The Calponin Family Member CHDP-1 Interacts with Rac/CED-10 to Promote Cell Protrusions

Cell protrusion defects in xd27 animals

BDU neurons are a pair of interneurons with cell bodies situated laterally in the anterior body of C. elegans. From its cell body, each BDU neuron projects an anterior process and a posterior process. The PLM neuron has its cell body located in the tail region and sends out an anterior process to the mid-body region of the animal. Our previous work demonstrated that the posterior process of BDU connects with the anterior process of PLM through gap junctions [26] (Fig 1A and 1B). During the formation of the BDU-PLM connection, both the BDU and PLM neurons produce extensively elaborated membrane protrusions to reach each other and finally form the neuronal connection. From a genetic screen to search for mutants which display a BDU-PLM disconnection phenotype, we identified xd27 (Fig 1C). In xd27 animals, the cell protrusions on both BDU and PLM cells are greatly reduced (Fig 1D and 1E). Although a single neurite can project out from the posterior BDU or anterior PLM cell bodies in xd27, the typical growth-cone-like membrane expansion is missing (Fig 1E). Actin filament dynamics are essential for cell surface protrusion but are not necessary for axonal elongation, which is thought to be mainly mediated by microtubule extension [27]. Intriguingly, the neurites from both BDU and PLM in xd27 can continuously extend (Fig 1F and 1G). In xd27 adults, the BDU neurite length is almost indistinguishable from wild type, while the PLM neurite is slightly shorter than wild type (Fig 1H). However, when we followed neurite growth during larval stages, we found that the PLM neurite elongates at a similar speed in both xd27 and wild-type animals (Fig 1I), suggesting that the formation of actin-mediated cell protrusions may be specifically affected by the xd27 mutation. In addition to BDU and PLM cells, the head neurons RMED and RMEV, and the D type motor neurons DDs and/or VDs, also display weak neurite extension defects (S1 Fig). However, the locomotion of xd27 is generally normal. Besides the neural deficits, xd27 animals display partial embryonic lethality and weak egg laying and distal tip cell migration defects (S1 Fig).

chdp-1 is required cell-autonomously for cell protrusion formation

Through genetic mapping and genomic DNA sequencing, we identified C10G11.7 (Fig 2A). C10G11.7 encodes a single type III CH domain-containing protein, which we named CHDP-1 (Fig 2A). It shares sequence similarity with calponin in mammals (S1F Fig). In addition to the CH domain, CHDP-1 contains two proline-rich motifs (P1 and P2) in the N-terminal region and one amphipathic helix motif (Helix) close to the C-terminus (Fig 2A). Proline-rich motifs widely participate in protein-protein interactions [28], while amphipathic helix motifs directly interact with membrane phospholipids [29]. A phenylalanine to leucine change at position 117 was identified in xd27 worms (Fig 2A). Another chdp-1 allele, tm4947, which removes the amphipathic helix domain and the majority of the CH domain (Fig 2A) and is likely a molecular null, displays a similar membrane protrusion defect to xd27 (Fig 2B and 2C), suggesting that xd27 may also act as a strong loss-of-function or null mutation. Introducing a wild-type copy of the chdp-1 gene into xd27 or tm4947 animals restores the proper morphology of developing BDU and PLM cells (Fig 2B and 2C). Thus, mutations of the chdp-1 gene are indeed responsible for the BDU-PLM connection defect.

The chdp-1 gene is widely expressed in almost every cell during the early embryonic stage (Fig 2D). From late embryonic to adult stages, however, chdp-1 expression was gradually restricted to certain sets of neurons including BDU, ALM, AVM, PLM, PVM and PVQ (Fig 2E). When chdp-1 cDNA was expressed in BDU or PLM alone, or BDU and PLM together, the BDU-PLM connection defect in chdp-1 mutants was efficiently rescued (Fig 2F). In contrast, expression of chdp-1 in hypodermis or muscle failed to rescue the BDU-PLM connection defect (Fig 2F), suggesting that chdp-1 functions cell-autonomously to regulate protrusion formation.

CHDP-1 localizes to the cell cortex

To understand how CHDP-1 regulates protrusion formation, we examined the subcellular localization of CHDP-1 in vivo. A functional translational CHDP-1 fusion green fluorescent protein (GFP) construct was introduced into worms. During the embryonic stage or after hatching, CHDP-1 is distributed specifically at the cell periphery (Fig 3A). After hatching, although some of the GFP::CHDP-1 becomes diffuse in the cytoplasm, most of the signal retains this membrane-like distribution (Fig 3B). Within the PLM neuron, CHDP-1 highlights the cell periphery (Fig 3C), including surface protrusions (Fig 3D). Further co-labeling experiments indicated that CHDP-1 co-localizes well with the plasma membrane marker Myr-mCherry (Fig 3C and 3D), suggesting that the CHDP-1 protein associates with the cell cortex.

The amphipathic helix motif has been implicated in membrane bilayer association [29]. We wondered whether the amphipathic helix motif hooks the CHDP-1 protein onto the plasma membrane. To test this hypothesis, we removed the amphipathic helix motif from CHDP-1 and examined the subcellular localization of the residual CHDP-1 fragment. We found that in the absence of the amphipathic helix motif, the cortex localization of CHDP-1 is completely lost (Fig 3E). In contrast, whenever the amphipathic helix motif is present, the peripheral distribution of CHDP-1 protein is largely preserved (Fig 3E). Thus, the cortex localization property of CHDP-1 is probably mediated by the amphipathic helix motif.

Next, we asked whether the cortex localization is important for CHDP-1 function. To address this question, we removed the amphipathic helix domain and performed rescue experiments. We found that deletion of the amphipathic helix domain largely abolished the rescue effect of CHDP-1 (Fig 3F). Therefore, localization to the cell cortex is crucial for CHDP-1 function.

CHDP-1 promotes cell protrusion

Without CHDP-1, robust cell protrusion is severely reduced. Furthermore, the amphipathic helix domain has been implicated in membrane deformation [16]. We therefore asked whether CHDP-1 can promote membrane protrusion. To test this possibility, we increased chdp-1 gene expression in PLM neurons using the Pmec-7 promoter. In wild-type animals, PLM cell bodies generally display smooth spindle-like shapes (Fig 4A). When chdp-1 gene expression was boosted, additional membrane protrusions of various sizes appeared on the surface of PLM cell bodies (Fig 4B–4D). The Pmec-7 promoter also drives chdp-1 expression in ALM, AVM, and PVM neurons, and we found that these cells also display ectopic membrane protrusions around the cell body region (S2 Fig). CHDP-1 localizes to the cell cortex. Double staining of CHDP-1::GFP and Myr::mCherry indicated that when it is overexpressed, the CHDP-1 protein still tightly associates with the cortex region (Fig 4E). This suggests that CHDP-1 may function at the cell margin to induce cell protrusion.

The formation of cell surface protrusions is a cooperative result of both membrane expansion and actin cytoskeleton rearrangement. The expanded plasma membrane must be stabilized by the underlying cortical actin network. To address whether the ectopic membrane protrusions are genuine cell surface protrusions, we co-labeled PLM cells with Myr-GFP and mCherry-actin. We found that most of the ectopically expanded plasma membrane is indeed supported by cortical actin (77%, n = 236) (Fig 4F and 4G). However, some protrusions do not contain any detectable underlying actin (23%, n = 236), and these tend to be smaller than those with actin (Fig 4F and 4G). We suspected that these actin-free protrusions may represent newly formed protrusions. Furthermore, in wild-type PLM cell bodies, the actin cytoskeleton is usually organized into stress fiber-like structures (Fig 4H) [30]. However, when ectopic protrusions were induced by chdp-1 over-expression, most of the actin signal was shifted to the cortex region underlying those ectopic protrusions and no stress fiber-like structures could be found in the center of the PLM cell bodies, where the nucleus is located (Fig 4I). Together, these results suggest that CHDP-1 promotes the formation of cell protrusions in vivo.

CHDP-1 does not bind to actin

Membrane expansion must be coupled with the actin network to efficiently promote protrusion. Can the cortex-localized CHDP-1 protein associate with actin, thus coupling membrane expansion to actin rearrangement? Interestingly, a tandem array of type I or II CH domains has been shown to possess actin-binding activity [23,24]. Therefore, we tested whether the CHDP-1 protein can self-associate by performing co-precipitation assays on 293T cells transfected with Flag- and Myc-tagged CHDP-1. We found that CHDP-1 molecules can indeed bind to each other (Fig 5A). Truncation analysis indicated that the self-association of CHDP-1 is mediated by the CH domain (Fig 5B). We next tested whether CHDP-1 can bind to actin. We firstly examined whether CHDP-1 binds to monomeric (G) actin. Flag-tagged CHDP-1 did not co-precipitate with worm actin ACT-1 (Fig 5C), suggesting that CHDP-1 does not associate with G-actin. Next, we purified the mCherry::CHDP-1 protein (Fig 5D) and performed co-sedimentation experiments with polymerized filamentous (F) actin. As shown in S2D Fig, the actin motor myosin co-sedimented with F-actin, and this myosin-actin association could be disrupted by ATP. In contrast, CHDP-1 was not sedimented with F-actin at all (Fig 5E). Together, these results indicate that CHDP-1 probably does not promote the formation of cell protrusions through direct actin binding.

CHDP-1 directly associates with Rac1/CED-10

To understand how CHDP-1 interacts with the actin cytoskeleton to regulate protrusion formation, we searched for CHDP-1-interacting molecules by performing immunoprecipitation (IP) followed by mass spectrometry (MS). With this approach, we identified the Rho small GTPase family member Rac1/CED-10 [31]. The worm Rac1 homolog, CED-10, is localized to plasma membranes [32] (Fig 6A). In developing PLM neurons, CED-10 co-localizes with CHDP-1 at the periphery of the PLM cell body as well as on the surface of the protrusion region (Fig 6B and 6C). This implies that CHDP-1 and CED-10 may interact with each other at the cell cortex in vivo.

To test whether CHDP-1 directly binds to CED-10, we performed the following experiments. Flag-tagged full-length CHDP-1 and Myc-tagged CED-10 were co-expressed in HEK293T cells. After affinity purification, the purified CHDP-1 and CED-10 were mixed together and co-immunoprecipitations were performed with Flag or Myc antibodies. In contrast to the mock-transfected samples, CED-10 was effectively co-precipitated with CHDP-1 (Fig 6D), and the purified CHDP-1 protein was co-precipitated by CED-10 (Fig 6E). Thus, CHDP-1 can directly bind to CED-10. To further identify the domain of CHDP-1 that interacts with CED-10, we performed co-IP experiments using truncated CHDP-1 fragments. We found that the CH domain of CHDP-1 is crucial for the interaction with CED-10 (Fig 6F). Taken together, these results indicate that CHDP-1 may bind to CED-10 through its CH domain.

CHDP-1 preferentially binds to GTP-CED-10

Coincidently, the BDU-PLM disconnection mutant screen identified a new ced-10 allele, xd33 (Fig 6G and 6H). Imaging analysis further indicated that formation of cell protrusions is considerably repressed in xd33 mutant animals (Fig 6H). xd33 is a C-to-T missense mutation resulting in a serine 22-to-phenylalanine change (S22F) in CED-10. To understand the role of CED-10 in BDU-PLM connection, we examined two other ced-10 mutants, n1993 and n3246 and found that they both display similar BDU-PLM disconnection defects to xd33 (Fig 6I). Previous report showed that the n3246 mutation is recessive to wild type, but homozygote ced-10(n3246) animals display defects not seen in ced-10(n1993) [33]. In xd33/n3246 trans-heterozygote animals, we found that BDU-PLM disconnection defect is similar to that of xd33 homozygote. Furthermore, when the wild-type ced-10 gene was introduced into xd33 animals, the BDU-PLM connection could be largely restored (S3 Fig), suggesting that the loss-of-function of ced-10 is responsible for the membrane protrusion defect in the corresponding ced-10 mutant worms. We further constructed chdp-1(xd27);ced-10(xd33) and chdp-1(tm4947);ced-10(xd33) double mutants and found that those double mutant animals displayed similar degree of BDU-PLM disconnection defect as chdp-1(xd27) animals (S3 Fig), implying that chdp-1 may function together with ced-10 to regulate cell protrusion.

As a member of the Rac family of GTPases, Rac1 cycles between the inactive GDP-bound form and the active GTP-bound form. We then asked how CHDP-1 interacts with different forms of CED-10. The glycine to valine mutation at position 12 (G12V), which is canonical for constitutive activation of Ras superfamily GTPases, mimics the GTP-bound active state. In contrast, the threonine to asparagine mutation at position 17 (T17N) mimics the GDP-bound inactive state. Co-immunoprecipitation experiments with these two mutant forms of CED-10 showed that the CHDP-1 protein precipitates much higher levels of the G12V mutant than the T17N mutant (Fig 7A and 7B). This suggests that CHDP-1 preferentially associates with GTP-bound active CED-10.

CHDP-1 facilitates the cell cortex localization of CED-10

Both CHDP-1 and CED-10 localize to the cell margin. How does the direct association of CHDP-1 contribute to regulation of CED-10 function? In chdp-1 mutant animals, we found that the cortex localization of CED-10 is partially lost (Fig 7C and 7D). In contrast, localization of CHDP-1 to the cell margin remains intact in various ced-10 mutants, including ced-10(xd33), ced-10(n1993) and ced-10(n3246) (Fig 7E and S3 Fig). Cell-fractionation assays further indicated that the proportion of membrane-bound CED-10 is reduced in chdp-1 mutants compared with wild-type samples (Fig 7F). The reduction of membrane-bound CED-10 suggested that the level of GTP-bound, active CED-10 protein maybe decreased in chdp-1 mutants. Therefore, we further tested the GTP-CED-10 level in vivo. Active GTP-Rac1 binds to the CRIB (Cdc and Rac interactive binding) domain of its effector PAK, while inactive GDP-Rac1 does not [34]. Thus, we performed pull-down assays on the PAK CRIB domain. In contrast to wild type, we found that the amount of GFP-tagged CED-10 pulled down by the PAK CRIB domain was considerably decreased in chdp-1 lysates (Fig 7G and 7H). Together, these data suggested that CHDP-1 facilitates the cell margin localization of CED-10 in vivo by stabilizing the active GTP-bound CED-10 protein.

CED-10 is required for CHDP-1-induced cell protrusion

As the critical regulator of actin dynamics, is CED-10 required for CHDP-1-induced cell protrusion? We showed that over-expression of CHDP-1 leads to extensive cell protrusion in PLM cell bodies. In three different ced-10 mutants, however, the ectopic protrusion on the surface of PLM cell bodies is significantly reduced. We measured the size of the PLM cell body (Fig 7I). In wild-type animals, the maximum area of a PLM cell body is about 26 μm². In chdp-1 over-expression animals, the area of the PLM cell body is expanded to around 138 μm². In ced-10 mutant animals, the area of the PLM cell body is 57 μm² (xd33), 72 μm² (n1933) and 64 μm² (n3246) respectively (Fig 7I).

We then examined how CED-10 coordinates with CHDP-1 on the ectopic protrusions. After co-injecting mec-7 promoter-driven rfp::chdp-1 and gfp::ced-10 constructs into wild-type animals, we found that most, if not all, CHDP-1 and CED-10 are co-distributed on the ectopic cell protrusions (Fig 7J). Very few protrusions contain either CHDP-10 (6.3%) or CED-10 (1.9%), suggesting a tight association of CHDP-1 and CED-10 on those ectopic protrusions. Next, we tested whether the previously described “actin-free” protrusions contain CED-10 or not. Co-labeling of PLM cells with mCherry::ACT-1 and GFP::CED-10 showed that while the majority of protrusions (69.2%) contain both CED-10 and actin, a significant proportion (23.2%) contain CED-10 without actin (Fig 7K). Thus, CED-10 is likely more closely associated with CHDP-1 than with actin to mediate protrusion formation (S3 Fig).

C. elegans genetics

Culture and manipulation of C. elegans strains were performed using standard methods. Mutants used in this studies are: LGI, chdp-1(xd27), chdp-1(tm4947); LGII, juIs76(Punc-25::GFP); LGIV, ced-10(n1993), ced-10(xd33), ced-10(n3246), kyIs262(Punc-86::MYR::GFP, Podr-1::dsRed). The xd27 and xd33 mutations were isolated from kyIs262 (Punc-86::Myr::GFP) animals treated with EMS. A total of 2,500 mutagenized haploid genomes were screened. The isolated strains were outcrossed with the N2 strain at least four times.

Generation of transgenic lines and constructs

Corresponding chdp-1 DNA fragments were amplified from N2 genomic DNA to perform the chdp-1 cloning experiment. chdp-1 cDNA was amplified by reverse transcription. ced-10 cDNA was obtained from Dr. Yuji Kohara. GFP sequence was inserted into Pchdp-1::CHDP-1 constructs. For protein co-localization, the RFP fragment was inserted into the Pmec-7::CHDP-1 construct. The mec-7 promoter was inserted into Pced-10::GFP::CED-10 construct to replace the ced-10 promoter. In general, plasmid DNAs of interest were injected at 10-50ng/μl and the co-injection markers Podr-1::RFP, rol-6 or Podr-1::GFP were injected at 10-50ng/μl. The corresponding transgenes and constructs are listed in (S1 Table).

Image collection and phenotypic quantification

Animals were mounted on 2% agar pads in M9 buffer containing 1% 1-phenoxy-2-propanol and examined by fluorescence microscopy unless indicated otherwise. Fluorescence photographs were taken using a Zeiss Axioimager A1 with an AxioCam digital camera and Axiovision rel. 4.6 software (Carl Zeiss) or an IX81 Olympus inverted confocal microscope. Contact was considered defective if the BDU neurite failed to touch the PLM neurite. Neurite and worm body lengths were measured with NIH ImageJ software. The length of each neurite was traced from the center of the cell body to the tip of neurite. The length of each animal was measured from the center of the PLM cell body to the tip of the nose. The relative BDU or PLM neurite length is defined as neurite length/animal length. The PLM cell body was measured using ImageJ software. To measure the co-distribution of actin, CED-10 and CHDP-1 on ectopic protrusions, fluorescence photographs were taken using confocal microscopy. The size of individual protrusions was measured with NIH Image J software using the line-tracing tool. All data are shown as mean ± SD. Statistical analyses were performed with Student’s t-test. For each genotype, more than 20 animals were analyzed.

Quantification of membrane protrusions

At different time points, eggs were mounted on thin 1% agar pads in M9 buffer; gentle pressure was applied to crack the egg shells, and embryos were examined immediately by fluorescence microscopy. To measure the membrane protrusion area, newly hatched L1 animals were collected and photographs were taken within 10 min of hatching. The expansion area was defined by linking the first branching or elaboration site of the neurite with the tips of all the branches or filopodia. The area was measured with NIH ImageJ software.

Quantification of membrane localization of CED-10

Some chdp-1(xd27) embryos die at the later elongation stage. Therefore we dissected gravid adults and obtained embryos at early developmental stages (from the 16-cell stage to gastrulation stage). Then the collected embryos were mounted on thin 1% agar pads in M9 buffer and examined immediately by fluorescence microscopy. Embryos in which at least half of the cells contained diffuse GFP::CED-10 or GFP::CHDP-1 signal were considered as showing defective localization of the GFP::CED-10 or GFP::CHDP-1 marker. All images were collected using Zeiss Axioimager A1 with an AxioCam digital camera and Axiovision rel. 4.6 software (Carl Zeiss). For each sample, at least 50 embryos were scored.

Time-lapse imaging

Newly hatched L1 animals were anesthetized with 0.1mM levamisole in M9 buffer and mounted on 2% agar pads at 22°C. Time-lapse images were captured every 1.5 min using a Delta-vision Core imaging system (Applied Precision) with an UPLSApo 100 6/1.40NAoil-immersion objective and a Photometrics CoolSnap HQ camera. Deconvolution and analysis of images were performed with Softworx (Applied Precision).

Immunoprecipitation for mass spectrometry analysis

Worm lysates were prepared from the N2 strain carrying either xdIs23(Pchdp-1::GFP) or xdIs48(Pchdp-1::GFP::CHDP-1). Specifically, worms were collected and lysed with a Dounce homogenizer (Cheng-He Company, Zhuhai, China) [43] in pre-chilled homogenizing buffer (50mM Tris-Cl pH8.0, NaCl 150mM, 0.5% sodium deoxycholate, 1% Triton-X 100, protease inhibitor [Roche]), then incubated for 15 min on ice, and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were incubated with GFP polyclonal antibody (Rabbit, Abcam, ab290, 1:1,000 dilution) overnight, and then incubated with protein A agarose beads (Cat#17-0780-01, GE) for 4 hr at 4°C. The pellet was washed three times in washing buffer (50mM Tris-Cl pH8.0, 150mM NaCl, 1% NP-40, 1 mM PMSF), and then boiled in sample buffer for 5 min. The boiled samples were resolved by SDS-PAGE gel and protein bands of interest were excised from SDS-PAGE gel and in-gel digestion was performed using a well-established protocol with slight modifications [44]. Briefly, the proteins embedded in gel slices were reduced with 10mM DTT and alkylated with 55mM iodoacetamide, and then digested overnight with sequencing grade trypsin (Sigma, USA) at 37°C. The tryptic peptides were analyzed by mass spectrometry using a TripleTOF 5600 mass spectrometer (AB SCIEX, Canada) coupled to an EksigentNanoLC. Peptide identification and quantification were performed with the ProteinPilot 4.2 software (AB SCIEX). The C. elegans proteome sequences (Uniprot) was used as the database and the mass tolerance was set to 0.05 Da for the database search. The false discovery rate (FDR) analysis was performed using the software PSPEP integrated with ProteinPilot. About 10 cytoskeleton-related proteins were identified in the mass spectrometry analysis. GST pull-down and co-immunoprecipitation assays were then performed to verify the putative protein-protein interactions. Among the tested proteins, CED-10 displayed consistent CHDP-1-binding activity in both GST pull-down and co-immunoprecipitation assays.

Co-immunoprecipitation

cDNA fragments were amplified and cloned into modified pcDNATM3.1/myc-HIS(-) or pFLAG-CMV-2 vectors through standard procedures. HEK293T cells were cultured in DMEM medium supplemented with 12% FBS. Plasmid transfections were carried out using Lipofectamine 2000 (Invitrogen). 24 hours after transfection, cells were harvested and lysed for 30 min at 4°C. After centrifugation, the supernatants were incubated with anti-FLAG M2 affinity gel beads at 4°C overnight and then washed three times with washing buffer and incubated with SDS-sample buffer. Samples were resolved by standard immunoblotting techniques. To quantify the binding affinity between CHDP-1 and different CED-10 mutant proteins (G12V and T17N mutant forms), the absolute signal intensity of individual protein bands was analyzed with ImageJ. The relative protein level was achieved by subtracting the respective FLAG control. For binding experiments with truncated CHDP-1, the following fragments were used: FN (AA 1–146), CH (AA 121–242) and FC (AA 243–338). All the co-immunoprecipitation experiments were repeated at least three times.

F-actin sedimentation

The Actin Binding Protein Spin-Down Biochem Kit (Cat. # BK001) from Cytoskeleton Inc was used to detect the F-actin binding activity of CHDP-1. The recombinant proteins Flag::mCherry::CHDP-1 and Flag::mCherry were expressed in HEK293FT cells. 24 hours after transfection, cells were harvested and lysed for 30 min at 4°C. After centrifugation, the corresponding supernatants were incubated with anti-FLAG M2 affinity gel beads at 4°C for purification of tagged proteins. F-actin was prepared from muscle actin and suspended in general actin buffer at room temperature for 1 hour. Proteins at a molar ratio of 1:3 (recombinant protein: F-actin) were incubated at 25°C for 30 minutes and pelleted at 15 0000g for 1.5 hours. Subsequently, protein samples were resolved by SDS-PAGE gel. The muscle myosin was used as a positive control for F-actin binding assay.

Cell fractionation

Wild-type and chdp-1(xd27) worms expressing Pced-10::GFP::CED-10 were collected and washed in M9 buffer. Then 500μl lysis buffer (250 mM sucrose, 50 mM Tris–HCl pH6.8, 1 mM EDTA) were added and the samples were homogenized with a Dounce homogenizer (Cheng-He Company, Zhuhai, China) (Chen et al., 2010) on ice for 15 min. The nuclear pellet was removed by centrifuging at 3,000 rpm for 10 min at 4°C. The supernatant was further centrifuged at 40,000 rpm for 1 hr. The new supernatant was collected as the cytosolic fraction. The pellet was further washed and centrifuged at 40,000 rpm for 45 min to obtain the membrane fraction. All samples were boiled with 2xSDS loading buffer then subjected to 10% SDS-PAGE gel analysis.

PAK-RBD pull-down assay

PAK-GST protein beads (human P21 activated kinase PBD) were used to detect the GTP-bound form of CED-10. Worm lysates were prepared from wild type and chdp-1(xd27) mutant animals carrying Pced-10::GFP::CED-10. Worms were lysed in pre-chilled homogenizing buffer with a Dounce homogenizer (Cheng-He Company, Zhuhai, China) and incubated for 15 min on ice, then centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were incubated with PAK-GST protein beads (Cat#PAK02, Cytoskeleton Inc) for 4 hr at 4°C. The pellet was washed three times and then boiled in sample buffer for 5 min. The boiled samples were resolved by SDS-PAGE and detected with anti-GFP antibody. The pull-down experiments were repeated three times. The absolute intensity of each protein band was quantified with ImageJ. The relative protein level was determined by normalizing each sample with the corresponding input, and then normalizing each chdp-1(xd27) sample with the paired WT sample.