RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes

EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation.


Introduction
Transmembrane proteins enter cells via several endocytic pathways including clathrin-dependent endocytosis (CDE) and a variety of less well understood clathrin-independent endocytosis (CIE) mechanisms [1][2][3]. After internalization some receptors will be recycled back to the plasma membrane via the endocytic recycling compartment (ERC) [4,5]. Recycling endosome transport is known to be essential for diverse biological processes, including cell migration, cytokinesis, and synaptic plasticity [5].
In the C. elegans intestine the small GTPase RAB-10 resides on a subset of basolateral endosomes where it regulates basolateral cargo recycling upstream of RME-1/EHD, a membrane remodeling protein with Dynamin-like features [6][7][8][9]. While the cargo-specificity of RME-1 is broad, RAB-10 appears more specific, with especially potent effects on the recycling of transmembrane proteins internalized by CIE, such as the model CIE cargo hTAC (the alpha-chain of the human IL2 receptor) [6,10]. Rab10 function in mammalian cells appears highly conserved, where Rab10 is highly enriched on the membranes of the common recycling endosomes and regulates basolateral recycling in polarized epithelial cells [11]. Likewise, in mammalian adipocytes, Rab10 functions in the insulin-stimulated recycling of glucose transporter GLUT4 [12]. The calponin homology (CH) domain protein Ehbp1 has also been reported to function in GLUT4 recycling in adipocytes, associated with the RME-1 homologs EHD1 and EHD2 [13,14].
In our previous work we determined that C. elegans EHBP-1 binds to the GTP-loaded conformation of RAB-10 through its C-terminal domain (a predicted coiled-coil) and functions with RAB-10 in the intestinal basolateral recycling of hTAC, and in the neuronal recycling of AMPA-type glutamate receptor GLR-1 [10,15]. EHBP-1 labels an extensive network of tubular endosomes in the intestine where it colocalizes with recycling cargo, and is also found on connected punctate endosomal membranes where it colocalizes with RAB-10. Loss of EHBP-1 produces phenotypes that strongly resemble those produced upon loss of RAB-10. These include RAB-10-specific phenotypes in polarized cells such as the intestinal epithelium, including accumulation of enlarged basolateral endosomes filled with fluid-phase markers and hTAC, and the abnormal accumulation of endosomal GLR-1 in interneurons [10,15]. ehbp-1 mutants or RNAi also produce phenotypes in non-polarized cells very similar to simultaneous loss of RAB-10 and its closest paralog RAB-8, including variable larval arrest, and fully penetrant adult sterility due to a failure in germline membrane transport and oocyte growth [15]. In Drosophila dEHBP1 has also been reported to act with Rab11 [16,17].
Our previous studies found that a truncated form of EHBP-1 lacking the RAB-10 interaction domain remained membrane associated, raising the question of how EHBP-1 associates with endosomal membranes [15]. Although not apparent in simple homology searches, a purely computational study using sequence profile searches with profile-profile comparison and fold recognition methods classified the EHBP-1 N-terminus as a putative C2-like domain (NT-C2) that could potentially mediate direct membrane binding [18]. It has been shown that endosomal recruitment of some conserved recycling regulators depends on the regulatory lipid phosphatidylinositol-4,5-bisphosphate [PI (4,5)P2] [9]. PI (4,5)P2 is enriched at the plasma membrane and recycling endosomes, and membrane bending proteins associated with recycling function such as RME-1/EHD and AMPH-1/Amphiphysin/BIN1 have been shown to associate with membrane structures enriched in PI (4,5)P2 [9,19,20]. In fact, we have previously shown that the PI(4,5)P2 level in basolateral recycling endosomes is modulated by RAB-10, in part through its effector CNT-1, an ARF-6 GAP [20]. Other reports also indicate a requirement for phosphatidylinositol-4-phosphate (PI4P) in recycling endosome function [21]. These findings imply that EHBP-1 could be targeted to recycling endosomes via PI (4,5) P2 and/or PI(4)P binding.
In addition to its N-terminal C2-like and C-terminal RAB-10-binding domains, EHBP-1 harbors a central CH domain. CH domains in different proteins are known to bind to the cytoskeleton, but vary in their specificity, with some binding to the microtubule cytoskeleton and others binding to actin filaments [22]. Requirements for the microtubule and actin cytoskeletons are well established in the endosomal system [23][24][25][26].
The actin cytoskeleton also plays essential roles along the endocytic pathway. First identified in studies of endocytosis in yeast, Arp2/3-dependent nucleation of actin at endocytic sites has been observed in many organisms, including mammals, and is thought to contribute to membrane fission [27][28][29][30][31]. Furthermore, certain forms of endocytic recycling are also actin-dependent. For instance, actin depolymerization results in the retention of TAC in tubular recycling endosomes together with Arf6, suggesting the necessity of actin function in Arf6-mediated recycling transport [32,33]. Retromer/WASH mediated local actin polymerization on endosomes has alternately been reported to enhance the fission of tubular cargo carriers from endosomes, or to stabilize tubular extensions for cargo loading prior to their release by fission [34,35].
Cargo carriers in the endosomal system are often tubular in nature, and their tubular shape has been proposed to help sort membrane intrinsic components away from lumenal content [4,36]. The endocytic recycling compartment in mammals is composed of a dense collection of endosomal tubules and vesicles [4]. In the C. elegans intestine the basolateral recycling compartment enriched in CIE cargo, EHBP-1, and RME-1, is highly tubular in nature and appears to have many interconnections [8,15,37]. The entire network collapses to vesicles upon loss of RAB-10 or EHBP-1, suggesting that they contribute to the formation and/or maintenance of such tubular endosomes [6,15].
To further dissect the function of RAB-10 effector EHBP-1, we studied individual domains of EHBP-1 in vitro and in vivo, and characterized EHBP-1 regulation by RAB-10. Here we demonstrate that the NT-C2 and CH domains are both indispensible for proper EHBP-1 function. We found that the EHBP-1 NT-C2 domain has an intrinsic ability to associate with endosomal membranes. RNAi-mediated knockdown of phosphoinositide kinases, colocalization assays with PI(4,5)P2 biosensor PH(PLCδ)-GFP, and liposome co-sedimentation assays revealed that the EHBP-1 NT-C2 domain preferentially associates with PI(4,5)P2 enriched endosomes via predicted patches of basic residues within the NT-C2 domain. Our biochemical studies indicate that the EHBP-1 CH domain preferentially binds to actin filaments and not microtubules, and EHBP-1 colocalizes with endosomal actin in vivo. Remarkably we find that the interaction of the EHBP-1 C-terminal domain with RAB-10(GTP) enhances the actin filament affinity of EHBP-1 via its central CH domain. Our data demonstrates that RAB-10 regulates EHBP-1 actin binding and suggests that RAB-10 and EHBP-1 function together with actin to create and/or maintain endosomal tubulation. cells, a phenotype very similar to rab-10 mutants. Such vacuoles are grossly enlarged early endosomes that can be labeled by fluid-phase endocytosis markers taken up from the basolateral surface (Fig 1B, S1A and S1A' and S1E Fig) [15]. This vacuole phenotype can be fully rescued by intestine-specific expression of tagged forms of full-length EHBP-1 ( Fig 1C and 1C', S1B-S1B'' and S1E Fig).
EHBP-1 contains three distinct protein domains, including an N-terminal C2-like domain (NT-C2), central CH (Calponin Homology) domain, and C-terminal predicted coiled-coil (CC) domain ( Fig 1A) [15,18]. Our previous studies showed that the predicted CC domain of EHBP-1 binds to RAB-10(GTP), and EHBP-1 missing the CC domain does not rescue the ehbp-1 mutant intestinal vacuole phenotype [15]. However, removal of the RAB-10-binding CC-domain does not cause redistribution of EHBP-1 to the cytoplasm. Rather, EHBP-1 lacking the CC-domain remains associated with misshapen endosomal membranes and acts as a dominant negative, impairing recycling [15]. These results indicated that while the CC-domain is important for function, EHBP-1 must have a mechanism for membrane association independent of the RAB-10 binding domain.
Notably, the residual EHBP-1(ΔNT-C2)-GFP labeled puncta were lost upon removal of RAB-10 (S2B Fig). Taken together these results suggest that the NT-C2 domain of EHBP-1 is important for EHBP-1 function and the recruitment to tubular endosomal membranes, with a contribution by the RAB-10-binding CC-domain in recruitment to punctate endosomes.
To further define the endosomal phosphoinositide association preference of EHBP-1, we tested the interaction of purified recombinant GST-NT-C2 with liposomes in sedimentation assays. We found that GST-NT-C2 preferentially pelleted with liposomes containing 5% PI (4,5)P2, with much less co-sedimentation with liposomes containing PI or PI(4)P (Fig 3M and  3N and S4C Fig).

Basic amino acid patches are required for NT-C2-domain recruitment to membranes
To determine residues that may contribute to membrane association, we analyzed evolutionary sequence conservation within the NT-C2 domain, and mapped conserved positively charged residues onto a homology model that we constructed. Our model suggests that the N-terminal 160 amino acids of EHBP-1 folds into a globular domain consisting of seven β-strands and an α-helical segment between strand-5 and strand-6. A patch of basic residues at the extreme N-Representative confocal images are shown for the EHBP-1(NT-C2 domain)-GFP labeling pattern in animals after RNAi-mediated depletion of PI-kinases involved in phosphatidylinositol metabolism. (B) Wild-type EHBP-1(NT-C2)-GFP localized to basolateral punctate and tubular endosomal structures in intestinal cells.

EHBP-1 in Endocytic Recycling
terminus of the fold prior to strand-1 is predicted in this model to contribute to the formation of a concavity in the β sheet, on the "upper surface" comprised of a constellation of basic and hydrophobic residues (Fig 4H).
We performed alanine substitution in three areas of the NT-C2 domain in the context of the NT-C2-GFP intestinal expression construct ( Fig 4G). Compared with wild type, modification of the four arginines within the patch prior to strand-1, predicted to line the concavity, results in loss of association with tubular endosomal membranes and diffusion of the mutated NT-C2-GFP within the cytosol (Fig 4A and 4B and Fig 4I). Mutation of pairs of arginines within this sequence reduced but did not eliminate membrane association, suggesting that all four arginines contribute to NT-C2 domain membrane binding (Fig 4E and 4F and Fig 4I).

EHBP-1 in Endocytic Recycling
Furthermore, we assayed the vacuole phenotype in ehbp-1(tm2523) mutants animals expressing EHBP-1(RRLRR6AALAA)-GFP. In contrast to WT EHBP-1 (S1B-S1B'' Fig), the number and size of vacuoles were not rescued by EHBP-1(RRLRR6AALAA)-GFP, indicating that the RRLRR motif is critical for EHBP-1 recycling function (S4D and S4E Fig). Mutation of nearby lysines, predicted to face away from the cleft, had no effect on the association of NT-C2-GFP with tubular endosomes (Fig 4D and Fig 4I). Another patch of basic residues (HRRRK of strand-2) on the predicted surface of the NT-C2 domain also appears to contribute to NT-C2 membrane association, as mutation of this sequence also produced a diffusive localization ( Fig  4C and Fig 4I). Collectively, our results suggest that EHBP-1 NT-C2 domain associates with PI (4,5)P2 enriched endosomal membranes through two patches of basic amino acids.
Unlike the NT-C2 domain above, which conferred robust localization to endosomes on its own, expression of the EHBP-1 CH-domain (aa260-510), fused to GFP, localized relatively diffusely in the intestinal cells. Sparse puncta were visible above background. These puncta partially overlapped with ARF-6 and RAB-10, indicating that they represent very weak recruitment to endosomes ( RAB-10 was required for this punctate recruitment, since the punctate labeling of EHBP-1-CC-GFP was lost in rab-10(ok1494) mutant animals (Fig 2E and 2E' and Fig 2F). This is distinct from the full length EHBP-1 protein that remains membrane associated in a rab-10 mutant background, presumably through the NT-C2 domain [15].
The EHBP-1 CH domain preferentially interacts with actin microfilaments CH domains have the potential to interact with cytoskeletal elements [22]. Structural studies on the kinetochore attached Ndc80 complex indicated that a CH-domain pair is involved in microtubule binding [38]. Mammalian EHBP1 was shown to colocalize with the cortical actin cytoskeleton in COS-1 cells, and overexpression of HA-EHBP1 induced cortical actin rearrangement [13]. Homology analysis suggests that the CH domain of C. elegans EHBP-1 most closely resembles the CH domain of β spectrin and the second CH domain of utrophin [39,40].
To determine if the EHBP-1 CH-domain interacts with actin microfilaments or microtubules, we assayed for interaction in vitro using co-sedimentation assays (Fig 5A-5F). First, we validated our co-sedimentation assays using human Utrophin actin binding CH-domains Similarly, we found that a purified fusion of GST to the EHBP-1 CH-domain increased its sedimentation by more than 5-fold in the presence of actin microfilaments, indicating that the EHBP-1 CH domain binds to polymerized actin (Fig 5A-5C). By contrast, addition of microtubules to the reaction failed to enhance GST-CH sedimentation (Fig 5D-5F). These results suggested that the EHBP-1 CH-domain functions to link EHBP-1 to polymerized actin.

Association of EHBP-1 labeled tubular recycling endosomes with actin in vivo
Previous work in C. elegans indicated that microtubules are required for the structure of tubular endosomes in the basolateral intestine [37]. Because we found binding of EHBP-1 to actin microfilaments in vitro, we asked whether actin polymerization is also important for the structure of these endosomes. Thus we injected the actin depolymerizing drug latrunculin B (LatB) into the worm pseudocoelom (body cavity) and assayed for effects on the EHBP-1-GFP labeled tubular endosomal meshwork. Indeed, LatB treatment greatly disrupted the EHBP-1-GFP pattern, converting many of the tubules to puncta (Fig 5G-5H and Fig 5J and 5K). Similar treatment with the microtubule-depolymerizing drug nocodazole (Noc) affected the EHBP-1-GFP meshwork in a different manner. The tubular network was still observed, but in a dotted line pattern (Fig 5I-5K). We also assayed the distribution of PH(PLCδ)-GFP labeled basolateral endosomal tubules after LatB and Noc treatments and observed similar results (S5A- S5D Fig).
These results indicate that formation or maintenance of basolateral tubular endosomes labeled by EHBP-1 requires both actin and microtubule cytoskeletal elements, although EHBP-1 itself is probably actin-specific in its interactions.
To further test the functional involvement of actin and microtubules in EHBP-1 mediated recycling we assayed Lat B and Noc treatments for effects on the well-defined recycling CIE cargo marker hTAC-GFP [6,15]. In our previous work we showed that loss of EHBP-1 specifically impaired hTAC-GFP recycling [15]. Our analysis indicates that hTAC-GFP accumulates intracellularly in intestinal epithelial cells after depolymerization of either actin or microtubules (S6A-S6D Fig).
If EHBP-1 links endosomal membranes to actin microfilaments then we would expect to find colocalization of EHBP-1-GFP with F-actin marker Lifeact-RFP. Indeed we found that many punctate regions of endosomes labeled by EHBP-1-GFP in intestinal cells were positive for Lifeact-RFP (Fig 5L-5L"). This is consistent with the localization of actin to endosomes microtubule cytoskeletons. (G) EHBP-1-GFP mainly localized to normal appearing tubular endosomes after injection of control DMSO. (H) The intestinal EHBP-1-GFP positive tubular meshwork was disrupted, and EHBP-1-GFP puncta number increased by about 2-fold, after LatB treatment. (I) Microtubule-depolymerizing drug nocodazole (Noc) treatment did not fully disrupt EHBP-1 labeled tubular network. (J-K) EHBP-1-GFP labeled puncta number (structure count) and total fluorescence area (total area) of these puncta within unit region were quantified respectively. Error bars are SEM (n = 18, 6 animals of each treatment were sampled in three different unit regions of each intestine defined by a 100 x 100 (pixel 2 ) box positioned at random). Asterisks indicate significant differences in the one-tailed Student's t-test (*p< 0.05, *** p< 0.001). (L-L") Actin marker Lifeact-RFP colocalizes well with EHBP-1-GFP on basolateral punctate endosomes in intestinal cells. (M-N" and O) The overlap between Lifeact-RFP and EHBP-1-GFP requires the CH domain. EHBP-1 (ΔNT-C2)-GFP colocalizes with actin marker Lifeact-RFP on punctate structures, however, loss of the EHBP-1 CH domain in a rab-10 mutant background resulted in the decrease of EHBP-1-GFP and Lifeact-RFP overlap percentage (from~47% to~8%). Percentage of GFP fluorescence area overlapping with Lifeact-RFP was sampled in three different regions of each intestine defined by a 100 x 100 (pixel 2 ) box positioned at random (n = 18 per genotype). Analysis of standard deviations was performed by the student's T-test. Error bars are SEM. Asterisks indicate significant differences in the one-tailed Student's t-test (*** p< 0.001). Scale bars represent 10 μm.

RAB-10(GTP) promotes the interaction of EHBP-1 with actin filaments
Our data suggested that EHBP-1 CH-domain associates with endosomal actin microfilaments in vitro and in vivo. To determine whether the RAB-10-binding CC-domain influences the actin affinity of the EHBP-1 CH-domain, we assayed for effects of RAB-10 on the ability of a GST-CH-CC fusion protein to co-sediment with F-actin. Without addition of RAB-10 to the reaction, an EHBP-1 fragment containing the CH and CC domains displayed a similar level of interaction with F-actin as the CH domain alone (Fig 5A and S7A and S7B Fig).
However, we detected elevated co-sedimentation of CH-CC with F-actin in the presence of active HA-RAB-10(Q68L), suggesting that RAB-10(GTP) interaction with the EHBP-1 CCdomain enhances the ability of EHBP-1 to bind to actin filaments (Fig 6A and Fig 6C and 6D).
In contrast, addition of HA-RAB-10(Q68L) did not enhance the ability of an EHBP-1 fragment lacking the RAB-10 binding CC-domain (C2-CH) to co-sediment with actin (S7D and S7E  Fig). As expected, we did not detect interaction between a GST control protein and F-actin ( Fig  6B and Fig 6C). Consistent with the in vitro data, we found that the EHBP-1(CH-CC)-GFP colocalized well with Lifeact-RFP in the C. elegans basolateral intestine (S6G-S6G " Fig).
Importantly, the augmented physical interaction between EHBP-1 and F-actin in the presence of RAB-10 was further confirmed in vivo by co-immunoprecipitation experiments between GFP-tagged EHBP-1 and endogenous actin in whole-worm lysates. EHBP-1-GFP was immunoprecipitated from worm lysates using an anti-GFP antibody and the precipitants were probed with an anti-actin antibody on western blots. The amount of actin co-immunoprecipitating with EHBP-1-GFP was strongly reduced in rab-10(ok1494) mutants, suggesting that RAB-10 promotes the interaction of EHBP-1 with F-actin (Fig 6E).

EHBP-1 mediated bridging of endosomal membranes and actin microfilaments promotes endosomal tubularity
Loss of EHBP-1 disrupts the tubular endosomal network as visualized by hTAC-GFP (Fig 6F  and 6G) or ARF-6-RFP (S8A and S8B Fig). Using the integrity of the hTAC-GFP labeled network as an assay, we sought to test the functionality of versions of EHBP-1 containing different combinations of domains. Importantly we found that overexpression of an EHBP-1 fragment including the membrane associating NT-C2 and F-actin binding CH domains can partially rescue the steady state tubular pattern of recycling cargo marker hTAC-GFP (Fig 6F and 6H and Fig 6K). This was in sharp contrast to the effects of expressing a CH-CC fragment or C2-CC fragment, neither of which could restore hTAC-GFP tubularity (Fig 6I and 6J and Fig 6K). This difference in rescuing ability was even more apparent in time-lapse imaging. Normally the hTAC-GFP labeled endosomal network in the basolateral intestine is highly dynamic, with frequent movement of puncta and tubules (Fig 6L and S1 Video). In ehbp-1 mutant animals the hTAC-GFP labeled endosomes are devoid of movement, appearing almost completely static (Fig 6L and S2 Video). This could be significantly rescued in an ehbp-1 mutant expressing the C2-CH fragment, but not upon expression of CH-CC or C2-CC fragments (Fig 6L and S3-S5 Videos). Compared with~18 tubule movement events (per unit area) /180 sec in wild-type animals, and~1 event/180 sec in ehbp-1(tm2523) mutant animals, C2-CH expression animals presented moderate dynamics with~7 events/180 sec (Fig 6L). These results are consistent with an important role for EHBP-1 in linking the endosomal membrane to the actin cytoskeleton, and the EHBP-1 CC domain-RAB-10 interaction acting as an enhancer for EHBP-1 CH domain-actin filaments binding during endocytic recycling, regulating membrane tubule formation and function.

Discussion
Our studies in C. elegans have demonstrated a requirement for EHBP-1 in basolateral recycling of CIE cargo in intestinal epithelia and postsynaptic recycling of AMPA receptors in interneurons, functioning with the small GTPase RAB-10 [10,15]. EHBP-1 is enriched in the intestinal cells on basolateral tubular and punctate endosomes, and loss of EHBP-1 results in reduced levels of interacting protein RAB-10 on endosomal membranes [15]. Loss of RAB-10 or EHBP-1 also completely disrupts the tubular character of these endosomes [15]. Our new data suggests that this loss of tubular character, which is closely linked with recycling endosome function, is due to a loss of EHBP-1-dependent linkage between endosomal membranes and F-actin.
The EHBP-1 N-terminal domain was predicted by bioinformatics to adopt a C2 domainlike fold (termed NT-C2) that might allow it to bind to membrane phosphatidylinositols, while the central CH domain suggested an interaction with the cytoskeleton [18,22]. In this study, we demonstrated the pivotal roles of EHBP-1 NT-C2 domain and CH domain in EHBP-1-mediated recycling regulation. Using in vitro and in vivo assays, we showed that the NT-C2 domain association with endosomal membranes requires two groups of basic residues (260-901aa) co-sediments with actin filaments in vitro. The co-sedimentation level of GST-CH-CC with actin filaments increased by~67% when complexed with HA-RAB-10(Q68L), a predicted constitutively active form of RAB-10. (B) control protein GST did not co-sediment with actin filaments. P/S ratio (pellet/supernatant) was quantified for GST-CH-CC and GST in (C), error bars are SEM (n = 3), asterisks indicate significant differences in the one-tailed Student's t-test, ** p<0.01, *** p<0.001). (D) Equilibrium binding of GST-CH-CC to F-actin measured by titrating 21 μM F-actin with 2.25 uM to 13.5 uM GST-CH-CC in the presence of HA-RAB-10(Q68L). (E) Co-immunoprecipitation of EHBP-1-GFP and endogenous actin in wild type and rab-10(ok1494) animals. EHBP-1-GFP was immunoprecipitated with anti-GFP antibody and precipitants were analyzed by immunoblotting using anti-actin antibody. Aliquots of total lysates (2% of the total input into the assay) were examined by immunoblotting using anti-actin and anti-GFP antibodies. predicted to form surface patches that could interact with phosphoinositides. We also found that the CH-domain associates with actin filaments but not microtubules, and that F-actin is important for developing the tubular character of these EHBP-1 associated endosomes. Remarkably, we found that the interaction of the EHBP-1 CC domain with RAB-10(GTP) enhanced the CH domain affinity for actin filaments. Thus our studies suggest that RAB-10 promotes bridging of recycling endosomes and actin filaments via EHBP-1 to create or maintain endosomal tubulation.
Recent phylogenetic analysis and structural modeling predicted an NT-C2 domain in the Ehbp1/EHBP-1 extreme N-terminus, providing a potential interface for EHBP-1 membrane lipid binding [18]. Studies focusing on the well known Ca 2+ -dependent C2 domain of PKC and the Ca 2+ -independent C2 domain of PI3K proposed that C2 domain lipid binding capacity involves two structural segments including a calcium binding pocket-like structure and a βsandwich surface respectively [41,42]. The negatively charged acidic residues in the pocket can coordinate Ca 2+ and lipid binding [41,43]. The clustered positively charged basic residues (H, R and K) within the β-sandwich regions of Ca 2+ -independent C2 domains participate in the interaction with negatively charged lipids [18]. However, bioinformatics predictions of the membrane binding mode of NT-C2 family proteins suggested that the NT-C2 extreme N-terminus, prior to strand-1, contains a patch of basic residues on the surface, contributing to lipid binding in parallel with the β-sandwich concave surface [18]. Our data revealed two regions of basic residues that appear to contribute to EHBP-1 NT-C2 membrane association. In our structural model these two basic regions appear to be located on opposite sides of the domain. Further structural dissection of the NT-C2 will be required to determine the true arrangement.
As reported in previous studies, PI (4,5)P2 and PI4P are both enriched in recycling endosomes and are important for recycling transport [20,21]. Our experimental results clearly indicated that the EHBP-1 NT-C2 domain is required for association with tubular endosomes and interacts with PI(4,5)P2. Within the limits of our assays, we did not observe obvious changes in the tubular endosomal network upon knockdown of PI3 kinases known to be important for early endosome function, such as type III PI3-kinase VPS-34 or type I PI3-kinase AGE-1, suggesting that they mainly affect other aspects of endosome function [44][45][46][47][48][49][50][51][52][53][54][55]. Phosphatidylserine (PS) is also known to be quite important for recruitment of many peripheral membrane proteins necessary for membrane traffic, including endocytic recycling, and phosphatidic acid (PA) has been implicated in recycling tubule formation in mammalian cells [56][57][58][59][60][61]. It will be important to test for roles of PS and PA in EHBP-1 function in the future.
C2 domains display a wide range of lipid selectivity, with preference for anionic PS and phosphatidylinositol-phosphates (PIPs) [41]. Unlike lipid binding PH domains [62], C2 lipid targeting often involves two recognition components, such as two lipids or a lipid/protein combination. For instance the protein kinase C (PKC) C2 domain uses its basic surface residues to bind plasma membrane PS and PI(4,5)P2 [63], while cytosolic phospholipase A2 (cPLA2) binds to the neutral lipid phosphatidylcholine (PC) and the anionic lipid ceramide-1-phosphate (C1P) through C2 domain Ca 2+ site charged hydrophobic side chains and a basic cluster [64]. Synaptotagmin utilizes two C2 domains to bridge the vesicular and plasma membranes, with the C2A domain binding vesicular PS and SNARE, while the C2B domain binds plasma membrane PI(4,5)P2 and SNARE [65,66].
The molecular basis for phosphoinositide-binding specificity of C2 and C2-like domains has been explored in recent years. Structural analysis of the PKCα C2 domain showed that PI (4,5)P2 binds to the concave surface of β3 and β4 strands. Intriguingly, aromatic residues Tyr 195 (strand 2) and Trp 245 (strand 5) interact directly with the inositol ring phosphate moieties of PI(4,5)P2. Loss of Tyr 195 and Trp 245 abrogated PI(4,5)P2 recognition and plasma membrane association of PKCα [67]. Phylogenetic analysis showed that Tyr 195 and Trp 245 are conserved among different C2 domains except in the DOCK-C2 and NT-C2 families [18]. However, Trp 71 of EHBP-1/Ehbp1 NT-C2 strand 4 is highly conserved among NT-C2 family members. One plausible possibility is that the Trp 71 residue participates, at least in part, in PI (4,5)P2 binding specificity. Further functional analysis will be required to test this model. Filamentous actin has long been known to be particularly important for the Arf6-mediated recycling of CIE cargo such as TAC and MHCI [5,32]. Colocalization assays and the presence of predicted actin-binding domains have indicated that NT-C2 proteins are involved in actin binding [13,18]. For instance Ehbp1 colocalizes with cortical actin filaments in cultured mammalian adipocytes and in Drosophila pII cells of the external mechanosensory organs [13]. Since certain CH domains have extensively documented actin binding and bundling functions, we hypothesized that EHBP-1 would link to the actin cytoskeleton via its CH domain [68][69][70]. Accordingly, our work strongly suggests that EHBP-1 promotes endosomal tubulation by linking PI(4,5)P2 enriched endosomal membranes to F-actin.
Microtubules are also critical players in many different intracellular trafficking processes. Although some CH domains bind to microtubules, C. elegans tubular endosomes aligned along microtubules, and hTAC recycling in the intestine is impaired upon microtubule disruption, no interaction of the EHBP-1 CH-domain with microtubules was detected in our assays. Thus we infer that while EHBP-1 is microfilament-specific, microtubules play an important role in C. elegans CIE cargo basolateral recycling, collaborating with the microfilament cytoskeleton to shape the endosomal network [37]. EHBP-1 may promote endosome tubulation by transducing force from growing actin filaments to endosomal membranes. Alternatively, EHBP-1 may anchor endosomal membranes to the actin cytoskeleton while other forces, such as pulling by microtubule motors, acts to deform the membranes.
CH-domain based actin binding structures, such as those found in alpha-actinin and spectrin, often present as a tandem arrangement of two CH domains (CH1-CH2) [22,71]. The CH1-CH2 dimer takes on a juxtaposed conformation, with weak F-actin affinity until the dimer adopts an open conformation [72,73]. Utrophin and dystrophin atomic structural models suggest a theme of tandem CH-domains, with one CH domain apposed to the other CH, within the same molecule or provided by two different molecules [74,75].
In the current study our experiments indicated that the EHBP-1 CH domain mediates the interaction with F-actin, and suggested that the RAB-10 interaction with the EHBP-1 CCdomain somehow potentiates the CH domain-F-actin interaction. Since we did not detect a difference in F-actin binding of the EHBP-1 CH only versus CH-CC fragments, and we also did not detect binding of the CH domain to the CC domain, we do not favor an auto-inhibition model for RAB-10 mediated activation of EHBP-1 actin binding activity (S8C Fig). Rather, since RAB-10 interacts with a predicted coiled-coil domain in EHBP-1, RAB-10 binding may potentiate EHBP-1 multimerization, producing a multivalent presentation of apposed CH domains from the dimerized EHBP-1 molecules. Further analysis will be required to test this model (S8D Fig).
We also note that in mammalian adipocytes Rab10 and Ehbp1 are key regulators of insulin stimulated GLUT4 recycling, but their relationship has not been tested [12,14,76]. Future investigation of the Ehbp1-mediated bridging of endosomal membranes and the actin cytoskeleton in human adipocytes could prove fruitful.

General methods and strains
All C. elegans strains were derived originally from the wild-type Bristol strain N2. Worm cultures, genetic crosses, and other C. elegans husbandry were performed according to standard protocols [77]. Strains expressing transgenes were grown at 20°C. A complete list of strains used in this study can be found in S1 Table. RNAi was performed using the feeding method [78]. Feeding constructs were either from the Ahringer library [79] or prepared by PCR from EST clones provided by Dr Yuji Kohara (National Institute of Genetic, Japan) followed by subcloning into the RNAi vector L4440 [78]. For most experiments, synchronized L1 or L3 stage animals were treated for 48-72 h and were scored as adults.
Liposome co-sedimentation assay 3ug GST-EHBP-1(aa1-223) or GST was mixed with 10ul 1mM Control PolyPIPosomes, PI PolyPIPosomes, PI4P PolyPIPosomes, PI(4,5)P2 PolyPIPosomes, respectively (Echelon Biosciences, Salt Lake City, UT) and rotated for 15 min at room temperature in 1 ml liposome binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM MgCl 2 ). The mixture was centrifuged at 90,000xg for 15 min, collecting the supernatant. The liposome pellet was resuspended in 1 ml liposome binding buffer and centrifuged at 90,000xg for 15 min to wash off unspecific bound proteins, this step was repeated three times. The pellet and 20ul supernatant samples were resolved by SDS-PAGE, and GST fusion proteins were detected by western analysis using anti-GST antibody.

F-actin co-sedimentation assay
Actin co-sedimentation assays were performed using an Actin-Binding Protein Biochem Kit: Non-Muscle Actin (BK013) (Cytoskeleton, Denver, CO), essentially as described by the manufacturer. Supplied α-actinin was used as a positive control. Briefly, protein preparations were incubated with 40ul freshly polymerized non-muscle actin (21 μM F-actin) or F-actin buffer alone. In order to test whether RAB-10(Q68L) enhances GST-EHBP-1(260-901aa) binding to F-actin, in vitro synthesized HA-RAB-10(Q68L) and HA-only were added to the mixture. After incubation for 30 minutes at room temperature, samples were centrifuged at 150,000x g for 1.5 h at 24°C to pellet F-actin and the co-sedimenting proteins. Supernatants were collected on ice, and pellets were resuspended on ice for 10 min. SDS-PAGE sample buffer was added to both supernatant and pellet fractions, and the entire fractions were then resolved by SDS-PAGE gel and processed for western blot or stained with coomassie blue. GST-EHBP-1 (260-510aa), GST-EHBP-1(260-901aa), GST-EHBP-1(1-510aa) and GST-hUtrophin(1-261aa) co-sedimentations with F-actin were quantified by densitometry using FluorChem FC3 version 3.4.0 (ProteinSimple, San Jose, CA).

Microtubule co-sedimentation assay
The microtubule-binding assays were performed using the Microtubule Binding Protein Spin-Down Assay Kit (BK029) (Cytoskeleton, Denver, CO). Microtubules were polymerized in cushion buffer (80 mM PIPES pH 7.0, 1 mM MgCl 2 , 1 mM EGTA, 60% glycerol) for 20 min at 35°C and stabilized with taxol. GST-EHBP-1(260-510aa) and the control proteins were mixed separately with microtubules (50μl final volume), incubated at room temperature for 30 min and centrifuged at 100,000x g for 40 min at room temperature on top of a 100 μl of cushion buffer supplemented with taxol. All supernatants and pellets were analyzed by SDS-PAGE as described above.

Whole-worm immunoprecipitation assay
Worms (9cm plates x 10) were collected and washed with M9 buffer. The worm pellet was lysed by French Press in ice-cold lysis buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl,1 mM EDTA, 0.5% NP-40, 1 mM PMSF, 1 mM Na 3 VO 4 , 1 μg/ml Pepstatin-A and 10 mM NaF) containing protease-inhibitor cocktail (Sigma, St. Louis, MO). The lysates were incubated at 4°C for 30 min and centrifugated at 13,000xg for 30min. Then, supernatant was incubated with 80μl Protein A+G Agarose (Beyotime, Shanghai, China) for 1h at 4°C to pre-clear non-specific bead-protein interactions. 2μl anti-GFP antibody (ab290) was added into pre-cleared supernatant and incubated at 4°C overnight, followed by incubation with 80μl Protein A+G Agarose (Beyotime, Shanghai, China) at 4°C for 4 hours. Precipitates were washed five times with lysis buffer and subjected to immunoblotting using anti-actin and anti-GFP polyclonal antibodies.

Confocal microscopy and imaging analysis
Live worms were mounted on 2% agarose pads with 10 mM levamisole. Multi-wavelength fluorescence images were obtained using an FLUOVIEW FV1000 microscope (Olympus, Tokyo, Japan) and captured using FV10-ASW Ver.3.1 software. Images taken in the DAPI channel were used to identify broad-spectrum intestinal autofluorescence caused by lipofuscin-positive lysosome-like organelles. Fluorescence images were obtained using an FV1000-IX81 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 60×N.A. 1.2 oil-immersion objective. Z series of optical sections were acquired using a 0.5μm step size.
To compare the subcellular distribution of GFP-tagged proteins, fluorescence data from GFP channel were analyzed by Metamorph software version 7.8.0.0 (Universal Imaging, West Chester, PA). The "Integrated Morphometry Analysis" function of Metamorph was used to detect the fluorescent structures that are significantly brighter than the background and to measure total puncta number (referred as "structure count") and total fluorescence area (referred as "total area") within unit regions. From total 6 animals of each genotype, "structure count" and "total area" were sampled in three different unit regions of each intestine defined by a 100 x 100 (pixel 2 ) box positioned at random (n = 18 each). In most cases, "total area" was used to compare tubularity, as the normal endosomal tubule network covers much more area than when the network collapses into puncta. Another parameter "structure count" was also sometimes used to assay this aspect, where the structure count increases as the network breaks down into puncta.
GFP and RFP-tagged proteins colocalization analysis were performed using "Measure colocalization" App of Metamorph software. After thresholding, the percentage of GFP fluorescence area (area A) overlapping with RFP fluorescence area (area B) in eighteen intestinal unit regions (3 regions per animal) was analyzed for each genotype. Most GFP/RFP colocalization experiments were performed on L3 and L4 larvae expressing GFP and RFP markers.