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First published online 2 September 2003
doi: 10.1242/jcs.00717

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Actin filament disassembling activity of Caenorhabditis elegans actin-interacting protein 1 (UNC-78) is dependent on filament binding by a specific ADF/cofilin isoform

Department of Pathology, Emory University, Atlanta, GA 30322, USA

View larger version (31K): [in a new window]   Fig. 1. Actin filament disassembling activity of the recombinant UNC-78 protein. (A) Purified bacterially expressed recombinant UNC-78 protein (3.2 µg). (B) UNC-60B-dependent filament disassembly by UNC-78. Rabbit muscle F-actin (10 µM) was incubated for 30 minutes with buffer (lanes 1 and 2), UNC-60B (10 µM) (lanes 3 and 4), UNC-78 (2 µM) (lanes 5 and 6), or both UNC-60B and UNC-78 (lanes 7 and 8) and separated into supernatants (s) and pellets (p) after ultracentrifugation. UNC-78 was treated in the absence of F-actin as a control (lanes 9 and 10). (C) Dose-dependence of filament disassembly by UNC-78 in the presence of UNC-60B. F-actin (10 µM) and UNC-60B (20 µM) were incubated for 30 minutes with the indicated concentrations of UNC-78 and analyzed by pelleting assays. Molecular mass markers in kDa are indicated on the left of A-C. (D,E) Quantitative analysis of UNC-78-induced filament disassembly. Rabbit muscle actin (D) or C. elegans actin (E) was incubated with various concentrations of UNC-60B and UNC-78 and subjected to pelleting assays. Percentages of actin in the pellets were quantified and plotted as a function of the UNC-78 concentration at different UNC-60B concentrations (0-30 µM). Data shown are mean±s.d. of three experiments.

View larger version (19K): [in a new window]   Fig. 2. The effects of UNC-78 and UNC-60B on the kinetics of light scattering of F-actin. F-actin (5 µM) was mixed with buffer (a, black circles), UNC-78 (1 µM) (b, white circles), UNC-60B (5 µM) (c, black triangles), or both UNC-78 and UNC-60B (d, black triangles) and the light scattering intensity (arbitrary units) was monitored over time. Time 0 was when monitoring started, which is after 20 sec of manual preparation of the samples and setting them in the instrument.

View larger version (15K): [in a new window]   Fig. 3. Effects of UNC-78 and UNC-60B on actin depolymerization as measured by a DNase I inhibition assay. 5 µM F-actin was incubated for 30 minutes with UNC-78 (0-0.6 µM) and UNC-60B (0-10 µM) and the G-actin concentrations were determined by a DNase I inhibition assay. Data shown are mean±s.d. of three experiments.

View larger version (21K): [in a new window]   Fig. 4. Effects of UNC-78 and UNC-60B on actin turnover as measured by nucleotide exchange. (A) F-actin (5 µM) was mixed with UNC-78 and/or UNC-60B as indicated in the figure in the presence of 40 µM ATP, and the fluorescence (arbitrary units) of ATP was monitored over time. (B) The data were fitted to exponential curves and the rates of increase in the fluorescence (F/second) were calculated and plotted as a function of concentration of UNC-60B. Data shown are mean±s.d. of four experiments.

View larger version (20K): [in a new window]   Fig. 5. Effects of UNC-78 and UNC-60B on spontaneous actin polymerization. G-actin (5 µM) was mixed with buffer (a), UNC-60B (5 µM) (b), UNC-78 (0.6 µM) (c), UNC-60B (5 µM) and UNC-78 (0.1 µM) (d), or UNC-60B (5 µM) and UNC-78 (0.6 µM) (e), and the turbidity (absorbance at 310 nm) was monitored over time.

View larger version (33K): [in a new window]   Fig. 6. Effects of ADF/cofilin isoforms and mutants on UNC-78-dependent actin filament assembly. F-actin (10 µM) was incubated for 30 minutes with UNC-78 (0-6 µM) and UNC-60B (A), UNC-60A (B), mouse M-cofilin (C), UNC-60B (A111V) (D), UNC-60B (S112F) (E), or UNC-60B (Q150*) (F), and subjected to pelleting assays. Percentages of actin in the pellets were quantified and plotted as a function of the concentration of ADF/cofilin proteins. Data shown are mean±s.d. of three experiments.

View larger version (64K): [in a new window]   Fig. 7. Specificity of anti-UNC-78 antibody and expression of UNC-78. Total worm lysates (25 µg proteins) of wild type (lane 1), unc-78 (gk27) (unc-78 null) (lane 2), and unc-60 (su158) (unc-60B null) (lane 3) and purified recombinant UNC-78 protein (0.1 µg) (lane 4 only in B) were resolved by SDS-PAGE (10% acrylamide gel) and visualized by Coomassie Blue (A) or subjected to western blot with anti-UNC-78 (B), anti-actin (C), or anti-UNC-60B (D). Molecular mass markers in kDa (lane M) are indicated on the left of A.

View larger version (74K): [in a new window]   Fig. 8. Localization of UNC-78 in wild-type embryos. Embryos at the 1.5-fold stage (A,C,E) and threefold stage (B,D,F) were stained by anti-UNC-78 (A,B) and anti-myoA (C,D) antibodies. Arrows in A,B,E,F indicate expression of UNC-78 in embryonic body wall muscle. Arrowheads in B indicate the pharynx. Merged images of double-staining of UNC-78 (green) and myoA (red) are shown in E and F. Scale bar: 20 µm.

View larger version (108K): [in a new window]   Fig. 9. Localization of UNC-78 in adult wild-type body wall muscle. Adult worms were stained by anti-UNC-78 (A,B) and anti-actin (C) or anti-myoA (D) antibodies. Parts of the body wall muscle cells are shown. Representative locations of UNC-78 are indicated by arrows in A,B,E,F. An arrow in c indicates a line of actin staining that overlaps with UNC-78. Arrowheads in D and F indicate myoA striations that are not co-localized with UNC-78. Merged images of double-staining of UNC-78 (green) and actin or myoA (red) are shown in E and F. Scale bar: 20 µm.

View larger version (71K): [in a new window]   Fig. 10. Localization of UNC-78 in body wall muscle of unc-60B null mutants. The unc-60 (su158) homozygotes (unc-60B null mutants) were stained by anti-UNC-78 (A) and anti-actin (B) antibodies. Arrows indicate aggregates of actin where UNC-78 is also localized. Arrowheads indicate residual striated myofibrils where weak localization of UNC-78 is detected. A merged image of UNC-78 (green) and actin (red) is shown in C. Scale bar: 20 µm.

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