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. 2012 Sep 1;125(Pt 17):4077-89.
doi: 10.1242/jcs.104950. Epub 2012 May 23.

CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

Kazumi Nomura  1 , Kanako Ono, Shoichiro Ono
Affiliations

CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

Kazumi Nomura et al. J Cell Sci. .

Abstract

Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.

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Figures

Fig. 1.
Fig. 1.
Expression and localization patterns of CAS-1 in C. elegans embryos and adult worms. (A,B) Expression patterns of CAS-1 in embryos at the 1.5-fold (∼420-min old; A) and 2-fold (∼450-min old; B) stages. Embryos were fixed and stained with anti-CAS-1 (left panels) and anti-MYO-3 (a marker for the body wall muscle; middle panels). Merged images are shown in the right panels (CAS-1 in red and MYO-3 in green). CAS-1 was expressed predominantly in the body wall muscle as indicated by arrows in A and B. (C–F) Expression patterns of CAS-1 in adult worms. (C) Adult worms were fixed and stained with anti-CAS-1 (left panel) and anti-MYO-3 (middle panel), and a region of the body wall muscle is shown. A merged image is shown in the right panel (CAS-1 in red and MYO-3 in green). Immunostaining of CAS-1 was also detected in the pharynx (D), the neurons (D), the intestinal lumen (E), and the spermatheca of the somatic gonad (F). (G–K) Sarcomeric localization of CAS-1 in C. elegans adult body wall muscle. Adult worms were fixed and stained with anti-CAS-1 (left panels) and anti-actin (G, middle), anti-MYO-3 (H, middle), anti-UNC-89 (I, middle), anti-vinculin (J, middle) or anti-α-actinin (K, middle) antibodies. Merged images are shown in the right panels (CAS-1 in red, and actin, MYO-3, UNC-89, vinculin and α-actinin in green). Arrowheads in G–I indicate positions of the M-lines, and arrows in G, J and K indicate positions of CAS-1 spots in the I-bands.
Fig. 2.
Fig. 2.
Effects of CAS-1 on actin polymerization. (A–D) Effects of CAS-1 on the initial phase of actin polymerization. G-actin (5 µM, 20% pyrene labeled) was polymerized by addition of salt at time 0 in the presence of 0–15 µM MBP–CAS-1 (A), MBP–CAS-1N (B) or MBP–CAS-1C (C), and intensity of the pyrene fluorescence (arbitrary units; AU) was monitored over time. (D) Relative rates of actin polymerization in the presence of MBP–CAS-1 (white circles), MBP–CAS-1N (white triangles), MBP–CAS-1C (white squares), and MBP (black circle) were determined as described in Materials and Methods. Data are means ± s.d. of three independent experiments. (E–H) Effects of CAS-1 on the steady state of actin polymerization. Varying concentrations of actin (20% pyrene labeled) were polymerized in the presence of 0–5 µM MBP (E), MBP–CAS-1 (F), MBP–CAS-1N (G) or MBP–CAS-1C (H) for 18 hr, and the intensity of the pyrene fluorescence (arbitrary units) was measured.
Fig. 3.
Fig. 3.
Binding of CAS-1 to G-actin and the UNC-60B–actin complex, examined by nondenaturing polyacrylamide gel electrophoresis. Various concentrations (1–10 µM) of MBP (A), MBP–CAS-1 (B), MBP–CAS-1N (C) or MBP–CAS-1C (D) were incubated with buffer only (lanes 4–7) or buffer with 10 µM G-actin (lanes 8–11) or 10 µM G-actin and 10 µM UNC-60B (lanes 12–15) and examined by nondenaturing acrylamide gel electrophoresis. G-actin alone (lane 1), UNC-60B alone (lane 2), and mixtures of G-actin and UNC-60B (lane 3) were also applied to determine positions of free G-actin, free UNC-60B and the actin–UNC-60B complex (asterisks).
Fig. 4.
Fig. 4.
Effects of CAS-1 on exchange of actin-bound nucleotides in the absence and presence of UNC-60B. ATP–G-actin (1 µM) was incubated with etheno–ATP in the presence of 0–0.5 µM MBP–CAS-1 (A,B), MBP–CAS-1N (C,D), or MBP–CAS-1C (E,F) without UNC-60B (A,C,E) or with 1 µM UNC-60B (B,D,F), and the fluorescence of etheno–ATP (arbitrary units) was monitored over time. (G) Rates of exchange of nucleotides (kobs) were determined from the data and plotted as a function of concentrations of the CAS-1 variants. Data are means ± s.d. of three independent experiments.
Fig. 6.
Fig. 6.
Effects of CAS-1 and UNC-60B on the lengths of actin filaments. Actin (2 µM, 20% DyLight549-labeled) was polymerized without an MBP fusion protein (A,B), or with 0.5 µM MBP (C,D), MBP–CAS-1 (E,F), MBP–CAS-1N (G,H) or MBP–CAS-1C (I,J) in the absence (A,C,E,G,I) or presence of 1 µM UNC-60B (B,D,F,H,J) and observed by fluorescence microscopy. Filament lengths (averages ± s.d., n = 50) are shown on the lower right corner of each panel.
Fig. 5.
Fig. 5.
Effects of CAS-1 and UNC-60B on actin filament turnover as determined by the rate of phosphate release. Final 5 µM F-actin was mixed with various concentrations of UNC-60B and MBP–CAS-1 (A), MBP–CAS-1N (B) or MBP–CAS-1C (C), and the rate of Pi release (arbitrary units) was determined and plotted as a function of concentrations of UNC-60B. (D) Rate of Pi release from 5 µM F-actin with 5 µM UNC-60B in the presence of 0–0.5 µM MBP–CAS-1 (circles), MBP–CAS-1N (triangles) or MBP–CAS-1C (squares) were plotted as a function of the concentrations of the CAS-1 variants. Data are means ± s.d. of three independent experiments.
Fig. 7.
Fig. 7.
cas-1 mutation causes disorganization of actin and UNC-60B in the body wall muscle. (A,B) F-actin organization in late larval body wall muscle. Wild-type (+/+; A) or cas-1(ok1523) homozygous (cas-1(ok1523)/cas-1(ok1523); B) worms were stained with tetramethylrhodamine–phalloidin, and regions of the body wall muscle are shown. Scale bar: 50 µm. (C–K) Localization of UNC-60B and actin in embryos. Wild-type (+/+; C–E), cas-1 heterozygous (cas-1(ok1523)/+; F–H) or cas-1 homozygous (cas-1(ok1523)/cas-1(ok1523); I–K) embryos were fixed and immunostained for actin (C,F,I) and UNC-60B (D,G,J). Merged images are shown in E, H and K (actin in green and UNC-60B in red). Arrows in C–H indicate positions of the body wall muscle. Arrowheads in I–K indicate abnormal aggregates of actin and UNC-60B.
Fig. 8.
Fig. 8.
UNC-60B deficiency does not affect myofibril localization of CAS-1. Immunolocalization of CAS-1 (A) and actin (B) in the body wall muscle of unc-60B(su158) adult worms. A merged image is shown in C with CAS-1 in red and actin in green. Arrows indicate positions of the M-lines, where CAS-1 remained localized. Arrowheads indicate actin aggregates where CAS-1 did not localize.
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