The α-arrestin SUP-13/ARRD-15 promotes isoform turnover of actin-interacting protein 1 in Caenorhabditis elegans striated muscle

Figures

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  Fig. 1.. The sup-13 mutation suppresses motility defects and sarcomere disorganization of the unc-78 null mutant. A) Motility of young adult worms was quantified by measuring beating frequency (beats per 30 s). An unc-78 null allele, unc-78(gk27), was used in this study and simply shown as unc-78 (n = 30, data are average ± standard deviation). ***, P < 0.001. n.s., not significant. B) Adult worms were stained for F-actin by tetramethylrhodamine-phalloidin, and regions of the body wall muscle are shown. Actin was organized in a striated manner in wild type but highly disorganized with aggregates in unc-78(gk27). This phenotype was suppressed in the sup-13(st210);unc-78(gk27) double mutant to nearly an appearance of wild type. The sup-13(st210) single mutant did not show a detectable phenotype in the F-actin organization. Bar, 10 μm. C) Worms at different developmental stages were stained for F-actin by tetramethylrhodamine-phalloidin, and percentages of worms with F-actin aggregates (counted as positive when >25% of total muscle cells contained aggregates) were analyzed. For each experiment, > 500 worms were examined for each stage, and average ± standard deviation of three independent…

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  Fig. 2.. sup-13 is a nonsense allele of arrd-15 encoding an α-arrestin. A) Domain organization of the C. elegans SUP-13/ARRD-15 protein (374 amino acids, NCBI Reference Sequence: NP_001254970.1) with a location of the sup-13(st210) mutation. Arrestin-N (Arr-N) and arrestin-C (Arr-C) domains are indicated by boxes. N- and C-termini are shown as N and C, respectively. For comparison, human ARRDC1/α-arrestin 1 (433 amino acids, NCBI Reference Sequence: NP_689498.1) and ARRDC5 (342 amino acids, NCBI Reference Sequence: NP_001073992.1) are shown. Multiple copies of PY motifs (PP × Y) are present in the C-termini of many α-arrestins but absent in SUP-13/ARRD-15 and human ARRDC5. B, C) Transgenic rescue of the sup-13 suppressor phenotype was demonstrated by expressing GFP::ARRD-15 in the body wall muscle of sup-13(st210);unc-78(gk27) using the myo-3 promoter. The worms were stained with tetramethylrhodamine-phalloidin, and sarcomeric actin filaments (F-actin) were examined in GFP::ARRD-15 positive [GFP (+)] and negative [GFP (−)] cells. In the merged image (GFP::ARRD-15 in green and F-actin in red), cell-cell boundaries are marked by white borders. B). Bar, 10 μm. The presence or absence of…

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  Fig. 3.. The second AIP1 gene aipl-1 is required for sup-13/arrd-15-dependent suppression of the unc-78 mutant phenotypes. A) Wild type, unc-78, or sup-13;unc-78 worms were treated with control RNAi or aipl-1(RNAi) from the L4 larval stage, and the phenotypes were examined on agar plates in the next generation. In control RNAi, all strains grew to adult worms. However, in aipl-1(RNAi), only wild type grew to adult worms, but unc-78 and sup-13;unc-78 were arrested as embryos. Bar, 1.0 mm. B) Wild type, unc-78, or sup-13;unc-78 worms were treated with control RNAi or aipl-1(RNAi) from the L1 larval stage, and stained for F-actin with tetramethylrhodamine-phalloidin when they became adults, and regions of the body wall muscle are shown. In adult muscle, aipl-1(RNAi) did not affect the F-actin organization in wild type and unc-78 but caused severe F-actin disorganization in sup-13;unc-78. Bar, 20 μm. C) Percentages of worms with F-actin aggregates (counted as positive when >25% of total muscle cells contained aggregates) were analyzed. For each experiment, >200 worms were examined for each sample, and average ± standard deviation of three independent experiments are shown (error bars are too…

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  Fig. 4.. The sup-13/arrd-15 mutation increases the protein levels of AIPL-1 in the body wall muscle. A, B) The AIPL-1::GFP protein levels in the body wall muscle were determined in the head region where the autofluorescence of the gut granules is absent. Fluorescence images of AIPL-1::GFP were captured using the same exposure settings for the four strains (A; bar, 20 μm), and the AIPL-1::GFP protein levels were determined as fluorescence intensity (arbitrary units) within the body wall muscle (BWM in A) in B. Data are average ± standard deviation (n = 10). *, 0.01 < P < 0.05. ***, P < 0.001. C, D) The UNC-78 protein levels in whole worm lysates were determined by Western blot using anti-UNC-78 antibody. Whole worm lysates of an equal number of young adults (30 worms) were loaded for each strain and probed with anti-UNC-78 antibody (C, top). Total proteins were stained with Coomassie Brilliant Blue (C, bottom) and used as a loading control to normalize the UNC-78 signals (arbitrary units) in D. Positions of molecular weight markers in kDa are shown on the left. Data are average ± standard deviation (n = 6). *, 0.01 < P < 0.05. **, 0.001 < P < 0.01. n.s., not significant.

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  Fig. 5.. Ubiquitinated AIPL-1 is accumulated in the sup-13/arrd-15 mutants. A, B) AIPL-1::GFP was immunoprecipitated with anti-GFP magnetic beads and probed with anti-Ub (Ub) antibody (A, top) and anti-DYKDDDDK (FLAG epitopes in AIPL-1::GFP) antibody (A, bottom). The anti-Ub signals (arbitrary units) were normalized to anti-DYKDDDDK signals and shown in the graph (B). Data are average ± standard deviation (n = 3). *, 0.01 < P < 0.05. **, 0.001 < P < 0.01. ***, P < 0.001. n.s., not significant. C, D) Whole lysates of adult worms were examined by Western blot using anti-Ub antibody (C, left). Total proteins on the blot were stained with Coomassie Brilliant Blue (CBB) (C, right) and used as a loading control to normalize the anti-Ub signals (arbitrary units) in D. Positions of molecular weight markers in kDa are shown on the left in A and C. Data are average ± standard deviation (n = 3). There were no significant differences among the data.

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  Fig. 6.. Model for the regulatory mechanism of AIP1 isoform turnover in the body wall muscle. The current study suggests that SUP-13/ARRD-15 specifically targets AIPL-1 for degradation in the body wall muscle. Therefore, in wild type, UNC-78 is the predominant AIP1 isoform that cooperates with UNC-60B (ADF/cofilin) to promote actin filament dynamics. In unc-78 single mutants, there is not enough AIP1 proteins to promote actin filament dynamics, causing sarcomere defects. However, in sup-13;unc-78 double mutants, AIPL-1 is not degraded and compensates for the absence of UNC-78. In sup-13 single mutants, both UNC-78 and AIPL-1 promotes actin filament dynamics and do not cause apparent sarcomere defects.