ and 15 μl of each mixture was spotted onto both a -2 plate and a -3 plate (-His) made using 27 g/L DOB Medium (MP, Cat. No. 4025–032), 20 g/L Bacto Agar (BD, Ref. 214010), and 0.62 g/L CSM-His-Leu-Trp (MP, Cat. No. 4530112). Both plates were incubated at 30°C for 72 h and imaged.

siRNA treatment

CRISPR/Cas9 gene-edited NIH3T3 cells expressing endogenous levels of EHD1 with GFP fused to the C-terminus were plated on fibronectin-coated coverslips and grown for 4 h at 37°C in 5% CO2 in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), 1x Penicillin Streptomycin (Gibco, 15140–122), 50 mg of Normocin (InvivoGen, NOL-40-09), and 2 mM L-Glutamine (Gibco, 25030–081). The cells were then treated with either mouse EHD4 siRNA oligonucleotides (Dharmacon, Custom Oligonucleotide, Seq: GAGCAUCAGCAUCAUCGACdTdT), mouse Rabenosyn-5 siRNA oligonucleotides (Dharmacon, On-TARGETplus SMARTpool, cat # L-056534-01-0010), mouse Syndapin 2 siRNA oligonucleotides (Dharmacon, On-TARGETplus SMARTpool, cat # L-045093-01-0005), or mouse MICAL-L1 siRNA oligonucleotides (Dharmacon, On-TARGETplus SMARTpool, cat # L-049952-00-0005) for 72 h at 37°C in 5% CO2 in 1x Opti-MEM 1 (Gibco, 31985–070) containing 12% heat inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150) and 2 mM L-Glutamine (Gibco, 25030–081) using Lipofectamine RNAiMax transfection reagent (Invitrogen, 56531), following the manufacturer’s protocol.

shRNA treatment

HeLa cells were plated on coverslips and grown for 24 h at 37°C in 5% CO2 in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), 1x Penicillin Streptomycin (Gibco, 15140–122), 50 mg of Normocin (InvivoGen, NOL-40-09), and 2 mM L-Glutamine (Gibco, 25030–081). The cells were then treated with pLKO.1-EHD4 shRNA [40] for 72 h at 37°C in 5% CO2 in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), and 2 mM L-Glutamine (Gibco, 25030–081), using FuGene 6 Transfection Reagent (Promega, E2691), following the manufacturer’s protocol.

Transfection

Immunofluorescence and LRP1 uptake

CRISPR/Cas9 gene-edited NIH3T3 cells expressing endogenous levels of EHD1 with GFP attached to the C-terminus were subjected briefly to LRP1 uptake. Uptake was performed by diluting Mouse anti-LRP1 (Novus, NB100-64808) (1:70) in DMEM/High Glucose (HyClone, SH30243.01) containing 10% heat inactivated Fetal Bovine Serum (Atlanta Biologicals, S1150), 1x Penicillin Streptomycin (Gibco, 15140–122), 50 mg of Normocin (InvivoGen, NOL-40-09), and 2 mM L-Glutamine (Gibco, 25030–081) in an ice water bath for 30 min, followed by 2 washes with 1x PBS. Pre-warmed DMEM media, as previously described, was added to these coverslips, which were incubated at 37°C in 5% CO2 for 30 min and washed once in 1x PBS. Following treatment, cells were then fixed in 4% paraformaldehyde (Fisher Scientific, BP531-500) in PBS for 10 min at room temperature. After fixation, cells were rinsed 3 times in 1x PBS and incubated with primary antibody in staining buffer (1x PBS with 0.5% bovine serum albumin and 0.2% saponin) for 1 h at room temperature. Cells were washed 3 times in 1x PBS, followed by incubation with the appropriate fluorochrome-conjugated secondary antibody diluted in staining buffer for 30 min. Cells were washed 3 times in 1x PBS and mounted in Fluoromount-G (SouthernBiotech, 0100–01). Z-stack confocal imaging was performed using a Zeiss LSM 800 confocal microscope with a 63x/1.4 NA oil objective. 10 fields of cells from each condition were collected from 3 independent experiments and assessed using NIH ImageJ.

Graphical and statistical analysis

NIH ImageJ was used to quantify particle count, total area, average size, % area, mean, and integral density. Size parameters were set for 0 –infinity. Circularity parameters were set to 0.0–1.0. Brightness parameters were set to either 75–255 or 125–255, though calculations for 100–255, 150–255, and 175–255 were also conducted. Brightness parameters were selected to eliminate recognition of background by ImageJ’s particle counter while optimizing selection of true positive fluorescent pixels. All statistical analyses were performed with significance using an independent sample two-tailed t-test under the assumption that the two samples have equal variances and normal distribution using the Vassarstats website (http://www.vassarstats.net/), or when comparing multiple samples, with a one-way ANOVA using post-hoc Tukey test for significance (https://astatsa.com). To address biological variations between individual tests, we have designed a modified version of the method described by Folks [41] for deriving a “consensus p-value” to determine the likelihood that the collection of different test/experiments collectively suggests (or refutes) a common null hypothesis, modified from the Liptak-Stouffer method [42]. All the graphics were designed using GraphPad Prism 7.

Results and discussion

Given the role of EHD1 in the regulation of endocytic recycling [21, 24, 25, 29, 36], and its sequence identity and relationship with EHD4 [32, 43], as well as the recent evidence supporting a role for EHD1 in endosomal fission [19, 20, 22, 23, 27, 44–46], we hypothesized that EHD4 coordinates fission and recycling with EHD1. To first test this idea, we assessed the ability of endogenous EHD1 and EHD4 to co-immunoprecipitate. As demonstrated in Fig 1A (left panel), EHD4 appeared in the cell lysates as a ~64 kDa band, and a band of the same size was co-immunoprecipitated by antibodies against EHD1, but not a beads-only control. Although we previously tested our EHD4 antibodies and demonstrated that they do not recognize EHD1 [32], to ensure that the ~64 kDa band was indeed EHD4 and not cross-recognition of EHD1 by the EHD4 antibody, we stripped the nitrocellulose filter paper and reblotted with antibodies to EHD1 (Fig 1A; right panel). Blotting with anti-EHD1 led to detection of a faster migrating ~60 kDa band that clearly migrated below the ~64 kDa EHD4 band, demonstrating that EHD1 and EHD4 reside in a complex in cells. Moreover, consistent with previous findings [32, 33], we found that HA-tagged EHD4 displayed partial co-localization (Pearson’s Coefficient 0.677 with standard deviation of 0.048) with EHD1-GFP in our CRISPR/Cas9 NIH 3T3 gene-edited cells expressing EHD1-GFP (S1 Fig).

We further analyzed the nature of EHD1-EHD4 interactions by instituting a series of truncations and/or mutations in EHD1 (Fig 1B) and testing whether it hetero-dimerizes with EHD4. As shown by yeast two-hybrid (Y2H) analysis, EHD1 both homo-dimerizes and hetero-dimerizes with EHD4, whereas EHD4 preferentially hetero-dimerizes with EHD1, but displays little propensity to homo-dimerize (Fig 1C). In addition, whereas the EH domain of EHD proteins is a well-characterized protein-binding module [28, 47–49], it remains superfluous for EHD dimerization. Since the structure of EHD1 is organized via several domains in addition to the C-terminal EH domain (Fig 1A), we addressed the role of these domains through a series of truncations and mutations. Indeed, truncations in the second helical domain (residue 309) or within the ATP-binding domain (residue 199) abrogated dimerization, whereas a coil-breaking valine-to-proline (V203P) in EHD1 [29] led to dramatically reduced EHD1-EHD4 hetero-dimerization, but had little effect on EHD1 homo-dimerization (Fig 1C). Moreover, EHD4 hetero-dimerized with EHD3 (which displays 86% identity with EHD1) and this interaction was similarly abrogated by the EHD3 V203P coil-breaking mutant (Fig 1D). However, EHD4 was unable to hetero-dimerize with EHD2 (which displays only 67% identity with EHD1 and diverges significantly from the functions of its other paralogs [50–60]) (Fig 1D).

Given the role of EHD1 in endosomal fission [19–26] and our previous study suggesting that EHD4 regulates endosomal size [32], we hypothesized that EHD4 might coordinate endosome fission and size with EHD1. To test this idea, we took untreated cells, mock-shRNA transfected cells, or cells subjected to EHD4-shRNA knock-down and examined EEA1-labeled SE after immunostaining (Fig 2A–2F). As demonstrated, EHD4-shRNA reduced EHD4 levels to almost non-detectable, with only a slight effect on EHD1 levels, potentially due to destabilization coming from the loss of hetero-dimerization (Fig 2G). While the EEA1-labeled SE were generally homogeneous in size and distribution in the untreated and mock-transfected cells (Fig 2; compare 2A and the inset in 2D to 2B and the inset in 2E), SE displayed a significant increase in size upon acute EHD4-depletion (Fig 2C; inset in 2F). Indeed, quantification of mean EEA1-labeled SE size demonstrated a 2-3-fold increase in the acute absence of EHD4 (Fig 2H), suggesting that EHD4 regulates endosomal fission, potentially through its interaction with EHD1.

Fig 2 — *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.

As we have recently demonstrated that EHD1-depletion impairs fission and induces enlarged SE [27], we next asked whether simultaneous depletion of EHD1 and EHD4 further impedes endosomal fission and leads to increased SE size. To this aim, we used CRISPR/Cas9 NIH3T3 cells that were gene-edited and chronically lack EHD1 (EHD1 KO), EHD4 (EHD4 KO) or both EHD1 and EHD4 (EHD1/EHD4 DKO) [34, 35] (Fig 3; protein expression validated in 3I). As demonstrated, in both the EHD1 and EHD4 single KO cell lines, SE size was modestly but significantly larger than in the wild-type parental NIH3T3 cell line (compare B and the inset in F, and C and the inset in G, to A and the inset E, and quantified in J). Moreover, in the EHD1/EHD4 DKO cell line, an additional increase in EEA1-labeled SE size was noted (Fig 3D and inset in 3H, and quantified in 3J). It is of interest that acute siRNA knock-down of EHD4 induces much larger endosome size than the more chronic EHD4 knock-out in the CRISPR/Cas9 NIH3T3 cells, suggesting that compensation may be occurring in the latter cells. Overall, these data are consistent with the role for EHD1 in vesiculation of tubular and vesicular recycling endosomes [19, 20, 22, 23, 45] and further support the notion that both EHD1 and EHD4 regulate endosomal fission, as their depletion leads to enlarged SE in cells.

Fig 3 — *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.

We have recently demonstrated that upon stimulation of receptor-mediated endocytosis, EHD1 can be recruited to SE to carry out fission and facilitate cleavage of budding vesicles and endocytic recycling [27]. However, despite the homology between the 4 EHD paralogs [18, 61], thus far only EHD1 has been directly implicated in fission. Accordingly, based on the interactions we characterized between EHD1 and EHD4, we hypothesized that EHD4 may be required for the recruitment of EHD1 to SE. To address this, we incubated CRISPR/Cas9 gene-edited NIH3T3 cells that express EHD1-GFP with antibodies to low-density lipoprotein receptor-related protein 1 (LRP1) to induce internalization of the receptor-antibody complexes (Fig 4). We have previously demonstrated that by inducing uptake of receptors such as LRP1 or transferrin receptor (unpublished observations) for 30 minutes, we can observe 2–3 fold increases in the recruitment of cytoplasmic EHD1 to SE [27]. The cells were either untreated (A and inset in D), mock-treated (B and inset in E) or EHD4-depleted with siRNA (C and inset in F). EHD4 siRNA knock-down was validated by immunoblotting, and EHD1-GFP levels were similar in untreated, mock and cells where EHD4 knock-down was effected by siRNA (Fig 4G). As demonstrated, in the untreated and mock-treated cells, LRP1 uptake led to the localization of EHD1-GFP to a smattering of vesicular and short tubular SE (Fig 4A and inset in 4D, 4B and inset in 4E; quantified in 4H). However, EHD4 knock-down led to a significant decrease in the number of vesicular and tubular SE marked by EHD1-GFP (Fig 4C and inset, 4F; quantified in 4H). These data suggest that EHD4 is required for the optimal recruitment of EHD1 to SE upon receptor-mediated internalization.

Fig 4 — *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.

EHD1 has been characterized as a protein that binds to motifs containing the tripetide asparagine-proline-phenylalanine (NPF) through its Eps15 homology (EH) domain, particularly when the motif is followed directly by negatively charged residues [28, 47–49, 62, 63]. However, its closest paralog, EHD3, displays more restricted binding to interaction partners [44], and the binding selectivity of EHD4 has not been well characterized. Accordingly, we hypothesized that EHD4 may interact with a subset of NPF-containing proteins that localize to SE and help anchor EHD1-EHD4 dimers to the cytoplasmic side of the SE membrane upon receptor-mediated internalization. To test this notion, we first assessed whether EHD4 could interact with several of the key EHD1-binding partners that contain NPF motifs and localize to SE. Initially, we used Y2H to assess interactions between EHD4 and both Rabenosyn-5 [29] and MICAL-L1, the latter which recruits EHD1 not only to endosomes [25, 31, 64] but also to the centrosome [65]. As demonstrated, similar to EHD1, EHD4 interacted with Rabenosyn-5 (Fig 5A). Somewhat surprisingly, despite being able to interact with EHD1, MICAL-L1 did not display binding to EHD4 (Fig 5A). We have previously shown that an alanine-aspartic acid pair within the EHD1 EH domain (at residues 519 and 520) was required for its selective binding to Rabankyrin-5, whereas the other EHD paralogs did not bind Rabankyrin-5 [44]. We also demonstrated that mutation of the asparagine-glutamic acid pair at the same residues (519 and 520) in the EHD3 EH domain to the alanine-aspartic acid pair found in that position in EHD1 altered its binding selectivity and facilitated an interaction with Rabankyrin-5 [44]. Accordingly, we now asked whether mutation of EHD4’s serine-serine to alanine-aspartic acid at the position that aligns with EHD1’s alanine-aspartic acid residues (residues 519 and 520) would allow it to bind to MICAL-L1 (Fig 5B and 5C). However, as demonstrated, EHD4 remained unable to interact with MICAL-L1, even after the SS to AD substitutions. Since we have shown that MICAL-L1 binds to another NPF-containing protein, Syndapin2 [31], we also tested EHD4-Syndapin2 binding (Fig 5D). As shown, both EHD1 and EHD4 were able to bind Syndapin2. These data suggest that EHD1 homo-dimers and EHD1-EHD4 hetero-dimers have multiple potential recruitment targets on SE.

Fig 5 — 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.

If the EHD1 and EHD4 NPF-containing binding partners are required for EHD1 recruitment to SE and subsequent fission, we rationalized that their depletion would cause reduced EHD1-GFP recruitment to SE upon stimulation of receptor-mediated internalization and increased endosomal size due to impaired fission. Accordingly, we first knocked-down Rabenosyn-5, a potential SE binding partner for both EHD1 and EHD4 (Fig 6). After validating Rabenosyn-5 knock-down efficacy (Fig 6M), we compared EEA1-labeled SE size and EHD1-GFP recruitment to SE in mock-treated cells (Fig 6A–6F) and Rabenosyn-5 knock-down cells (Fig 6G–6L) upon stimulation of receptor-mediated internalization. As demonstrated, upon Rabenosyn-5 knock-down, significantly less EHD1-GFP was observed on SE (compare Fig 6H and 6K to 6B and 6E; quantified in 6O). Moreover, EEA1-labeled SE were significantly larger in Rabenosyn-5 knock-down cells (compare Fig 6G and 6J to 6A and 6D; quantified in 6N). One possibility was that EHD4 helps mediate an interaction between Rabenosyn-5 and EHD1; however, upon EHD4 knock-down we still observed interactions between EHD1 and Rabenosyn-5 (S2 Fig), suggesting that this is not the case. Overall, these data support the notion that Rabenosyn-5 plays a role in the recruitment of EHD1 homo-dimers and EHD1-EHD4 hetero-dimers to SE.

Fig 6 — *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.

We next tested whether Syndapin2, another EHD1 and EHD4 endosomal binding partner, was similarly required for EHD1 recruitment to SE and endosomal fission (Fig 7). Syndapin2 knock-down efficacy was first verified by immunoblotting (Fig 7M). As demonstrated, Syndapin2 knock-down led to dramatically reduced recruitment of EHD1-GFP to endosomes (compare Fig 7H and 7K to 7B and 7E; quantified in 7O). Indeed, impaired recruitment of EHD1-GFP led to enlarged EEA1-labeled SE (compare Fig 7G and 7J to 7A and 7D; quantified in 7N). These data suggest that Syndapin2 is involved in recruitment of EHD1 to SE.

Fig 7 — *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.

Finally, we assessed whether MICAL-L1 is required for EHD1 recruitment and SE fission (Fig 8). As noted, unlike Rabenosyn-5 and Syndapin2, MICAL-L1 bound only to EHD1 and not EHD4 (Fig 5). Nonetheless, upon MICAL-L1 knock-down (validated by immunoblotting in Fig 8M), significantly less EHD1 was recruited to endosomal membranes (compare Fig 8H and 8K and 8B and 8E; quantified in O). Furthermore, EEA1-labeled SE size was enhanced in the MICAL-L1 knock-down cells compared to mock-treated cells (compare Fig 8G and 8J to 8A and 8D; quantified in 8N). Despite the inability of EHD4 to interact with MICAL-L1, these findings may result from the tight interaction between MICAL-L1 and Syndapin2, since degradation of either protein occurs in the absence of its binding partner [31]. Nonetheless, these data indicate that despite being unable to interact directly with EHD4, MICAL-L1 also serves as a potential docking/recruiting site for EHD dimers at the SE membrane.

EHD4 is perhaps the most poorly characterized of the C-terminal EHD family of proteins. Although a variety of physiologic functions have been proposed for it, including within cardiac and kidney cells [66–68], testis development [69], neurons [43, 70, 71] and the extracellular matrix [72], to date its mechanistic function in endocytic membrane trafficking has not been addressed extensively. In this study, we have characterized EHD4’s ability to hetero- and homo-oligomerize and identified several EHD4 interaction partners that also interact with EHD1.

Although the precise stoichiometry of EHD dimers and interaction partners on SE remains unclear, several possibilities exist (Fig 9). For example, EHD1-EHD4 hetero-dimers could either bind to Syndapin2-MICAL-L1 complexes, with EHD1 interacting directly with MICAL-L1 and EHD4 interacting with one of the Syndapin2 NPF motifs. Alternatively, the hetero-dimeric EHD1-EHD4 proteins could dock by binding two adjacent Rabenosyn-5 proteins on the cytoplasmic face of SE membrane, bound to phosphatidylinositol-3-phosphate via its Fab 1, YOTB, Vac 1, and EEA1 (FYVE) domain [73]. On the other hand, EHD1 homo-dimers can bind MICAL-L1-Syndapin2 complexes or Rabenosyn-5 in an unrestricted manner, facilitating recruitment.

Whereas previous studies have addressed the localization of EHD4 to SE [32, 33], these studies predated the concept of EHD1 as a major endosomal fission protein [19, 22, 23, 45, 74]. Moreover, while previous studies demonstrated that EHD4 was in part recruited to SE [32], those studies did not quantify the degree of recruitment, and in our current study we observe significantly impaired recruitment of EHD1 to SE. Our current study supports a role for EHD4 as a dimeric partner with EHD1, facilitating its recruitment to SE and thus similarly implicating EHD4 as a protein intimately connected to the SE fission machinery. These findings are significant, especially since a role for EHD proteins was initially hypothesized in membrane curvature rather than directly in fission [75].

While the stoichiometry of EHD dimers and the precise mode of their recruitment will require further examination, our study helps clarify the complex mechanisms by which EHD1 is recruited to SE to carry out fission and facilitate recycling. We have demonstrated a role for EHD4 in the recruitment of EHD1 to SE, along with at least 3 SE proteins that interact with either EHD1, or both EHD1 and EHD4, namely Rabenosyn-5, Syndapin2 and MICAL-L1. Whether additional SE proteins are also involved in docking EHD dimers on SE remains to be determined.

Supporting information

S1 Fig. Partial co-localization between EHD1 and EHD4.

A-F, HA-EHD4 was transfected into CRISPR/Cas9 gene-edited cells expressing endogenous levels of EHD1-GFP on coverslips, fixed and stained with primary antibodies against HA and secondary Alexa-568 antibodies and imaged to detect HA-EHD4 (red; A and inset in D), EHD1-GFP (green; B and inset in E) and then merged to show both channels (C and inset in F). G, Immunoblot shows expression of the correct-sized HA-EHD4 band at ~65 kDa. The Pearson’s Coefficients were calculated with the NIH ImageJ plugin JACoP, and averaged to provide a value of 0.677 (~68%) with a standard deviation of 0.048.

(TIFF)

Click here for additional data file.^{(2.6MB, tiff)}

S2 Fig. EHD1 immunoprecipitates Rabenosyn-5 in the absence of EHD4.

A, NIH3T3 parental cells were grown on a culture dish, pelleted, lysed and either subject directly to SDS PAGE (lysate; right lane), or first immunoprecipitated with beads only (control; left lane) or with anti-EHD1 coupled beads (middle lane) before immunoblotting with anti-Rabenosyn-5 and anti-EHD1. B and C, CRISPR/Cas9 gene-edited NIH3T3 cells knocked out for EHD4 were grown on a culture dish, pelleted, lysed and either subject directly to SDS PAGE (lysate; right lane), or first immunoprecipitated with beads only (control; left lane) or with anti-EHD1 coupled beads (middle lane) before immunoblotting with anti-Rabenosyn-5 and anti-EHD1. C is a darker exposure of the immunoblot depicted in B.

(TIFF)

Click here for additional data file.^{(2.6MB, tiff)}

S3 Fig. Full-sized gels and repetitions from all of the experiments in the manuscript.

(PDF)

Click here for additional data file.^{(1.4MB, pdf)}

Acknowledgments

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or any funding agency. The authors wish to thank Eylon Caplan for his discussions and assistance in adopting and modifying a robust consensus p-value test.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

S.C. was supported by R01 GM123557 from the National Institutes of General Medical Sciences at the National Institutes of Health. The authors thank the University of Nebraska Medical Center Advanced Microscopy Core Facility, which receives partial support from NIGMS: INBRE P20 GM103427 and COBRE P30 GM106397 grants, as well as support from the National Cancer Institute for The Fred & Pamela Buffett Cancer Center Support Grant P30 CA036727, and the Nebraska Research Initiative. The funders had no role in study design, data collection, analysis, decision to publish or manuscript preparation.

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PLoS One. doi: 10.1371/journal.pone.0239657.r001

Decision Letter 0

Ruben Claudio Aguilar

7 Apr 2020

PONE-D-20-06631

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

PLOS ONE

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Reviewer #2: Partly

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: The study aimed to unveil the role of EHD4 in the fission process that occurs in sorting endosomes (SE). The interaction between EHD1 and EHD4 was reported previously in Sharma et al., 2008. This work unfortunately has not clearly elucidated further the role of EHD4 in the process. Few key questions need to be answered for acceptance of this manuscript.

Specific comments:

1. Fig. 1A: Please address the extra bands seen in the Input blot.

2. Fig. 1B seems incomplete without showing the input levels for EHD1.

3. Fig. 1C: What is the minimal region required for interaction between EHD1 and EHD4? If only the ATP-binding G-domain is required, why do we not see nay interaction when 1-309 fragment is used? Why not include a mutant which lacks completely the EH domain to test its role in the interaction? Seems from your results that something between the residues in EHD1 between 309-439 is required for binding. Kindly discuss this in the text.

4. Fig. 2G: What is the level of EHD1 in these cells? I think it is critical to address this given the discrepancy in the average size of endosome between EDH4 KD and KO in Fig. 2H and Fig. 3H

5. Fig. 3G: How many times was this blot repeated? Are actin levels always much lower than EHD1 and EHD4 levels.

6. Do EHD1 and EHD4 co-localize in cells? If yes, on what compartment and under what conditions? For example, what happens to their co-localization upon internalization of LRP1?

7. The above question will help clarify results from Figure 4. What compartment is EHD1 recruited to? Are the number of these compartments affected or only the recruitment of EHD1 to these structures?

8. Fig. 6-8: These figures do not reveal the role of EHD4 in recruitment of EHD1 to the SE for fission. What is the overlap, if any, between EEA1 and EHD1-GFP? How is this altered in the KD of Rabenosyn-5, Syndapin-2 and MICAL- L1? What is the localization of EHD4 in mock versus the KD of Rabenosyn-5, Syndapin-2 and MICAL- L1? Is the co-localization between EHD1 and EHD4 affected in KD of Rabenosyn-5, Syndapin-2 and MICAL- L1?

9. If EHD4 plays a role in the recruitment of EHD1 to SE via Rabenosyn-5, Syndapin-2 and MICAL- L1, an immunoprecipitation of EHD1 with these proteins in the presence/ or absence of EDH4 will clarify this. Without these experiments, the role of EHD4 in the recruitment of EHD1 via interaction with partners, since they have common partners, is not convincing.

Reviewer #2: This manuscript by Jones et al explores the mechanisms by which EHD4 (a C-terminal EH domain-containing ATPase) contributes to endosome tubulation and fission. These authors and other groups previously found that EHD4 can vesiculate membranes, that it controls endosome size and sorting function, that it binds to its better-characterized homolog EHD1, and that EHD4 is required for normal EHD1 localization to endosomes (George 2007, Sharma, 2008, Cai 2013).

In this manuscript, the authors further define the functional relationship between EHD1 and EHD4. They narrow down the domains through which EHD4 heterodimerizes with EHD1. They extend on the previous cellular studies by using new CRISPR EHD1 and EHD4 single and double knockout cell lines and an EHD1-GFP knockin cell line, showing that EHD4 is involved in EHD1 recruitment to tubular structures. Finally, they find a role for a variety of EHD1/EHD4 binding partners in this process including Rabenosyn-5, Syndapin-2 and MICAL-L1 (though they cannot at present distinguish if these effects are via EHD4 or EHD1 interactions). The data presented opens up new mechanisms for EHD4 function and the authors hypothesize on possible mechanisms for how EHD4 promotes EHD1 mediated fission of sorting intermediates from the endosome. Overall, this work adds more mechanistic understanding of the role of EDH4 to the puzzle of molecular players involved in endosomal fission. To improve the paper, the authors should provide additional clarification and explanations for their rationale and methods as outlined below, and add some more context to place their results in the literature.

Major Points

1. I have several points relating to putting the manuscript in the context of the field.

a. The authors should note more explicitly which of the figures replicate theirs and others’ previous findings (e.g. co-IP of overexpressed EHD1 and EHD4 and endogenous EHD4 by EHD1, some of the characterization of EHD protein domain interactions (e.g. V203P), endosome enlargement upon EHD4 depletion (George 2007, Sharma 2008)), MICAL-L1 being required for EHD1 recrtuiment (Sharma 2009) to put the work in the context of the literature and most importantly to guide the reader to what is new.

b. In particular, the authors previously reported (Sharma 2008) that EHD4 KD led to redistribution of EHD1 to large early endosomes marked with Rab5, whereas in this report, using LRP1-labeled compartments to mark the sorting endosome, they see a “loss” of EHD1 from structures (Fig 4). Can they please introduce this previous result in the text and discuss the difference in these assays (and see point below regarding overall EHD1 levels in these cells)?

c. The introduction could be expanded to include more clarification on the connections between the known players involved in budding vesicle formation. Further explanation on known binding partners (Rabenosyn-5, MICAL-L1, syndapin2) would increase the salience of identifying EHD4s interaction with these proteins. Also making a connection between known regulators of vesicle fission (WASH, Retromer, FERARI) and EHD1 would enhance the introduction/discussion.

2. I have several points relating to experimental design and statistical analysis

a. The authors should justify their rationale for choosing one-tailed t-tests for significance as opposed to other statistical methods, particularly for data sets including more than two conditions. Even if a change in a particular direction is expected, it would be more rigorous for the authors to use a two-tailed test, and even better to use ANOVA as opposed to a t-test for experiments with three or more conditions.

b. The authors should justify the use of experiment (n=3) rather than cells within a representative experiment as their biological replicates, and comment on the spread of the data, particularly with respect to endosomal size. In Figure 2 and 3 in particular, one of the experimental averages is much lower than the others. Does the lower point for each condition correspond to the same experiment?

3. I have several points relating to experimental interpretation

a. The double KD analysis needs clarification. The effect of each of EHD1 and EHD4 single KO on endosome size was previously published, but EHD1 KO is not included in the current experiments for comparison. It is especially relevant to understand how the increase in sorting endosome size in the EHD1 and EHD4 double knock down compares to the EHD1 single knock down. How does the single EHD1 KD compare to EHD4 KOD and EHD1/EHD4 double KD? Does this analysis indicate that in addition to a shared function through heterodimerization, that EHD1 or EHD4 also have functions independent of each other?

b. In Figure 4 was there an overall decrease in EHD1-GFP signal or just a decrease in the localization to vesicles and tubules? If the overall EHD1-GFP signal is decreased, is possible that the decrease in localization to vesicles and tubules is due to an overall decrease in EHD1-GFP?

Minor Points

1. If possible, IPs should show input (on the same blot without lane cropping, with an indication of fraction loaded relative to IP) to show how much of lysate EHD4 immunoprecipitates with EHD1.

2. The authors should add clarification and rationale for choosing the various truncation mutations of EHD1 used to identify EHD1-EHD4 interactions (Figure 1B) and clarification of the LRP1 uptake assay and how this method is used to detect increases in EHD1 localization to vesicles and tubes (Figure 4). This would increase the accessibility of the paper to a wider audience.

3. The authors should comment in the text on why gene edited cells might exhibit a much more subtle EHD4 endosome enlargement phenotype than the KD cells.

4. Authors should describe secondaries and method used to detect immunoblots so the reader can understand if there may be IgG bands.

5. Authors should list the name of the FIJI macro/plugin used for particle analysis, and explain the rationale/use of different brightness settings.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 23;15(9):e0239657. doi: 10.1371/journal.pone.0239657.r002

Author response to Decision Letter 0

20 Aug 2020

Point-by-Point Rebuttal:

Specific comments:

1. Fig. 1A: Please address the extra bands seen in the Input blot.

We apologize for not previously marking the 50 kDa band observed in the HeLa lysate lane as “non-specific,” but we have now done so. The only other “extra” band is the weak one observed above the EHD4, which could be a phosphorylated form, but at present this is unknown.

2. Fig. 1B seems incomplete without showing the input levels for EHD1.

We now provide the input levels for EHD1 for the right-panel of Fig. 1A as requested.

The minimal region required for interaction between EHD1 and EHD4 is within residues 1-439, meaning that from amino acid 439 to the C-terminus at residue 534 (the EH-domain) the residues are superfluous. Unfortunately, the arrows indicating amino acid positions in Fig. 1B were misaligned and we apologize for the confusion; this has now been corrected. The reviewer is correct that our data suggest that the region between residues 309-439 may be responsible for binding, and we speculate that in addition, the V203P mutant, which weakens EHD1 homo-oligomerization, has an even more deleterious effect on the EHD1-EHD4 heterodimers and their interaction. We have now amended the text to reflect these points.

4. Fig. 2G: What is the level of EHD1 in these cells? I think it is critical to address this given the discrepancy in the average size of endosome between EDH4 KD and KO in Fig. 2H and Fig. 3H

The reviewer has raised a good point, and we now provide an immunoblot showing EHD1 levels upon EHD4 acute KD, and have commented on the slight decrease in EHD1 expression under these conditions. With regard to the discrepancy in endosome size between acute EHD4 siRNA knock-down and chronic CRISPR/CAS9 gene-edited EHD4 knock-out cells, we respectfully speculate that long-term chronic knock-out is likely met with mechanisms of compensation, thus the effects are less dramatic. We have amended the text to discuss and highlight this possibility.

5. Fig. 3G: How many times was this blot repeated? Are actin levels always much lower than EHD1 and EHD4 levels.

The immunoblots and experiments in Fig. 3 were repeated 3 times (note a new immunoblot has been included with both EHD1 and EHD4 KO in addition to EHD1/EHD4 KO); this information has now been included in the figure legend. With regard to the actin levels observed—they are not “lower” than the EHD levels, but rather result from a lower concentration of primary and secondary antibodies so that actin, the most highly expressed protein in the cell, can be viewed in the same exposure as the EHD proteins.

6. Do EHD1 and EHD4 co-localize in cells? If yes, on what compartment and under what conditions? For example, what happens to their co-localization upon internalization of LRP1?

This is an excellent question, but unfortunately there are no commercial antibodies available that specifically mark EHD4 by immunofluorescence (without cross-reacting with EHD1). A decade ago, we had prepared rabbit serum in-house to selectively detect EHD4, but once the serum had been used up, we have since been unable to generate an effective new anti-EHD4 serum. To address this important question, we first sought to generate new EHD4 antibodies. While the new antibodies are excellent for immunoblotting, even following affinity purification they are not suitable for immunostaining experiments. Although we recognize it is not ideal and would prefer to immunostain endogenous EHD proteins, to address the concern we have transfected EHD4 into our CRISPR/Cas9 gene-edited EHD1-GFP expressing cells to determine the degree of co-localization, and provide this new information in the form of new Supplemental Fig. 1.

We have previously shown that EHD1 is recruited to the early endosome/sorting endosome (EE/SE), tubular recycling endosomes (TRE), as well as to the ciliary pocket. Upon EHD4 depletion, the number of these compartments is slightly decreased (data not shown), as well as the recruitment of EHD1 to these structures. The EE/SE are still present, though presumably due to a loss of fission, there are slightly fewer, yet larger, structures. This slight reduction in number would not account for the significant loss of EHD1 seen in Fig 4.

We agree with the reviewer that Figures 6-8 don’t necessarily represent direct recruitment of EHD1 by EHD4 to SE; however, since EHD4 appears to exist preferentially in heterodimeric form with EHD1, these experiments reflect the combined recruitment of the dimer pairs. While it would be ideal to address EHD4 localization with the SE markers, EHD1 nonetheless provides an informative surrogate. We have recently demonstrated that EHD1 can be recruited to EEA1-containing SE (Dhawan et al., 2020, J. Biol. Chem.), and in Figs. 6-8, the failure to recruit EHD1 to these structures in the absence of Rabenosyn-5, Syndapin2 and MICAL-L1 likely reflects impaired recruitment of both EHD1-EHD1 dimers and EHD1-EHD4 dimers.

We thank the reviewer for this suggestion and we have performed immunoprecipitations of EHD1 with Rabenosyn-5 in the presence and absence of EHD4, since EHD1 and Rabenosyn-5 display the most robust interaction that can be observed by immunoprecipitation. We provide this new information in the form of new Supplemental Fig. 2. Accordingly, we find that there is no significant difference in interaction.

Major Points

1. I have several points relating to putting the manuscript in the context of the field.

We appreciate the reviewer’s suggestion and have amended the text to make such clarifications. In particular, we have included a new paragraph (second to last in the Results and Discussion) that addresses these issues directly.

We thank the reviewer for this comment; a major difference between the study by Sharma et al. and our current study is that the former was done under “steady-state” conditions, whereas the current study was done upon LRP1 uptake, which we recently described as a trigger to induce endosomal recruitment of EHD1 (Dhawan et al.). Consistent with our previous study, EHD4 knock-down indeed induces larger SE. In addition, also consistent with our previous study, fewer tubular structures were observed containing EHD1. The reviewer is correct that we previously did observe some EHD1 on the enlarged endosomes, but the former study was not quantified, and in the present study while we do observe EHD1-GFP on the enlarged SE, there is quantifiably less. We have included this explanation in the Discussion (amended to the second last paragraph).

We thank the reviewer for this comment; we have modified the Introduction accordingly to include these points and broaden the overall introduction.

2. I have several points relating to experimental design and statistical analysis

We have taken the comments regarding our statistical analyses with utmost seriousness, and have made the following changes: 1) We have applied one-way ANOVA where necessary (Figs. 2, 3 and 4) along with the post-hoc Tukey HSD test for significance. 2) We have moved to independent two-tailed t-tests where ANOVA analysis is not needed. 3) Most importantly, given the concerns about assessing statistical significance with biological samples that often present trends, but have wide variations in absolute values between individual experiments (but not between samples within individual experiments), we adopted the use of a Consensus p-value. This method allows researchers to use statistical calculations to derive an “overall” or consensus p-value from 3 or more experiments. The method we adopted is derived from the papers by Folks (reference 41) and Rice (reference 42), and we believe that inclusion of these statistical methods greatly improves our manuscript.

As discussed in our response to the reviewer’s point above (2a), we have now used a consensus p-value that addresses the very valid concerns of the reviewer. To address biological variations between individual tests, we have designed a modified version of the method described by Folks for deriving a “consensus p-value” to determine the likelihood that the collection of different test/experiments collectively suggests (or refutes) a common null hypothesis, modified from the Liptak-Stouffer method. Such consensus p-values put less emphasis on absolute values, and more on the trends between individual experiments.

3. I have several points relating to experimental interpretation

We appreciate the suggestion by the reviewer and have added the EHD1 KO condition for comparison to EHD4 KO and the EHD1/EHD4 DKO (see revised Fig. 3).

The number of EHD1-GFP punctae was decreased upon EHD4 KD. EHD4 siRNA was assessed to ensure that EHD1 protein levels are not impacted. We have included an immunoblot illustrating that EHD1-GFP protein levels are not impacted upon EHD4 KD.

Minor Points

1. If possible, IPs should show input (on the same blot without lane cropping, with an indication of fraction loaded relative to IP) to show how much of lysate EHD4 immunoprecipitates with EHD1.

We now provide the input levels for EHD1 for the right-panel of Fig. 1A as requested.

We thank the reviewer for helping us to clarify, and have included this in the text.

3. The authors should comment in the text on why gene edited cells might exhibit a much more subtle EHD4 endosome enlargement phenotype than the KD cells.

We have now addressed this issue in the Results and Discussion.

4. Authors should describe secondaries and method used to detect immunoblots so the reader can understand if there may be IgG bands.

We have added additional explanation describing secondary antibodies and immunoblotting.

5. Authors should list the name of the FIJI macro/plugin used for particle analysis, and explain the rationale/use of different brightness settings.

No macros or plugins were used for particle analysis, only using tools available in the base version. We have added further explanation of the particle analysis and the rationale behind using particular brightness settings. For the co-localization analysis in the new Supplemental Fig. 2, please see the figure legend for the macro/plugins used.

Attachment

Submitted filename: Rebuttal- SC-Aug10-2020.docx

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PLoS One. doi: 10.1371/journal.pone.0239657.r003

Decision Letter 1

Ruben Claudio Aguilar

11 Sep 2020

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

PONE-D-20-06631R1

Dear Dr. Caplan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: Overall, we feel our concerns were addressed and we recommend this paper for publication.

Major points

1. The authors did a good job contextualizing their findings in the field as well as with their previous publication.

2. The authors addressed concerns about the statistical tests and recalculated statistical measures for all experiments in the manuscript. However, they do not detail how they “modified” the Folks and Liptak-Stouffer methods. This should be clarified in the methods.

3. The authors added the additional data which we requested. We note that identifying the specific compartment on which EHD1 and EHD4 interact may be complex and is likely beyond the scope of this paper.

The authors addressed all of our minor concerns.

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PLoS One. doi: 10.1371/journal.pone.0239657.r004

Acceptance letter

Ruben Claudio Aguilar

14 Sep 2020

PONE-D-20-06631R1

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

Dear Dr. Caplan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Partial co-localization between EHD1 and EHD4.

(TIFF)

Click here for additional data file.^{(2.6MB, tiff)}

S2 Fig. EHD1 immunoprecipitates Rabenosyn-5 in the absence of EHD4.

(TIFF)

Click here for additional data file.^{(2.6MB, tiff)}

S3 Fig. Full-sized gels and repetitions from all of the experiments in the manuscript.

(PDF)

Click here for additional data file.^{(1.4MB, pdf)}

Attachment

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

Tyler Jones

Naava Naslavsky

Steve Caplan

Roles

Abstract

Introduction

Materials and methods

Cell lines

Antibodies

DNA constructs, cloning, and site-directed mutagenesis

Co-immunoprecipitation

Yeast two-hybrid assay

siRNA treatment

shRNA treatment

Transfection

Immunofluorescence and LRP1 uptake

Graphical and statistical analysis

Results and discussion

Fig 1. Interaction between endogenous EHD1 and EHD4.

Fig 2. EHD4 depletion induces enlarged sorting endosomes.

Fig 3. EHD1 and EHD4 coordinately control endosome size.

Fig 4. Reduced EHD1 recruitment to endosomes upon EHD4 knock-down.

Fig 5. EHD4 interacts with sorting endosome resident proteins.

Fig 6. Increased sorting endosome size and decreased EHD1 recruitment upon Rabenosyn-5 knock-down.

Fig 7. Increased sorting endosome size and decreased EHD1 recruitment upon Syndapin2 knock-down.

Fig 8. Increased sorting endosome size and decreased EHD1 recruitment upon MICAL-L1 knock-down.

Fig 9. Model depicting potential mechanisms for EHD1 endosomal recruitment.

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ruben Claudio Aguilar

Roles

Author response to Decision Letter 0

Decision Letter 1

Ruben Claudio Aguilar

Roles

Acceptance letter

Ruben Claudio Aguilar

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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