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First published online 7 August 2007
doi: 10.1242/jcs.013516

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UNC-87, a calponin-related protein in C. elegans, antagonizes ADF/cofilin-mediated actin filament dynamics

¹ Department of Pathology, Emory University, Atlanta, GA 30322, USA
² Unit of Actin Cytoskeleton Regulation, Consorzio Mario Negri Sud, Department of Cell Biology and Oncology, Via Nazionale 8a, 66030 Santa Maria, Imbaro, Italy

View larger version (36K): [in this window] [in a new window]   Fig. 1. UNC-87 or UNC-60B interacts with Ce-actin in a mutually exclusive manner. (A) 10 µM Ce-actin was pre-incubated with 0-10 µM UNC-87 for 30 minutes, and then, various concentrations of UNC-60B (0-20 µM) were added to the mixtures. After 30 minutes, the samples were centrifuged at 285,000 g for 20 minutes, and the supernatant (s) and pellets (p) were analyzed by SDS-PAGE (12% acrylamide gel). Representative gels from experiments in the absence (a) or in the presence of 10 µM UNC-87 (b) are shown. Quantitative analysis of the results by densitometry is shown in c, in which the amounts of actin-bound UNC-60B (mol/mol actin) were plotted as a function of total UNC-60B concentrations (µM). (B) 10 µM Ce-actin was pre-incubated with 0-20 µM UNC-60B for 30 minutes, and then, various concentrations of UNC-87 (0-20 µM) were added to the mixtures. After 30 minutes, the samples were centrifuged at 285,000 g for 20 minutes, and the supernatant (s) and pellets (p) were analyzed by SDS-PAGE. Representative gels of experiments in the absence (a) and in the presence of 20 µM UNC-60B (b) are shown. Quantitative analysis of the results by densitometry is shown in c, in which the amounts of actin-bound UNC-87 (mol/mol actin) were plotted as a function of free UNC-87 concentrations (µM). (d) The quantitative results were further analyzed using a Scatchard plot.

View larger version (74K): [in this window] [in a new window]   Fig. 2. UNC-60B inhibits actin-bundling activity of UNC-87. (A) 10 µM rabbit F-actin was pre-incubated with 0-20 µM UNC-60B for 30 minutes, and varied concentrations of UNC-87 (0-20 µM) were added to the mixtures. After 30 minutes, the samples were centrifuged at 18,000 g for 10 minutes, and the supernatant (s) and pellets (p) were analyzed by SDS-PAGE. Representative gels of the experiments in the absence (a) and presence of 20 µM UNC-60B (b) are shown. Quantitative analysis of the results by densitometry is shown in (c), in which the percentages of sedimented (bundled) actin were plotted as a function of total UNC-87 concentrations (µM). Data are means ± s.d. of three experiments. (B) 1 µM Alexa Fluor 488-biotin-labeled rabbit F-actin was incubated with buffer only (a), 0.4 µM UNC-87 (b), 1 µM UNC-60B (c), or 1 µM UNC-60B and 0.4 µM UNC-87, and the filaments were observed by fluorescence microscopy. UNC-87 induced actin bundling (b, arrows), but the bundling was inhibited by the presence of UNC-60B (d). Bar, 20 µm.

View larger version (158K): [in this window] [in a new window]   Fig. 3. UNC-87 and tropomyosin inhibit actin filament severing by UNC-60B. Alexa Fluor 488-biotin-labeled rabbit actin filaments in perfusion chambers were treated in two sequential perfusion steps with a buffer containing proteins as indicated on the figure. Arrows in j indicate severed filaments. Micrographs of the same fields were taken after both perfusion steps. Bar, 20 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 4. UNC-87 and tropomyosin inhibit UNC-60B-mediated actin turnover. (A) Simultaneous binding of UNC-87 and CeTM to Ce-actin. (a) Ce-actin (10 µM) was pre-incubated with 0-10 µM UNC-87 for 30 minutes and subsequently incubated with 2 µM CeTM for 30 minutes. The reactions were analyzed by pelleting assays (s, supernatants; p, pellets). (b) Ce-actin (10 µM) was pre-incubated with 0-5 µM CeTM for 30 minutes and subsequently incubated with 5 µM UNC-87 for 30 minutes. The reactions were analyzed by pelleting assays as shown in a. (B) Effects of UNC-87 or CeTM on UNC-60B-accelerated actin turnover were measured by nucleotide exchange. F-actin (5 µM) from rabbit muscle (a) or C. elegans (b) was mixed with UNC-60B (2.5 µM) and UNC-87 (0, 0.25, 0.5, 1, or 2 µM; black circles), UNC-60B (2.5 µM) and CeTM (0, 0.5, 1, or 2 µM; white circles), or UNC-60B (2.5 µM), CeTM (0.5 µM) and UNC-87 (0, 0.5, 1, or 2 µM; black triangles) in the presence of 40 µM etheno-ATP, and the fluorescence of etheno-ATP was monitored over time. The data were fitted to exponential curves and the rate of increase in the fluorescence (kobs: 1/second) were calculated and plotted as a function of concentration of UNC-87 or CeTM. Values are mean ± s.d. of three experiments. (C) Co-pelleting assay of 10 µM Ce-actin with 10 µM UNC-60B after pre-incubation with various concentrations of UNC-87 in the absence (a; black circles in c) or presence of 5 µM CeTM (b; white circles in c). Relative amounts (%) of actin-bound UNC-60B in the pellets were plotted as a function of total UNC-87 concentrations (c). Values are means ± s.d. of three experiments.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Localization of UNC-87, CeTM and UNC-60B in adult body wall muscle. Wild-type adult nematodes were double-stained with anti-UNC-87 (a,d,g) and anti-actin (b) or anti-UNC-60 (e) or anti-CeTM (h) antibodies. Merged images are shown in c,f,i. Bars, 10 µm. (j) Schematic representation of locations of UNC-87, CeTM and UNC-60B in the thin filaments.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Genetic interaction between UNC-87 and UNC-60B in adult body wall muscle. (A) Adult body wall muscle of wild-type (a-c,g-i), unc-87(e1216) (d-f), or unc-60B(r398) (j-l) animals was double stained with anti-actin (a,d,g,j) and anti-UNC-60B (b,e) or anti-UNC-87 (h,k) antibodies. Merged images are shown in c,f,i,l (red: actin, green: UNC-60B, or UNC-87). (B) Actin filament organization in adult body wall muscle was visualized by staining with tetramethylrhodamine-phalloidin. Micrographs of wild-type (a), +/+; unc-60B(r398) (b), unc-87(e1216)/+; +/+ (c), unc-87(e1216)/+; unc-60B(r398) (d), unc-87(e1459)/+; +/+ (e), and unc-87(e1459)/+; unc-60B(r398) (f) muscle. Bars, 10 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Genetic interaction between UNC-87 and CeTM in body wall muscle. (A) Adult body wall muscle of wild-type (a-c) or unc-87(e1216) (d-f) animals was double stained with anti-actin (a and d) and anti-CeTM (b,e) antibodies. Merged images are shown in c and f (red: actin, green: CeTM). (B) Actin filament organization in adult body wall muscle was visualized by staining with tetramethylrhodamine-phalloidin. Wild-type (a-c), unc-87(e1216) homozygous (d-f), or unc-87(e1459) homozygous (g-i) animals were treated with control RNAi (a,d,g), CeTMI,II (RNAi) (b,e,h), or CeTMI,II,III,IV (RNAi) (c,f,i). Bars, 10 µm.