Alpha spectrin is essential for morphogenesis and body wall muscle formation in Caenorhabditis elegans

Abstract

A common feature of multicellular animals is the ubiquitous presence of the spectrin cytoskeleton. Although discovered over 30 yr ago, the function of spectrin in non-erythrocytes has remained elusive. We have found that the spc-1 gene encodes the only alpha spectrin gene in the Caenorhabditis elegans genome. During embryogenesis, alpha spectrin localizes to the cell membrane in most if not all cells, starting at the first cell stage. Interestingly, this localization is dependent on beta spectrin but not beta(Heavy) spectrin. Furthermore, analysis of spc-1 mutants indicates that beta spectrin requires alpha spectrin to be stably recruited to the cell membrane. Animals lacking functional alpha spectrin fail to complete embryonic elongation and die just after hatching. These mutant animals have defects in the organization of the hypodermal apical actin cytoskeleton that is required for elongation. In addition, we find that the process of elongation is required for the proper differentiation of the body wall muscle. Specifically, when compared with myofilaments in wild-type animals the myofilaments of the body wall muscle in mutant animals are abnormally oriented relative to the longitudinal axis of the embryo, and the body wall muscle cells do not undergo normal cell shape changes.

Figure 1.

spc-1 encodes α spectrin. (A) The genomic structure of the spc-1 gene and the encoded protein structure. The exons are indicated by the black boxes, and the introns are indicated by the thin black lines. The nonsense mutation is shown (ra409), and the Tc1 insert is shown by the inverted triangle in the 10th exon. The cDNA clones used for generating dsRNA are shown above the genomic structure. Spectrin repeats are illustrated by ovals. The nonrepetitive domains are indicated by boxes. † and * indicate where the spc-1(ra409) mutation and the Tc1 introduce premature stop codons, respectively. (B) Deduced amino acid sequence of C. elegans α spectrin aligned to Drosophila and human nonerythroid α spectrin. The C. elegans α spectrin (Ce_α; sequence data available from GenBank/EMBL/DDBJ under accession no. AAB53876) is 61% identical and 76% similar to Drosophila α spectrin (Dm_α; sequence data available from GenBank/EMBL/DDBJ under accession no. P13395), and it is 57% identical and 72% similar to the human nonerythroid α spectrin (Hs_α; sequence data available from GenBank/EMBL/DDBJ under accession no. NP_003118). Identity is boxed, and similarity is shaded.

Figure 2.

α spectrin localizes to the cell membrane of most cells during embryogenesis. (A) Four representative wild-type embryos at different developmental stages labeled with AS1 and examined by confocal immunofluorescence microscopy. The small arrowhead indicates a four-cell stage embryo where a spectrin is localized to the cell junctions. An ∼100-cell stage embryo is shown with the large arrow; note cell membrane immunofluorescence. The large arrowhead indicates intestinal localization of a spectrin in a comma stage embryo. α spectrin localization in the nervous system of a threefold embryo is indicated by the arrow. (A') Western blot analysis of a mixed population of wild-type animals. Lane 1 indicates a ∼240-kD band recognized by AS1 on wild-type worm extracts. (Lane 2) Preincubation of AS1 with the GST–α spectrin fusion protein shows no reactivity to wild-type worm extracts. (B) A representative 1.5-fold embryo labeled with AS1. Basolateral and apical localization of a spectrin is shown. Arrow indicates apical region, and the arrowhead indicates basolateral region of the intestine. (C) The body wall muscle cells (identified by counter staining with myosin antibody [unpublished data]) of a representative 1.5-fold embryo are outlined by a spectrin (arrows). Bar, 10 μM.

Figure 3.

α and β spectrin localization in spectrin mutants. Representative (A) unc-70(s1639) and sma-1(ru18) (B) embryos labeled with AS1 and examined by confocal immunofluorescence microscopy. In unc-70 (β spectrin) mutant embryos, (A) α spectrin fails to localize to the membrane except to the apical region of the intestine (arrow) and the hypodermis (arrowhead). (B) α spectrin localization is normal in sma-1 (β_H spectrin) mutant embryos (the arrow indicates the apical region of the intestine, and the arrowhead indicates the apical region of the hypodermis). Wild-type (C) and spc-1(ra409) (D) embryos labeled with β spectrin antisera. (C) β spectrin localizes to the cell membrane of all cells including the nervous system (arrowheads). However, β spectrin does not localize to the apical region of the intestine (arrow) and hypodermis. (D) In the absence of α spectrin, β spectrin is not stably localized to the membrane as is seen by the faint immunofluorescence in the spc-1 mutant embryo. However, stronger β spectrin immunofluorescence is detected in the nervous system of spc-1 mutants (arrowheads). Bar, 10 μM.

Figure 4.

α spectrin is required for embryonic elongation. Nomarski images of representative wild-type (A and B) and spc-1(ra409) (C) embryos. (A) A wild-type embryo that has completed embryonic elongation (arrows indicate fully elongated pharynx). (B) A 1.5-fold wild-type embryo that has partially completed elongation (arrows indicate developing pharynx). (C) A spc-1 mutant embryo at the same stage as the wild-type embryo shown in A. spc-1 mutant embryos undergo elongation at a slower rate than wild-type animals and fail to complete elongation. Arrows indicate the abnormal morphology of the pharynx. Bar, 10 μM. (D) Lengths of N2 (n = 7), spc-1 (n = 5), egl-19 (n = 4), sma-1 (n = 6), let-502 (n = 5), and mlc-4 (n = 4) embryos measured every 15 min from first cleavage at 24°C. All the embryos initiated elongation at the same time. N2 and egl-19 continue at the same rate until twofold in length where egl-19 ceases elongation. spc-1, sma-1, let-502, and mlc-4 undergo elongation at a slower rate. Additionally, body wall muscle contraction commenced at the same time (∼400 min) in all the embryos except egl-19, which failed to display muscle contraction. Data is shown as the mean ± SD.

Figure 5.

α spectrin is required for the organization of the hypodermal apical actin cytoskeleton. Representative wild-type (A), spc-1(ra409) (B), sma-1(ru18) (C), let-502(h738) (D), and mlc-4(or253) (E) embryos were stained with FITC-phalloidin and examined by confocal fluorescence microscopy to visualize the circumferentially oriented actin cytoskeleton in the hypodermis. Arrowheads indicate the circumferential actin fibers, and arrows indicate the thin filaments of the body wall muscle. In wild type (A), the actin filaments are organized in a circumferential pattern in parallel bundles (arrowheads) that run perpendicular to the body wall muscle quadrant (arrows). Ai and Aii are threefold embryos (same developmental age as embryos shown in B–E, and Aiii is a 1.5-fold embryo. The inset shows parallel organization of the actin fibers. In spc-1 (α spectrin) embryos (B), several defects are observed in the highly regular pattern of the hypodermal actin cytoskeleton. Note the gaps (Bi and Biii, arrowheads), discontinuities, and clumping (Bii, arrowhead). Additionally, the thin filaments of spc-1 mutant embryos are abnormally oriented, and the body wall muscle quadrants are wider than the muscle quadrants in wild-type animals (Bii and Aii, arrows). The inset highlights the disorganization of the actin fibers. (C) Although slightly less severe, similar defects are observed in sma-1(β_H spectrin) embryos. The arrowhead indicates a gap in the highly organized actin cytoskeleton. The hypodermal actin cytoskeleton in both let-502 (D) and mlc-4 (E) mutant embryos appears mostly wild type. In some instances, the spacing between some parallel actin bundles is abnormal in let-502(h738) embryos (see text). The same body wall muscle defect is observed in sma-1, let-502, and mlc-4 mutant embryos that is observed in spc-1 mutant embryos. All of these mutants have abnormally oriented thin filaments, and the body wall muscle is wider than normal (C and D, arrows). Bar, 10 μM.

Figure 6.

Myosin filament organization is abnormal in elongation-defective mutants. Representative wild-type (A), spc-1(ra409) (B), sma-1(ru18) (C) , and egl-19(st556) (D) embryos were labeled with myosin antisera. The right dorsal muscle is shown in A–D, and anterior is up. In wild-type embryos (A), the myosin filaments are organized into a double row of A bands that runs approximately parallel with the longitudinal axis of the embryo. In both spc-1 (α spectrin) (B) and sma-1 (β_H spectrin) (C) mutant embryos, the myosin filaments are organized into double rows of A bands, but the filaments are abnormally oriented. The myosin filaments in these mutants are almost oriented at a 20° angle to the longitudinal axis of the embryo. Additionally, the muscle quadrants of the α (B) and β_H spectrin (C) mutant embryos are wider than normal (compare with A). Arrowheads in B and C indicate a large gap between the myofilament lattice in neighboring muscle cells. In egl-19 mutants (D), the body wall muscle quadrants and the myofilaments are normal (compare with A). Bar, 5 μM. (E) Width of the right dorsal muscle quadrant of N2 (n = 6), egl-19 (n = 5), spc-1 (n = 6), sma-1 (n = 5), let-502 (n = 6), and mlc-4 (n = 5) embryos. The width of a muscle quadrant in wild-type animals is ∼3 mm. Similarly, the muscle quadrants from the Pat mutant, egl-19, are ∼3-μM wide. The slow elongation mutants, spc-1, sma-1, let-502, and mlc-4, all have muscle quadrants that are ∼6-mm wide. Data is shown as the mean ± SD.

Figure 7.

Body wall muscle cells fail to undergo cell shape changes in the slow elongation mutants. Representative wild-type (A and B), let-502(h738) (C), and egl-19(st556) (D) embryos double labeled with myosin antisera (red) and β spectrin antisera (green). In wild-type embryos, the body wall muscle cells change shape from round to spindle-shaped cells. (A) Comma stage wild-type embryo showing the initial shape of the body wall muscle cells at the start of embryonic elongation. (B) Threefold wild-type embryo indicating the cell shape change that the body wall muscle cells undergo during embryonic elongation. (C) In the slow elongation mutants (let-502), the body wall muscle cells fail to undergo normal cell shape changes and remain similar to the body wall muscle cells in the wild-type comma stage embryo (C compared with A and B). (D) Normal cell shape changes occur in the Pat mutant, egl-19. Arrowheads indicate cell width. Bar, 10 μM.

Figure 8.

The basement membrane and hemidesmosomal-like structures have an expanded distribution in the slow elongation mutants. Representative wild-type (A and E), spc-1(ra409) (B and F), sma-1(ru18) (C and G), and egl-19(st556) (D and H) embryos double labeled with perlecan antisera and MH4, an antibody that recognizes the intermediate filaments of the hypodermal hemidesmosomal-like structure. In wild-type embryos (A), perlecan is evenly distributed under each of the body wall muscle quadrants. Similarly, in spc-1 (α spectrin) (B) and sma-1 (β_H spectrin) (C) mutants, perlecan is distributed evenly under each of the body wall muscle quadrants. However, the spatial distribution of perlecan in spc-1 and sma-1 mutants is wider than normal, reflecting the defects observed in the body wall muscle (Figs. 6 and 7). In egl-19 mutants (D), the distribution of perlecan is normal. In wild-type embryos, the hemidesmosomes have assemble under each of the body wall muscle quadrants (E). In spc-1 (F) and sma-1 (G) mutant embryos, the hemidesmosomes have assembled normally under each body wall muscle quadrant (F and G). However, the hemidesmosomes are assembled in an area wider than normal. In egl-19 mutants (H), the distribution of the hemidesmosomes is normal. Arrowheads indicate width of basement membrane (A–D) and hemidesmosomes (E–H). Bar, 10 μM.