The basolateral localization of the LET-23 EGFR in the VPCs of C. elegans larvae is necessary for the efficient binding of the LIN-3 EGF ligand secreted by the AC (Figure 1A; Kaech et al., 1998; Whitfield et al., 1999). After ligand-induced receptor endocytosis from the basolateral plasma membrane, LET-23 accumulates on the apical cortex of the VPCs (Haag et al., 2014). In late L2/early L3 larvae before the VPCs have started dividing, a translational LET-23::GFP reporter was up-regulated in the 1° VPC P6.p, while expression faded in the other, more distal VPCs. Most of the LET-23::GFP protein in P6.p was detected, approximately at equal levels, on the basolateral and apical plasma membranes (Figure 1B). Only a faint and diffuse intracellular LET-23::GFP signal could be observed in the VPCs of wild-type animals. After the first round of VPC divisions, LET-23::GFP continued to be strongly expressed on the plasma membrane of the 1° P6.p descendants (Figure 1B’). A systematic RNA interference screen for genes controlling LET-23 trafficking had previously identified the chp-1 gene as a regulator of LET-23 localization (Haag et al., 2014). chp-1 encodes a conserved CHORD-containing protein homologous to human CHORDC1/Morgana (Ferretti et al., 2011). RNAi against chp-1 leads to a strong reduction in plasma membrane localization and to the intracellular accumulation of the LET-23::GFP reporter in P6.p and its descendants (Figure 1C,C’). To confirm the RNAi-induced phenotype, we examined LET-23 localization in chp-1(tm2277) deletion mutants (chp-1(lf)). Since homozygous chp-1(lf) mutants are sterile as adults, we analyzed LET-23 EGFR localization in the homozygous offspring of heterozygous chp-1(lf)/hT2 balanced mothers (Supplementary file 1). In the following experiments, we compared chp-1(lf) homozygous larvae to balanced chp-1(lf)/hT2 heterozygous control siblings, since LET-23::GFP localization in chp-1(lf)/+ heterozygotes was indistinguishable from wild-type animals (for example, compare Figure 1B with Figure 3A). Homozygous chp-1(lf) L2 and L3 larvae exhibited a completely penetrant intracellular mislocalization of LET-23 EGFR in the VPCs and their descendants (Figure 1D,D’). The distinct plasma membrane signal observed in the VPCs of wild-type animals was absent in chp-1(lf) larvae.

Taken together, the CHORD-containing protein CHP-1 is required for the plasma membrane localization of the EGFR in the VPCs.

In mammalian cells, CHORDC1/Morgana forms a complex with the heat-shock protein 90 (HSP90) and was proposed to function as a co-chaperone for a subset of the HSP90 clients (Gano and Simon, 2010). We therefore tested if a loss-of-function mutation in the C. elegans hsp-90 gene affects LET-23 EGFR localization in as similar manner as chp-1(lf). The hsp-90(ok1333) allele contains a 1258 bp deletion resulting in the insertion of a Ser followed by a stop codon after amino acid 208, thereby truncating the remaining 494 residues of the protein (www.wormbase.org). The ok1333 allele was previously reported to eliminate hsp-90 function (Lissemore et al., 2018). Homozygous, hsp-90(ok1333) larvae segregated by heterozygous nT1 balanced mothers (Supplementary file 1) displayed a paralyzed Unc phenotype and arrested at the mid to late L3 stage. We thus examined LET-23::GFP expression in the VPCs of homozygous hsp-90(ok1333) larvae shortly before they arrested, at the late L2 to early L3 stage (Pn.p to Pn.px stage). While the overall intensity of the LET-23::GFP signal in the VPCs of homozygous hsp-90(ok1333) mutants was reduced compared with their heterozygous siblings, the LET-23::GFP signal remained localized at the plasma membrane (Figure 1E–F’). Especially, we did not observe the intracellular LET-23::GFP accumulation seen in chp-1(lf) mutants (Figure 1D). A similar reduction in LET-23::GFP expression with persisting membrane localization was observed in larvae carrying the hsp-90(p673) missense mutation (Figure 1—figure supplement 1A,A’). Even though the p673 allele acts as a gain-of-function allele with respect to the constitutive Dauer (Daf-c) phenotype (Birnby et al., 2000), our data suggest that for LET-23 membrane localization p673 rather represents a reduction-of-function allele.

The C. elegans genome encodes an hsp-90 paralog, called enpl-1, which is homologous to the human HSP90B1 chaperone (also called GP96 or GRP94) (Natarajan et al., 2013). Since mammalian HSP90B1 controls the trafficking of Toll-like receptors through the endoplasmic reticulum (ER) (Randow and Seed, 2001), we tested whether ENPL-1 instead of HSP-90 might act together with CHP-1 to regulate LET-23 localization. For this purpose, we used a 1613 bp enpl-1deletion allele ok1964 that removes most of the coding sequences and eliminates gene function (Natarajan et al., 2013). Homozygous enpl-1(ok1964) larvae did not exhibit a change in LET-23::GFP expression or localization in the VPCs (Figure 1G–H’), indicating that ENPL-1 is not required for LET-23 membrane localization. Finally, we performed RNAi knock-down of other known HSP90 co-chaperones, cdc-37, daf-41 and sgt-1 (Li et al., 2012), but observed no change in LET-23::GFP localization (Figure 1—figure supplement 1B–F’).

We conclude that CHP-1 acts independently of the HSP90A and HSP90B1 chaperones to control the plasma membrane localization of the LET-23 EGFR in the VPCs.

To investigate if chp-1 plays a general role in membrane trafficking, we examined the expression pattern of three other transmembrane receptors expressed in the VPCs; LIN-12 NOTCH (Shaye and Greenwald, 2002), LIN-18 RYK (Inoue et al., 2004) and the β-integrin subunit PAT-3 (Hagedorn et al., 2009). The translational LIN-12::GFP reporter was expressed on the apical membrane of the VPCs (Figure 1G), while the LIN-18::GFP and PAT-3::GFP translational reporters localized predominantly to the basolateral compartment (Figure 1H,I). chp-1 RNAi did not alter the apical localization of the LIN-12::GFP reporter (Figure 1G’). Furthermore, the chp-1(lf) mutation did not affect the basolateral localization of the LIN-18::GFP or the PAT-3::GFP reporter (Figure 1H’,I’).

Thus, CHP-1 does not play a general role in the apical or basolateral secretion of transmembrane receptors, but it is rather specifically required for the membrane localization of the EGFR.

We next tested if chp-1 acts cell-autonomously in the VPCs. First, we performed tissue-specific RNAi to downregulate chp-1 expression in the VPCs. For this purpose, we used a transgenic line expressing the rde-1(+) gene under control of the Pn.p cell-specific promoter/enhancer in an rde-1(lf) RNAi-resistant background carrying the let-23::gfp reporter (Haag et al., 2014) (strain AH2417 in Supplementary file 1). This approach has previously been shown to downregulate gene expression in a tissue-specific manner (Qadota et al., 2007). RNAi against chp-1 in the Pn.p cell-specific RNAi strain caused an intracellular accumulation of LET-23::GFP in 31% of the cases, while control animals treated with the empty RNAi vector showed no change in LET-23::GFP localization (Figure 2A–B’).

In addition, we used a tissue-specific CRISPR/Cas9 approach to inactivate chp-1 in the VPCs (Shen et al., 2014). For this purpose, we expressed the Cas9 endonuclease under control of a 1° VPC-specific egl-17 promoter fragment (Inoue et al., 2002) together with two single-guide (sg) RNAs that target the first exon of chp-1 and were expressed under control of the ubiquitous eft-3 promoter (array zhEx558 in strain AH4609 in Supplementary file 1). In around 5% of zhEx558 animals, we observed an intracellular accumulation of LET-23::GFP (Figure 2D,D’). In all these cases, LET-23::GFP was mislocalized only in the 1° VPC P6.p and its descendants. Control sibling lacking the zhEx558 array showed a wild-type membrane localization of LET-23::GFP (Figure 2C,C’). The relatively low penetrance of the CRISPR/CAS9-induced mislocalization phenotype compared to the tissue-specific RNAi could be due to an inefficient binding of the sgRNAs to the target sequence, to mosaic expression of the extrachromosomal array or to the perdurance of the CHP-1 protein in the VPCs.

Together, these experiments indicated that CHP-1 acts cell-autonomously to regulate LET-23::GFP localization in the VPCs.

The intracellular accumulation of LET-23::GFP in chp-1(lf) mutants appeared granular and unevenly structured, while in chp-1(lf)/+ control animals, LET-23::GFP was localized predominantly at the plasma membrane of the 1° VPC P6.p and its descendants (Figure 3A–C; note that single mid-sagittal confocal sections through the VPCs are shown in Figure 3, whereas Figures 1, 2 and 4 shows wide-field images of the entire VPCs.) In order to determine the intracellular compartment, in which LET-23::GFP accumulates in chp-1(lf) mutants, we generated two reporters that mark the Golgi and the ER of the VPCs, respectively. To label the Golgi apparatus, we expressed an alpha-mannosidase 2A AMAN-2::mCherry fusion protein in the VPCs under control of the pan-epithelial dlg-1 promoter. AMAN-2 has previously been shown to localize to the Golgi network in the C. elegans intestine (Chen et al., 2006). In contrast to vertebrate cells that contain one large juxta-nuclear Golgi stack, invertebrate cells contain many small Golgi stacks (Golgi ‘ministacks’) dispersed throughout the cytoplasm (Ripoche et al., 1994). Accordingly, the AMAN-2::mCherry reporter labeled punctate structures scattered throughout the cytoplasm of the VPCs (Figure 3A’–F’). In chp-1(lf)/+ animals, LET-23::GFP showed on average 26% co-localization with the AMAN-2::mCherry reporter when analyzed on a voxel per voxel basis in confocal optical sections of the VPCs (Figure 3A–C’’ and Figure 3—figure supplement 1). In homozygous chp-1(lf) mutants, the co-localization with AMAN-2::mCherry was slightly increased to 34.4% (Figure 3D–F’’ and Figure 3—figure supplement 1). However, the strongest LET-23::GFP signal was detected at the plasma membrane, indicating that only a minor fraction of LET-23::GFP is found in the Golgi apparatus.

To label the ER compartment of the VPCs, we created a reporter consisting of the dlg-1 promoter driving expression of an mCherry tag C-terminally fused to the C34B2.10 gene, which encodes the 12 kDa subunit (SP12) of the C. elegans ER signal peptidase complex (Rolls et al., 2002). Translational SP12 reporters have previously been shown to localize to a reticular tubular network that extends to the cortex in various cell types of C. elegans and resembles the ER architecture in yeast and mammalian cells (Voeltz et al., 2002). In confocal sections through the VPCs of chp-1(lf)/+ animals, we observed 34% co-localization between the SP12::mCherry and LET-23::GFP reporters, though the strongest LET-23::GFP signal intensity was found at the cell cortex where no SP12::mCherry was detected (Figure 3G–I’’ and Figure 3—figure supplement 1). By contrast, in homozygous chp-1(lf) mutants, we observed a strong overlap between the LET-23::GFP and SP12::mCherry signals inside the cells, resulting in 64% co-localization between the two reporters (Figure 3J–L’’ and Figure 3—figure supplement 1).

Therefore, the loss of chp-1 function leads to the intracellular retention of LET-23 predominantly in the ER.

Since LET-23::GFP accumulated mainly in the ER compartment of chp-1(lf) mutants, we tested if loss of chp-1 function causes ER stress triggering the unfolded protein response (UPR) pathway. The hsp-4 gene encodes a homolog of the mammalian Grp78/BiP protein that is upregulated upon ER stress via the XBP-1 transcription factor and the IRE-1 kinase/endoribonuclease (Calfon et al., 2002). The expression of an hsp-4::gfp reporter thus serves as an in vivo readout for the UPR (Taylor and Dillin, 2013). As a positive control, we treated animals with tunicamycin, which induces UPR by blocking the formation of N-acetylglucosamine lipid intermediates necessary for the glycosylation of newly synthesized proteins in the ER (Taylor and Dillin, 2013). Untreated chp-1(lf) mutants did not show elevated hsp-4::GFP expression when compared to the wild-type (Figure 3—figure supplement 2A,C,E). A 4 hr exposure of young adult wild-type animals to 25 µg/ml tunicamycin caused an approximately eightfold increase in hsp-4::GFP fluorescence intensity (Figure 3—figure supplement 2B,E). Interestingly, tunicamycin-treated chp-1(lf) mutants exhibited a stronger induction of hsp-4::GFP expression (Figure 3—figure supplement 2D,E).

Taken together, the intracellular accumulation of LET-23 in the chp-1(lf) mutants does not activate the UPR pathway under standard conditions. However, chp-1(lf) mutants are slightly hypersensitive to ER stress induced by tunicamycin-treatment.

Binding of the LIN-3 EGF ligand to the LET-23 EGFR on the basolateral cortex of the VPCs induces rapid receptor endocytosis (Haag et al., 2014). The endocytosed LET-23 may be recycled to the basolateral compartment, accumulate in the apical membrane compartment or undergo lysosomal degradation (Stetak et al., 2006; Skorobogata and Rocheleau, 2012). In lin-3(e1417) mutants that lack LIN-3 expression in the AC (Hwang and Sternberg, 2004), LET-23::GFP accumulated on the basolateral cortex of the VPCs, while the apical LET-23::GFP signal was reduced (Figure 4C), as reported previously (Haag et al., 2014). We thus examined whether the intracellular accumulation of LET-23 in chp-1(lf) mutants depends on ligand-induced receptor endocytosis. In lin-3(e1417); chp-1(lf) double mutants, we observed the same intracellular accumulation of LET-23::GFP as in chp-1(lf) single mutants (Figure 4B,D). Thus the intracellular accumulation of LET-23::GFP in chp-1(lf) mutants does not depend on ligand-induced receptor endocytosis.

Next, we asked whether chp-1 acts at the level of the tripartite LIN-2/LIN-7/LIN-10 complex, which is required for the basolateral retention of LET-23 and facilitates ligand binding (Kaech et al., 1998; Whitfield et al., 1999). Mutations in lin-2, lin-7 or lin-10 cause a penetrant vulvaless (Vul) phenotype because LET-23 is mislocalized to the apical membrane compartment, where it cannot bind to LIN-3 secreted by the AC on the basal side of the VPCs (Simske et al., 1996; Whitfield et al., 1999). In lin-2(lf) single mutants, the LET-23::GFP signal was detected almost exclusively on the apical membranes of the VPCs (Figure 4E). In chp-1(lf); lin-2(lf) double mutants, most of the LET-23::GFP signal was found in the intracellular compartment similar to chp-1(lf) single mutants (Figure 4F). However, we did observe a faint LET-23::GFP signal on the apical cortex in chp-1(lf); lin-2(lf) double mutants, indicating that a small fraction of LET-23::GFP can be secreted to the apical plasma membrane in the absence of chp-1. Finally, RNAi against sar-1, which encodes a small GTP-binding protein required for ER to Golgi transport, caused the same intracellular mislocalization of LET-23::GFP as observed in chp-1(lf) mutants (Figure 4G).

We thus conclude that CHP-1 neither regulates the ligand-induced endocytosis nor the basolateral retention of LET-23, but rather controls the secretion of the receptor from the ER to the plasma membrane.

The basolateral membrane localization of LET-23 is necessary for efficient ligand binding and activation of the downstream RAS/MAPK signaling pathway in the VPCs (Kaech et al., 1998; Hoskins et al., 1996; Whitfield et al., 1999). To quantify the output of the RAS/MAPK pathway in the VPCs, we quantified the expression of a transcriptional P_egl-17::cfp reporter as a marker for the 1° cell fate (Burdine et al., 1998). egl-17 encodes a fibroblast growth factor (FGF)-like protein, which is upregulated by RAS/MAPK signaling in the 1° VPC (P6.p) and its descendants until the late L3 stage. P_egl-17::cfp expression levels in chp-1(lf) mutants at the Pn.pxx stage were decreased around five-fold when compared to wild-type larvae at the same stage (Figure 5A,B,E). We further examined the activity of the lateral NOTCH signaling pathway using a transcriptional P_lip-1::gfp reporter that is upregulated in 2° VPC in response to LIN-12 NOTCH activation (Berset et al., 2001). Expression of the P_lip-1::gfp reporter was unchanged in chp-1(lf) mutants (Figure 5C,D). Thus, CHP-1 acts as a positive regulator of RAS/MAPK signaling, while the activity of the lateral NOTCH pathway is not affected by chp-1(lf).

Despite the strong reduction in RAS/MAPK reporter expression, the VPCs in chp-1(lf) mutants were induced to proliferate and differentiate into vulval cells. In most chp-1(lf) single mutants, the three proximal VPCs P5.p, P6.p and P7.p differentiated as in wild-type animals (Figure 4F,G). However, the vulval invagination formed by the descendants of the three induced VPCs had an abnormal shape and the vulval cells formed two separate invaginations, indicating that CHP-1 performs additional functions during vulval fate execution or morphogenesis (Figure 5G). To quantify vulval induction, we determined the vulval induction index (VI) by counting the average number of VPCs per animals that were induced to differentiate, as described (Schmid et al., 2015). A VI of 3 indicates wild-type differentiation, while a VI > 3 signifies over- and a VI < 3 under-induction. The VI thus serves as a quantitative readout to examine genetic interactions between signaling pathway components.

Most chp-1(lf) mutants showed a VI of 3, though we observed over- as well as under-induced animals (Figure 5L). To investigate the interaction between chp-1 and the EGFR/RAS/MAPK signaling pathway, we constructed double mutants between chp-1(lf) and core EGFR/RAS/MAPK pathway components. The lin-3(e1417rf) allele caused a strong reduction in the VI of chp-1(lf) mutants, approximately to the level of lin-3(e1417) single mutants (Figure 5H,I,L; Hwang and Sternberg, 2004). This indicated that the VPCs in chp-1(lf) mutants are at least partially sensitive to the inductive AC signal. The let-23(sy1) allele specifically prevents the interaction of LET-23 EGFR with the LIN-2/LIN-7/LIN-10 receptor localization complex and causes a similar apical receptor mislocalization and partially penetrant Vul phenotype as the lin-2(lf) mutation (Kaech et al., 1998; Whitfield et al., 1999). Double mutants between chp-1(lf) and let-23 egfr(sy1) or lin-2(lf) exhibited a significantly stronger Vul phenotype than either of the single mutants (Figure 5L). Thus, chp-1(lf) enhanced the Vul phenotype caused by apical mislocalization of LET-23 EGFR. By contrast, the Muv phenotype caused by the n1046 gain-of-function (gf) mutation in the let-60 ras gene (Beitel et al., 1990) was significantly enhanced by chp-1(lf) (Figure 5J–L). Similar to the apical mislocalization in lin-2(lf) mutants, the intracellular mislocalization of LET-23 in chp-1(lf) mutants likely results in decreased ligand sequestering by P6.p, which allows more LIN-3 signal to diffuse to the distal VPCs (Hajnal et al., 1997). In combination with the hyper-sensitive let-60(n1046gf) background, reduced ligand sequestering results in the induction of the distal VPCs and a hyperinduced phenotype (Hajnal et al., 1997).

In summary, our genetic analysis indicated that chp-1 positively regulates EGFR/RAS/MAPK signaling in the VPCs. However, the VPCs in chp-1(lf) mutants are capable of differentiating into vulval cells because they remain partially sensitive to the inductive LIN-3 EGF signal.

Besides VPC fate specification, LIN-3 to LET-23 signaling is also required for the proper alignment between the AC and the 1° VPC P6.p (Grimbert et al., 2016). In wild-type L2 stage larvae, the relative position between the AC and VPCs is highly variable. However, by the early L3 stage the 1° VPC P6.p has migrated toward the AC such that the AC and P6.p are precisely aligned with each other. In wild-type larvae at the mid-L3 (Pn.pxx) stage, the AC was located at the vulval midline above the two inner 1° P6.p descendants (the VulF cells) (Figure 5M,M’). By contrast, in chp-1(lf) mutants the AC was often misplaced and occasionally located between VulF and VulE (Figure 5N,N). To quantify the AC to VulF alignment, we measured the distance between the AC nucleus and the midpoint between the two VulF cells at the Pn.pxx stage. In chp-1(lf)/+ heterozygous control animals, the AC to mid-VulF distance was around 1 µm or smaller, while most chp-1(lf) mutants exhibited a distance greater than 1 µm (Figure 5O).

Thus, in addition to VPC fate specification chp-1 is also required for the precise alignment between the AC and the VPCs, which is also mediated by LIN-3/LET-23 signaling.

To examine if the role of CHP-1 in controlling EGFR localization and signaling is conserved in mammalian cells, we generated a CRISPR/Cas9-mediated knock-out of the mammalian chp-1 homolog CHORDC1 in cultured cells. We used the human vulva epidermoid carcinoma cell line A431 because these cells express high levels of wild-type EGFR and respond strongly to EGF stimulation (Van de Vijver et al., 1991). A431 cells were transduced with lentiviral particles that deliver Cas9 and two sgRNAs targeting the first exon of CHORDC1 (see Materials and methods). As negative control, cells were transduced with a lentivirus delivering a scrambled sgRNA. After bulk puromycin selection to eliminate uninfected cells, two-cell populations were generated, subsequently termed A431 KO and A431 control cells, respectively. Western blot analysis revealed a 96% reduction of CHORDC1 protein levels in A431 KO cells (Figure 6A).

A431 KO cells displayed a reduced growth rate and a tendency to grow in smaller, scattered patches when compared to A431 control cells (Figure 6B,C). Furthermore, loss of CHORDC1 resulted in cell lethality approximately 16 days post lentiviral transduction, which made it impossible to establish A431 KO lines from single-cell clones. Therefore, the following experiments were performed with populations of A431 KO and control cells 10 to 14 days post-lentiviral transduction. To further characterize the morphological defects of A431 KO cells, we visualized the actin cytoskeleton by phalloidin staining. The numerous F-actin rich filopodia protruding from the plasma membrane of A431 control cells were absent in A431 KO cells (Figure 6D,E). Instead, A431 KO cells contained densely packed cortical F-actin filaments arranged in a circumferential manner. A similar phenotypic switch was observed after treatment of A431 cells with the Hsp90 inhibitor geldanamycin (GA) (Figure 6F; Ahsan et al., 2012; Gano and Simon, 2010).

Since EGFR signaling induces the remodeling of the actin cytoskeleton in migratory cells (Appert-Collin et al., 2015), we tested if the absence of filopodia in A431 KO cells might be due to reduced EGFR signaling. Serum starvation of A431 control cells caused a strong reduction in filopodia formation (Figure 6G). Stimulation of serum starved A431 control cells with recombinant EGF induced the reappearance of actin-rich filopodia within 10 min (Figure 6G’). By contrast, EGF stimulation of serum starved A431 KO cells did not induce filopodia formation (Figure 6H,H’).

To directly measure the activity of the EGFR/RAS/MAPK pathway, we quantified ERK1/2 activity after EGF stimulation of serum-starved cells using a phospho-ERK1/2 specific antibody to probe western blots of total cell lysates (Gabay et al., 1997). In A431 control cells, phospho-ERK1/2 levels reached the maximum levels 5 min after EGF stimulation and declined almost to baseline levels within 30 min (Figure 6I). The total ERK1/2 levels did not change during the EGF stimulation. By contrast, phospho-ERK1/2 levels in A431 KO cells increased to around half of the maximal levels observed in A431 KO cells and decreased more rapidly.

Taken together, our results show that the human CHP-1 homolog CHORDC1 is required for EGF-induced filopodia formation and sustained ERK1/2 activation in A431 cells. Analogous to the results obtained for C. elegans chp-1, loss of CHORDC1 function does not eliminate but rather attenuates EGFR signaling in A431 cells.

Since chp-1 is required for the membrane localization of the LET-23 EGFR in C. elegans, we investigated if CHORDC1 also regulates EGFR localization in A431 cells. We analyzed receptor localization by immunofluorescence staining of fixed cells with an antibody against the extracellular domain of the EGFR. In A431 control cells, most of the EGFR signal was detected together with the actin-rich filopodia at the cell cortex, whereas only a small amount of EGFR staining was detected inside the cells (Figure 7A). In A431 KO cells, on the other hand, most of the EGFR staining was observed in intracellular punctae (Figure 7B). Orthogonal xz-projections through the cells revealed that most of the EGFR staining in A431 control cells overlapped with the cortical actin signal, while in A431 KO cells part of the signal was detected inside the cells and a fraction near the cortex underneath the cortical actin (Figure 7A’–B’’). Moreover, A431 KO cells appeared significantly flatter than A431 control cells.

To examine if the loss of CHORDC1 results in a similar mislocalization of the EGFR to the ER as in C. elegans chp-1(lf) mutants, we co-stained the cells with antibodies against the EGFR and the ER marker PDI (protein disulfide isomerase) (Jaronen et al., 2013). While in A431 control cells only 5% of the EGFR signal colocalized with the PDI marker, 51% of the EGFR signal in the A431 KO cells overlapped with the PDI staining (Figure 7C–E).

Since CHORDC1 has been reported to act as a co-chaperone (Gano and Simon, 2010), we hypothesized that loss of CHORDC1 function might result in the incorrect folding of the EGFR and thereby cause its accumulation in the ER. To test this hypothesis, we performed trypsin sensitivity assays of the EGFR in A431 control versus KO cells. Since misfolded proteins are usually more susceptible to trypsin degradation, this assay can detect changes in protein folding (Ninagawa et al., 2015). Total protein extracts of A431 control and KO cells were incubated with varying concentrations of trypsin, and the amount of full-length EGFR was quantified by western blotting. As internal control, the EGFR signal intensities were normalized to the tubulin levels in the same lysates, as tubulin was degraded at approximately the same rate in the two cell populations. This assay revealed a faster degradation and thus an increased trypsin sensitivity of the EGFR in A431 KO cells (Figure 7F).

Consistent with earlier reports (Ahsan et al., 2012), the inhibition of HSP90 by geldanamycin caused an approximately two-fold reduction in total EGFR levels, while loss of CHORDC1 in A431 KO cells reduced EGFR levels only by 20% (Figure 7G). Notably, geldanamycin treatment further reduced the EGFR levels in A431 KO cells, below those detected in geldanamycin-treated A431 control cells. Thus, HSP90 stabilizes the EGFR at least in part independently of CHORDC1.

Taken together, we have found that the EGFR accumulates in the ER of A431 cells lacking CHORDC1, analogous to the mislocalization of LET-23 in C. elegans chp-1(lf) mutants. The increased trypsin sensitivity of the mislocalized EGFR in CHORDC1 mutant cells could be due to incorrect protein folding.

Unless specified otherwise, C. elegans strains were maintained at 20°C on Nematode Growth Medium (NGM) agar plates as described (Brenner, 1974). The C. elegans Bristol N2 strain was used as wild-type reference, and all strains generated through genetic crosses were derived from N2. A complete list of the C. elegans strains used can be found in the Supplementary file 1.

RNAi feeding experiments were performed as described previously (Kamath and Ahringer, 2003). The strain of interest was fed with E. coli HT115 expressing dsRNA against a specific target mRNA. Twenty synchronized L1 larvae were transferred to NGM plates containing 3 mM IPTG and 50 ng/ml ampicillin seeded with the indicated RNAi bacteria. The F1 progeny of the 20 P0 animals was analyzed at the L3 stage to score LET-23::GFP localization or at the L4 stage to examine vulval induction.

Vulval induction was scored by examining 20–40 worms of the indicated genotypes at the L4 stage under Nomarski optics. Animals were mounted on 4% agarose pads and anesthetized with 20 mM tetramisole in M9 buffer as described (Sternberg and Horvitz, 1986; Schmid et al., 2015). The vulval induction index (VI) was scored by counting the induced VPCs in 20–40 animals and calculating the average number of induced VPCs per animal. Statistical analysis is described in the legend to Figure 4.

The tunicamycin treatment to induce ER stress was carried out as described in Taylor and Dillin (2013). Briefly, animals expressing the hsp-4::gfp reporter were synchronized at the L1 stage and allowed to develop on NGM plates until the first day of adulthood. Then, they were incubated for 4 hr at room temperature in 25 µg/ml tunicamycin solution in M9 buffer. Control animals were incubated in an equivalent dilution of DMSO, which was used as a solvent for tunicamycin. After the treatment, HSP-4::GFP expression was observed with a 10x lens on a Leica DM RA wide-field microscope. Images were analyzed using Fiji software (Schindelin et al., 2012), and the average intensity of the whole body in each animal was measured to make the box plot in Figure 3—figure supplement 2.

Plasmid constructs were made using Gibson Assembly cloning (Gibson et al., 2009) and verified by DNA sequencing. A list of the oligonucleotide primers used for plasmid construction can be found in the Supplementary file 2. To construct plasmid pAHE3 (Pdlg-1::aman-2::mCherry::unc-54 3‘UTR, the plasmid the pCFJ151 backbone (Frøkjær-Jensen et al., 2008) was recombined with the Pdlg-1 promoter and the unc-54 3’ UTR, amplified as two individual fragments using the primers OEH153 and OEH158 and OEH159 and OEH156. The aman-2 (F58H1.1) genomic sequence encoding the first 82 amino acids, including the signal sequence and transmembrane anchor, amplified from genomic DNA using the primers OEH152 and OEH155, and the mCherry coding sequence, amplified with the primers OEH154 and OEH157, were then inserted after the Pdlg-1 promoter.

To construct pAHE6 (Pdlg-1::mCherry::C34B2.10(SP12)::unc-54 3‘UTR), the pCFJ151 backbone (Frøkjær-Jensen et al., 2008) was recombined with the Pdlg-1 promoter and the unc-54 3’ UTR, amplified as two individual fragments using the primers OAHE22 and OEH159 and OEH158 and OEH153. The mCherry coding sequence, amplified with the primers OAHE19 and OAHE20, and the genomic sequence of C34B2.10 (SP12) containing the stop codon, amplified with the primers OAHE21 and OAHE8, were then inserted after the Pdlg-1 promoter. The primer OAHE21 contained an additional linker sequence encoding three Alanines for the N-terminal fusion with mCherry. For each of the two reporter plasmids, single copy insertion transgenes were generated by the MosSCI method as described (Frøkjær-Jensen et al., 2008).

Two target sites in the first exon of the genomic chp-1 locus (sgRNA #1: CAG TGC TAT CAT AAA GGA TG and sgRNA #2 CGG TCT CCT TTT CGA TCC CA) were identified using the CRISPR design tool (http://crispr.mit.edu/) (Hsu et al., 2013). Double-stranded oligonucleotides were synthetized and cloned into the pDD162 (Addgene) to produce pAHE4 (sgRNA #1) and pAH5 (sgRNA #2). Plasmid pEV5 (Pegl-17-Δpes-10::cas9) (gift by Evelyn Lattmann) was used for 1° VPC-specific expression of the CAS9 protein. The plasmids pAHE4 and pAHE5 were co-injected into the gonads of wild-type animals as described (Mello et al., 1991) at a concentration of 50 ng/μl each together with the plasmid pEV5 at 100 ng/μl and the transformation markers pGH8 (Prab-3::mCherry) at 10 ng/ μl, pCFJ104 (Pmyo-3::mCherry) at 5 ng/μl and the pCFJ90 (Pmyo-2::mCherry) at 2.5 ng/μl to create the extrachromosomal array zhEx558.

The human vulva epidermoid carcinoma cell line A431 was obtained from Sigma Aldrich (85090402), tested to be free of mycoplasm and cultured in Dulbecco’s Modified Eagle Medium (Gibco 41966–029) according to standard mammalian tissue culture protocols and sterile technique. DMEM was supplemented with 10% FCS (Gibco 10500–064) and 1% Pen-Strep (Gibco 15140–122).

CHORDC1 guide RNAs targeting the first exon of CHORDC1 (CHORDC1 sgRNA #1: TTA CCG TCG GAA TTG GTC TC and CHORDC1 sgRNA #2: AGA CCA ATT CCG ACG GTA AG), as well as the scramble sequence GCA CTA CCA GAG CTA ACT CA, were identified using the CRISPR Design Tool (http://crispr.mit.edu/) (Hsu et al., 2013). Double-stranded oligos were generated and cloned into the lentiCRISPRv2 vector (Addgene), which was then transfected in combination with pVSV-G, pMDL and pREV into HEK293T cells to produce lentiviral particles (vMW6_CHORDC1 sg#1, vMW7_CHORDC1 sg#2, vMW9_scramble). Four days following transfection, the media from cells was collected, clarified by centrifugation, and filtered through a 0.45 μM filter to collect lentiviral particles. Subsequently, the particles were concentrated in Amicon Ultra tubes (Ultracel 100 k, Millipore). The titer of viral particles was determined before vMW6_CHORDC1 sg#1 and vMW7_CHORDC1 sg#2 were used to transduce 180’000 A431 cells in a 12-well plate at a combined MOI of 10. A431 cells were supplemented with DMEM media containing the lentiviral mix and 10 μg/ml polybrene and cultured under normal conditions. In parallel, cells were transduced with vMW9_scramble at a MOI of 10. Three days after transduction, cells were grown in the presence of 1.2 μg/ml puromycin. One week after puromycin selection, the puromycin-resistant populations were frozen and kept as stocks used in the subsequent experiments.

For western blot analysis, cells were lysed in lysis buffer on ice (100 mM Tris/HCl, 150 mM NaCl, 1% Triton X, 1 mM EDTA, 1 mM DTT, 10 ml lysis buffer + 1 tablet protease inhibitor), scraped with a cell scraper and snap frozen in liquid nitrogen. 100 µl of this mix was sonicated in a Bioruptor sonicator device (Diagenode), before 100 µl of 2x SDS loading dye were added. About 10 μg of protein extract were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% dried milk in 1x PBS plus 0.2% Tween 20 and then incubated with the diluted primary antibodies overnight at 4°C. Secondary anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (HRP) were used as the secondary antibodies. The HRP was detected by incubating the membrane with the SuperSignal West Pico or Dura Chemiluminescent Substrate (Thermo Scientific) for 4 min, before the signals were measured on a digital western blot imaging system. The antibodies used for Western blot analysis were: anti-CHORDC1 (HPA041040 Atlas Antibodies), anti-Tubulin (ab18251 abcam), anti-EGFR (HPA018530 Atlas Antibodies), anti-ERK1/2 (M5670 Sigma Aldrich), anti-ERK1/2 activated (M8159, Sigma Aldrich), HRP anti-Rabbit (111-035-144 Jackson Immuno Research) and HRP anti-Rabbit (115-035-146 Jackson Immuno Research). Quantification of western blots was done by measuring the band intensities in Fiji.

Cells were grown on glass slides in 24-well plates under standard conditions for 48 hr. Slides were then rinsed in PBS and fixed for 15 min in 4% PFA at 37°C. After washing with PBS, cells were permeabilized with PBS containing 0.2% Triton X-100% and 0.5% BSA for 5 min, and then blocked for 1 hr in PBS containing 0.5% BSA and 0.2% gelatine. Primary antibodies were added in blocking solution for 1 hr at room temperature in a humid chamber. The cells were rinsed in PBS three times before being incubated for 40 min in the dark with secondary antibodies and Phalloidin 568 (B3475 Thermo Scientific). After three washes with PBS, cells were stained for 5 min with PBS containing 0.1 µg/ml DAPI, followed by three washes with PBS. Glass slides were mounted with ProLong Gold Antifade Mountant (Thermo Scientific). The antibodies used for immunocytochemistry were: anti-CHORDC1 (HPA041040 Atlas Antibodies), anti-Tubulin (ab18251 abcam), anti-EGFR (MA5-13269 Thermo Scientific), anti-PDI (MA3-019 Thermo Scientific), Alexa Fluor 488 (A11034 Thermo Scientific), and Alexa Fluor 647 (A21236 Thermo Scientific). For all antibody stainings, at least three biological replicates were made.

250’000 A431 cells were grown in 12-well plates under standard conditions for 24 hr. Thereafter, growth medium was replaced with DMEM lacking FCS. After 15 hr of starvation, cells were stimulated with 100 ng/ml human EGF (E9644 Sigma) for 10 min at 37°C, before they were lysed and prepared for western blot analysis.

750’000 A431 cells were seeded into a 25 cm² flask and grown until they reached confluency. After washing twice with cold PBS, cells were scraped with a cell scraper, lysed in lysis buffer (100 mM Tris pH = 8, 1% NP-40, 150 mM NaCl, 1 mM DTT) for 10 min on ice, sonicated for 10 min at 4°C, and clarified by centrifugation at 17,000 x g for 10 min at 4°C. Aliquots containing 50 µg of cleared protein samples were incubated with 0.1, 0.15, 0.2, 0.4, 0.6, 0.8, 1, 1.25 and 1.5 µg/ml Trypsin (Sigma EMS0004) and incubated for 15 min at 25°C while shaking. Thereafter, 2x SDS loading dye was added and samples were boiled prior to resolving the proteins by SDS-PAGE.

To examine the expression pattern of fluorescently tagged proteins, a Leica DM RA wide-field microscope equipped with a Hamamatsu ORCA-ER camera using a 40x/1.3 NA or 63x/1.4 NA oil immersion objective was used. Fluorescent and Nomarski mages were acquired with the Openlab 4.0 or VisiView 2.1 software. To observe the localization of GFP reporters, around 40 worms at the L3 stage were mounted on 4% agarose pads and anesthetized with 20 mM tetramisole in M9 buffer. In each experiment, the illumination intensity, camera exposure time and other software settings were kept constant.

Around 40 larvae at the Pn.p to Pn.pxx stage were mounted on 4% agarose pads in M9 buffer containing 2 mM tetramisole. z-stacks at 0.2 to 0.3 μm spacing were taken using a Plan-Apochromat 63x/1.4 NA oil immersion objective on a Zeiss LSM710 confocal laser scanning microscope equipped with an 458/488/514 nm argon laser and a 594 nm helium-neon laser. Images were acquired by using the LSM710 ZEN 2012 software (Zeiss). GFP was excited with a 488 laser excitation and emission was detected in a range of 493–566 nm. mCherry was excited with a 594 nm laser excitation and emission was detected in a range of 599–696 nm. Images were captured with a variable frame size, a pinhole equivalent to 1 Airy and a pixel size of 0.08 μm. Identical camera gain settings were used for all live animal imaging (AMAN-2::mCherry: GFP 600, mCherry 500/mCherry::SP12: GFP 600, mCherry 600 in Figure 3). Images of antibody-stained A431 cells (Figure 6 and 7) were taken on a Leica CLSM SP8 upright microscope equipped with 405/488/552/638 nm solid state diode lasers. z-stacks at 0.16 µm spacing were taken using a 63x/1.4 HCX PL APO CS2 oil immersion objective with a variable frame size and a pinhole equivalent to 1 Airy.

Images were analyzed and processed with Fiji software (Schindelin et al., 2012) to adjust brightness and contrast. Images in Figures 3, 6 and 7 were deconvolved using the Huygens deconvolution software (Scientific Volume imaging). To quantify co-localization in Figure 3 and 7, raw images (before deconvolution) were processed in Fiji using the Subtract Background command (default settings, rolling ball radius = 0.3). To quantify co-localization, z-stacks were analyzed with the ImarisColoc module in Imaris 8.3. (Bitplane). The GFP channel of each image was thresholded to define a ROI using the mask channel function. An automatic threshold implemented in Imaris was set for both channels based on a statistical significance algorithm (Costes et al., 2004). With this approach, the extent of co-localization of two fluorescent-labeled proteins in an image is automatically quantified, without the bias of visual interpretation. Based on an automatically identified threshold calculated by the Coste’s approach, a Manders Colocalization Coefficient (MCC) indicating the fraction of total probe fluorescence that co-localizes with the fluorescence of a second probe was calculated (Manders et al., 1993). The calculated MCC values were averaged, and statistical analysis was performed using a a two-tailed Student’s t-test for independent samples.