Chen Y , Bharill S , Altun Z , O'Hagan R , Coblitz B , Isacoff EY , Chalfie M .

R01 GM030997 NIGMS NIH HHS , R37 GM030997 NIGMS NIH HHS , GM30997 NIGMS NIH HHS , R01 NS035549 NINDS NIH HHS , NS35549 NINDS NIH HHS , R01 GM117051 NIGMS NIH HHS

	FIGURE 1:. TRN expression of MEC-6 and POML-1. Confocal sections of TRN cell bodies of (A) MEC-6::TagRFP and the ER marker YFP::TRAM-1, (B) POML-1::TagRFP and the ER marker YFP::PISY-1, (C) MEC-6::TagRFP and POML-1::YFP, and (D) MEC-6::TagRFP and the Golgi marker AMAN-2::YFP and their correlation coefficient (E). Scale bars, 5 μm (A–D, F–H, J–L). The number of examined TRNs is given in parentheses (E, I). Symbols for significance here and in all subsequent figures are described in Materials and Methods. Neurite expression of (F) POML-1::YFP and MEC-6::TagRFP, (G) POML-1::YFP and MEC-4::TagRFP, and (H) POML-1::YFP and MEC-2 and their correlation coefficient (I). Anti-GFP and anti-MEC-2 antibodies (Ab) were used to label the proteins in (H). (J) MEC-6::3XFLAG expression as detected by an anti-FLAG antibody in intact (top) and permeabilized (bottom) cultured TRNs. (K) POML-1::YFP expression as detected by an anti-GFP antibody in intact (top) and permeabilized (bottom) TRNs in culture. The faint immunofluorescence in intact cells (J, K) was not specific because it was often observed in cells that did not express MEC-6::3XFLAG or POML-1::YFP. Images in J and K are representative of 40 cells examined in two independent experiments. (L) MEC-4 expression detected with an anti-MEC-4 antibody that recognizes the extracellular domain in intact (top) and permeabilized (bottom) cultured TRNs. Images are representative of 20 cells examined in two independent experiments.
	FIGURE 2:. POML-1 is required for touch sensitivity and mec-4(d)–induced TRN degeneration. (A) poml-1(u882) reduced the touch response to 10 touches in sensitized backgrounds (30 animals were examined in three independent experiments). (B) poml-1 and mec-6 mutations suppress mec-4(d) degenerations. n, number of animals examined. TRNs labeled with GFP in L4 and young adult animals were scored as having survived. uIs83 is an integrated array that overexpresses mec-4(d). The rescue experiments used three to five stable lines.
	FIGURE 3:. The effect of POML-1 on MEC-4(d) channel activity and their physical interaction in Xenopus oocytes. (A) Effect of POML-1 (white bars) on the MEC-4(d) amiloride-sensitive current at −85 mV in oocytes. The number of oocytes tested is given in parentheses. The oocytes were from at least two frogs. (B) Immunoprecipitation (IP) of POML-1 with MEC-4(d) and MEC-6 expressed in oocytes. IB, immunoblot probe. Images are representative of two or three independent experiments. Molecular weights (kilodaltons) of the protein markers are indicated on the right. EGFP::POML-1 is functional, since its coexpression with MEC-4(d) generated amiloride-sensitive currents (EGFP::POML-1 and Myc::MEC-4(d), Iamil = −1.4 ± 0.3 μA, n = 5) that were similar to those of coexpressing untagged POML-1 and MEC-4(d) (Iamil = −1.6 ± 0.6 μA, n = 5) 6 d after cRNA injection. The negative control (–) is EGFP with the same tag.
	FIGURE 4:. MEC-4 expression in cultured TRNs with mec-6(u450) or poml-1(ok2266) mutation. Images (A) and quantification (B) of MEC-4 expression as detected by an anti–MEC-4 antibody in intact (left) and permeabilized (right) TRNs in culture. MEC-4 immunofluorescence intensity was normalized to that of wild-type (WT) TRNs. Scale bars, 5 μm (A, C). Because most cell bodies leaked after immunostaining for intact cells (as evident by the staining for MEC-18, a cytoplasmic protein), immunofluorescence was only measured for MEC-4 surface expression in intact neurites (which did not show MEC-18 staining; Materials and Methods). Statistical significance is indicated for comparison with the WT cells by one-way ANOVA with Tukey post hoc (B, D). The number of cell bodies tested from two or three experiments is given in parentheses (B, D). The subcellular localization of MEC-4 and markers for the ER, endosome, and Golgi (C) in cultured TRNs and their correlation coefficient (D).
	FIGURE 5:. Effect of mec-6, poml-1, and crt-1 mutations on MEC-4 and MEC-2 expression in the TRNs. Unless noted, the following mutations were used: crt-1(ok948), mec-4(u253), mec-5(u444), mec-6(u450), uba-1(it129), and poml-1(ok2266). (A) MEC-4::TagRFP expression. Left, merged images of expression at 10 focal planes; right, images of the single plane showing the best-focused image of the same cell body. Scale bar, 5 μm (A–C). (B) Confocal images of MEC-4::TagRFP and POML-1::YFP in the TRN cell body of WT animals (top) and mec-6(u450) mutants (bottom). Their correlation coefficient is 0.2 ± 0.04 and 0.7 ± 0.04 in WT and mec-6 mutants (10 TRNs), respectively. (C) MEC-2 expression in the TRN neurite. Representative of 20–30 TRNs examined in two experiments. (D) MEC-4::TagRFP fluorescence intensity (normalized to WT) in PLM cell bodies of L4 larvae and young adults of controls and mec-6(u450), poml-1, and crt-1 without (black) or with (white) a uba-1 mutation. For each pair of white and black bars, the effect of the uba-1 mutation was significant at p < 0.001, except for WT, for which it was at p < 0.01. The comparison to the control within each group (with or without uba-1) was significant at p < 0.001 by two-way ANOVA with Bonferroni posttest. The number of examined PLM cells collected from three independent experiments is given in parentheses (D, E). (E) Fluorescence intensity of MEC-4::TagRFP (normalized to WT control) in PLM cell bodies of WT and mutants either untreated (black) or treated (white) with 50 μM bortezomib for 8 h. Statistical comparisons are as in D, with the exception that the difference between treated and untreated WT animals was not significant.
	FIGURE 6:. MEC-6, POML-1, and CRT-1 may function as chaperones. The mutations mec-6(u450), poml-1(ok2266), and crt-1(ok948) were used. (A) The effect of sec-24.1 and sec-24.2 overexpression, uEx[sec-24(+)], on the suppression of mec-4(d) deaths by poml-1, crt-1, and mec-6 mutations. In some strains, the cargo-binding sites of sec-24.1 and sec-24.2 were mutated (sec-24(m)). n, number of animals examined. The results with uEx[sec-24(+)] and uEx[sec-24(m)] were collected from two to five stable lines. (B) Effect of overexpressing sec-24(+) on MEC-4::TagRFP fluorescence intensity in the PLM cell bodies and proximal neurites of L4 larvae and young adults with the poml-1 mutation. Data for uEx[sec-24(+)] were collected from two stable lines. The number of PLM cells examined is given in parentheses. Fluorescence intensity was normalized to that of PLM in poml-1 mutants (control). Scale bar, 5 μm. (C) Schematic of CFP::MEC-4::YFP protein (left) and images of CFP::MEC-4::YFP in the TRN cell body taken with the CFP (blue), FRET (red), and YFP (yellow) channels, respectively (right). The Net FRET signal is given by a pseudocolored image to show the relative intensity. (D) The normalized FRET signal (see Materials and Methods) of CFP::MEC-4::YFP either in the TRN cell bodies (white bars) of WT animals and mutants or in the puncta of WT animals (black bar). The number of cell bodies or strongly fluorescent puncta tested (from two or three stable lines with extrachromosomal arrays collected from three of four experiments) is given in parentheses. Statistical significance is indicated for comparison with the FRET signal in the WT cell body. (E) CFP::MEC-4::YFP intensity (measured in the YFP channel and normalized to that WT controls) and FRET signals in the TRN cell body of WT, mec-6, poml-1, and crt-1 animals treated with bortezomib. The number of examined cells bodies here and in F is indicated in parentheses. These experiments used cells from an integrated line, which produced similar FRET signals to the stable lines with extrachromosomal arrays used in D. Bortezomib treatment had a significant effect on CFP::MEC-4::YFP intensity (left, F(1, 58) = 33.56, p < 0.0001) but no effect on the FRET signal (right, F(1, 58) = 0.01, p = 0.9162, by two-way ANOVA with Bonferroni posttests). The value above the bracket is that of the pairwise comparison. The values below the bracket are for the comparison to the WT of each untreated (black bars) or treated group (white bars) by two-way ANOVA with Bonferroni posttests. The difference in CFP::MEC-4::YFP fluorescence intensity (left) between control WT and bortezomib-treated mec-6, poml-1, and crt-1 mutants was not significant. (F) FRET signals in mec-6 and poml-1 animals overexpressing sec-24(+) in TRNs.
	FIGURE 7:. Effect of MEC-6 and POML-1 on MEC-4 surface expression in Xenopus oocytes. (A) Images and (B) quantification of MEC-4::EGFP fluorescent spots on the oocyte surface visualized by TIRF imaging (19–29 patches from 14–16 cells from two different batches) 2 d after cRNA injection. Values are compared with the expression of MEC-4::EGFP alone using the Mann–Whitney test. The field dimensions are 13 μm × 13 μm. (C) Western blot of Myc::MEC-4(d) on the surface of oocytes as detected by biotinylation (top) and the expression of Myc::MEC-4(d) in total lysate of oocytes (middle) at 2 d after cRNA injection. β-Actin detected in total lysate was used as an input control (bottom). Molecular weights (kilodaltons) of the protein markers are indicated on the right. (D) Quantification of changes in surface Myc::MEC-4(d) detected by biotinylation at 2 d after cRNA injection (the number of independent experiments is given in parentheses). All data are normalized and compared with Myc::MEC-4(d) expression alone by the one-sample t test. MEC-6, MEC-2, and POML-1 did not affect Myc::MEC-4(d) levels in total lysates at 2 d after injection (MEC-6, 1.0 ± 0.1; MEC-2, 1.0 ± 0.1; POML-1, 0.9 ± 0.1; four or five independent experiments, normalized and compared with the expression of Myc::MEC-4(d) alone; not significant by one-sample t test). The normalized amount of total Myc::MEC-4(d) differed by no more than 25% in any of the experiments. (E) The amiloride-sensitive MEC-4(d) current at −85 mV (12–18 oocytes [2 d after cRNA injection] or 6–12 oocytes [other times] of three batches) on its own and in the presence of MEC-2, MEC-6, and POML-1 at various times after cRNA injection. p < 0.001 for Iamil at −85 mV between oocytes 2 d after injected with MEC-4(d) and MEC-6 vs. MEC-4(d) alone, MEC-4(d) and MEC-2, or MEC-4(d) and POML-1; no statistically significance was found between oocytes 2 d after injection with MEC-4(d) alone vs. MEC-4(d) and MEC-2, or MEC-4(d) and POML-1. p < 0.001 for Iamil at −85 mV between oocytes 1 d after injected with MEC-4(d) and MEC-6 vs. MEC-4(d) alone; p < 0.01 between MEC-4(d) and MEC-6 vs. MEC-4(d) and MEC-2, or MEC-4(d) and POML-1. One-way ANOVA with Tukey post hoc.

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