UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle

Abstract

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.

Figure 5

Effects of mutant UNC-60B on the nucleating activity of F-actin. C. elegans F-actin was mixed with wild-type or mutant UNC-60B and used as nuclei to induce polymerization of pyrene-labeled G-actin. The fluorescence of pyrene was measured over time and the initial rate of the increase in the fluorescence was taken as a nucleation rate. The inset is the same data with different scaling. The data are expressed as relative nucleation rates to that of F-actin alone. Data represent the average of two separate experiments.

Figure 1

Motility of unc-60 mutants and identification of mutation sites. (A) Motility of the adult unc-60 mutant animals were scored by their beating frequencies in liquid. Values are the means ± SD, n = 10. (B) Sequence alterations in the unc-60 mutants. The mutations are designated as the original residue, the position, the converted residue. The asterisk designates a stop codon. The gray region shows a putative actin-binding α-helix that was predicted from the homology between UNC-60B and yeast cofilin for which a structure has been solved.

Figure 6

Changes in UNC-60B protein levels in unc-60 mutants. (A) Specificity of anti–UNC-60A and anti–UNC-60B antibodies. Total lysates of wild-type nematodes were separated by SDS-PAGE, transferred onto a membrane and reacted with anti– UNC-60A or anti–UNC-60B antibodies. Each antibody specifically reacted with a single band of expected size, which was further confirmed by reactivity with either recombinant UNC-60A or UNC-60B (data not shown). Molecular mass markers in kD are indicated on the left. (B) Levels of UNC-60A and UNC-60B proteins in the unc-60 mutant alleles. Only the level of UNC-60B protein varied among the alleles. The intensities of the bands of UNC-60B were quantified and shown in Table I. No alteration in the level of actin was observed.

Figure 2

Effects of the mutant UNC-60B proteins on F-actin. C. elegans F-actin (10 μM) was incubated with various concentrations of wild-type or mutant UNC-60B proteins (horizontal axes; note that the scaling of C and D is different from the others) and the mixtures were examined by copelleting assays. On the vertical axes are plotted the sedimented portions (expressed as concentrations assuming that the pellets were reconstituted in the original volumes) of actin (closed circles with solid lines) and wild-type or mutant UNC-60B (open circles with dashed lines). Nonspecific sedimentations of wild-type or mutant UNC-60B were determined by control experiments as described in Materials and Methods and subtracted from the data of assays with actin. Data represent the average of two separate experiments.

Figure 3

Effects of the mutant UNC-60B proteins on actin polymerization. C. elegans G-actin (5 μM) was incubated with the indicated molar ratios of wild-type or mutant UNC-60B, polymerization was started by adding salt (time 0), and the rates of polymerization were measured as changes in turbidity (absorbance at 310 nm) over time.

Figure 4

Effects of the mutant proteins on the concentration of unassembled actin. C. elegans actin at 5 μM was polymerized in the presence of wild-type or mutant UNC-60B for 4 h and the concentration of unpolymerized actin was quantified with a DNase I inhibition assay. Data represent averages of three experiments. Error bars are not included in the figure because they make the graph difficult to read. SDs of these data are <0.17 μM.

Figure 7

Expression of UNC-60B in embryonic body wall muscle. (a–d) Specific expression of UNC-60B in embryonic body wall muscle cells. 1.5-fold (a and b) and threefold (c and d) stage embryos were double-stained for UNC-60B (a and c) and body wall muscle–specific myosin heavy chain A (myoA) (b and d). An arrow in (c) indicates a region where UNC-60B is located in a striated pattern. Bar, 10 μm.

Figure 8

Disorganization of embryonic myofibrils in unc-60 mutants. Immunofluorescent localization of actin (a, c, e, and g) and UNC-60B (b, d, f, and h) in threefold embryos of wild-type (a and b), unc-60(e677) (c and d), unc-60(r398) (e and f), and unc-60(s1307) (g and h) were observed by double staining and the same fields from each image are shown. Actin in wild-type (a) is continuous along the arrays of the body wall muscle cells. However, in the unc-60 mutants (c, e and g), actin is found in discrete thick bundles. The extent of disorganization of myofibrils was indistinguishable between the most severe (c, e677) and second mildest (e, r398) mutants. The weakest mutant, s1307 (g), showed somewhat larger bundles. Bar, 10 μm.

Figure 9

Disorganization of adult myofibrils in unc-60 mutants. Localizations of myosin (myoA), actin, and UNC-60B in adult body wall muscle of the unc-60 mutants were observed by immunofluorescence microscopy. Actin and UNC-60B were observed by double staining and the same fields from each image are shown. Bar, 10 μm.