Myotactin, a novel hypodermal protein involved in muscle-cell adhesion in Caenorhabditis elegans - PubMed (original) (raw)

Myotactin, a novel hypodermal protein involved in muscle-cell adhesion in Caenorhabditis elegans

M C Hresko et al. J Cell Biol. 1999.

Abstract

In C. elegans, assembly of hypodermal hemidesmosome-like structures called fibrous organelles is temporally and spatially coordinated with the assembly of the muscle contractile apparatus, suggesting that signals are exchanged between these cell types to position fibrous organelles correctly. Myotactin, a protein recognized by monoclonal antibody MH46, is a candidate for such a signaling molecule. The antigen, although expressed by hypodermis, first reflects the pattern of muscle elements and only later reflects the pattern of fibrous organelles. Confocal microscopy shows that in adult worms myotactin and fibrous organelles show coincident localization. Further, cell ablation studies show the bodywall muscle cells are necessary for normal myotactin distribution. To investigate myotactin's role in muscle-hypodermal signaling, we characterized the myotactin locus molecularly and genetically. Myotactin is a novel transmembrane protein of approximately 500 kd. The extracellular domain contains at least 32 fibronectin type III repeats and the cytoplasmic domain contains unique sequence. In mutants lacking myotactin, muscle cells detach when embryonic muscle contraction begins. Later in development, fibrous organelles become delocalized and are not restricted to regions of the hypodermis previously contacted by muscle. These results suggest myotactin helps maintain the association between the muscle contractile apparatus and hypodermal fibrous organelles.

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Figures

Figure 1

Figure 1

(A) Schematic representation of a cross-section through a threefold stage embryo depicting the spatial relationships between the contractile apparatus of the bodywall muscle (oval-like shapes), the muscle basement membrane (black) and hypodermal fibrous organelles (hatched areas). Dorsal is toward the top of the page. The four muscle quadrants are shown, each consisting of two rows of muscle cells, with the long axis of the cells perpendicular to the page. The contractile apparatus is restricted to the portion of the muscle cell closest to the muscle cell membrane facing the hypodermis. Hypodermal fibrous organelles are restricted to the region of the hypodermis adjacent to bodywall muscle cells. A specialized basement membrane, positioned between muscle cells and the hypodermis, helps to anchor muscle cells. Myotactin is thought to be an integral membrane protein expressed on the basal hypodermal membrane with its large extracellular domain extending into the basement membrane toward the adjacent muscle cells. (B) Schematic representations of the localization of muscle, fibrous organelle–associated intermediate filaments, the MH5 protein and myotactin in dorsal views of progressively older embryos. Intermediate filament proteins and the MH5 protein, an immunologically defined protein that localizes near fibrous organelles, show the same distribution at all developmental stages. Schematics a, d, and g represent an early stage of development (just before the start of elongation); b, e, and h represent an intermediate stage (twofold); and c, f, and i represent a later stage (threefold). In each schematic (a–i) a portion of the two dorsal muscle quadrants is shown. Each quadrant consists of two rows of mononucleated muscle cells with the long axis of the cells oriented along the anterior-posterior axis of the worm. Anterior is toward the top of the page. Wavy, black, vertical lines mark the lateral edges of the dorsal hypodermis. Horizontal, dashed black lines in a, d, and g represent the boundaries between adjacent dorsal hypodermal cells. These lines do not appear in b, c, e, f, h, and i because at these later developmental stages the dorsal hypodermal cells have fused to form a syncytium. Gray areas represent the localization of components of the muscle cell contractile apparatus (a–c), myotactin (d–f), and the fibrous organelle–associated intermediate filaments and MH5 protein (g–i). Schematics a–c are focused on the plane of the muscle cells, while d–i are focused on the dorsal hypodermal cells. Although we discuss only the dorsal surface of the embryo, similar events occur on the ventral surface. At stages earlier than those shown, muscle cells are adjacent to lateral (seam) hypodermis, and muscle proteins are diffusely distributed in muscle cells. Similarly, the hypodermal proteins are diffusely distributed in dorsal hypodermal cells. As muscle cells migrate to contact dorsal hypodermal cells, muscle components accumulate at the membrane where muscle cells contact each other and the hypodermis (a). At about the same time, myotactin, intermediate filament proteins and the MH5 protein are recruited to specific regions of the hypodermis adjacent to some muscle cells. Myotactin accumulates adjacent to where the contractile apparatus is forming (d) while intermediate filaments and the MH5 protein accumulate in a single broad patch in each hypodermal cell adjacent to one muscle quadrant (g). By the twofold stage, the sarcomeric organization of the obliquely striated muscle is observed (b). At this stage myotactin is still adjacent to the forming contractile apparatus and in fact is organized into rows of dots running oblique to the long axis of the worm and following the oblique striations of the muscle (e). In contrast, intermediate filaments and the MH5 protein are organized in circumferentially oriented bands that are restricted to the small regions of the hypodermis adjacent to muscle (h). During the threefold stage, myotactin colocalizes with intermediate filaments and the MH5 protein (f and i).

Figure 1

Figure 1

(A) Schematic representation of a cross-section through a threefold stage embryo depicting the spatial relationships between the contractile apparatus of the bodywall muscle (oval-like shapes), the muscle basement membrane (black) and hypodermal fibrous organelles (hatched areas). Dorsal is toward the top of the page. The four muscle quadrants are shown, each consisting of two rows of muscle cells, with the long axis of the cells perpendicular to the page. The contractile apparatus is restricted to the portion of the muscle cell closest to the muscle cell membrane facing the hypodermis. Hypodermal fibrous organelles are restricted to the region of the hypodermis adjacent to bodywall muscle cells. A specialized basement membrane, positioned between muscle cells and the hypodermis, helps to anchor muscle cells. Myotactin is thought to be an integral membrane protein expressed on the basal hypodermal membrane with its large extracellular domain extending into the basement membrane toward the adjacent muscle cells. (B) Schematic representations of the localization of muscle, fibrous organelle–associated intermediate filaments, the MH5 protein and myotactin in dorsal views of progressively older embryos. Intermediate filament proteins and the MH5 protein, an immunologically defined protein that localizes near fibrous organelles, show the same distribution at all developmental stages. Schematics a, d, and g represent an early stage of development (just before the start of elongation); b, e, and h represent an intermediate stage (twofold); and c, f, and i represent a later stage (threefold). In each schematic (a–i) a portion of the two dorsal muscle quadrants is shown. Each quadrant consists of two rows of mononucleated muscle cells with the long axis of the cells oriented along the anterior-posterior axis of the worm. Anterior is toward the top of the page. Wavy, black, vertical lines mark the lateral edges of the dorsal hypodermis. Horizontal, dashed black lines in a, d, and g represent the boundaries between adjacent dorsal hypodermal cells. These lines do not appear in b, c, e, f, h, and i because at these later developmental stages the dorsal hypodermal cells have fused to form a syncytium. Gray areas represent the localization of components of the muscle cell contractile apparatus (a–c), myotactin (d–f), and the fibrous organelle–associated intermediate filaments and MH5 protein (g–i). Schematics a–c are focused on the plane of the muscle cells, while d–i are focused on the dorsal hypodermal cells. Although we discuss only the dorsal surface of the embryo, similar events occur on the ventral surface. At stages earlier than those shown, muscle cells are adjacent to lateral (seam) hypodermis, and muscle proteins are diffusely distributed in muscle cells. Similarly, the hypodermal proteins are diffusely distributed in dorsal hypodermal cells. As muscle cells migrate to contact dorsal hypodermal cells, muscle components accumulate at the membrane where muscle cells contact each other and the hypodermis (a). At about the same time, myotactin, intermediate filament proteins and the MH5 protein are recruited to specific regions of the hypodermis adjacent to some muscle cells. Myotactin accumulates adjacent to where the contractile apparatus is forming (d) while intermediate filaments and the MH5 protein accumulate in a single broad patch in each hypodermal cell adjacent to one muscle quadrant (g). By the twofold stage, the sarcomeric organization of the obliquely striated muscle is observed (b). At this stage myotactin is still adjacent to the forming contractile apparatus and in fact is organized into rows of dots running oblique to the long axis of the worm and following the oblique striations of the muscle (e). In contrast, intermediate filaments and the MH5 protein are organized in circumferentially oriented bands that are restricted to the small regions of the hypodermis adjacent to muscle (h). During the threefold stage, myotactin colocalizes with intermediate filaments and the MH5 protein (f and i).

Figure 2

Figure 2

Confocal images of a wild-type adult worm fragment stained for intermediate filaments (green) and myotactin (red). (A) Intermediate filaments are concentrated in thin bands running circumferentially around the worm (arrows). The intermediate filament-dependent staining within each band is not uniform but is associated with discrete punctate structures within the band. (B) Like intermediate filament-dependent staining, myotactin-dependent staining is associated with punctate structures that are organized into bands (arrows). (C) Merging of the images shown in A and B shows the punctate patterns seen with the two antibodies are coincident and thus produce the yellow fluorescence.

Figure 3

Figure 3

Schematic diagram of the partial myotactin cDNAs and genomic clones. The top two lines represent C. briggsae and C. elegans genomic clones, respectively. Exons are depicted as filled boxes and introns as lines. pG-cc9 is the genomic clone isolated using the MH46 antibody as a probe (see Materials and Methods) and pC-ccXX(X) are the overlapping cDNA clones. RT-PCR-1, 2 and 3 (see Materials and Methods) were sequenced to confirm the intron–exon boundaries between exons 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 12 and 13, and 13 and 14. The open boxes at the 3′ ends of pC-cc214, pC-cc307, and the C. elegans and C. briggsae genes represent noncoding sequence. The positions of the predicted translational start (ATG) and the stop codon (TAA) are indicated. pC-cc503 contains eight bases at the 5′ end identical to the eight 3′ bases of the SL1 spliced leader sequence. Note that exons 6–9, 13, 19A, 19B, 24 and 25 are all differentially spliced. The combinations with which these exons are used has not been determined. The complete sequence of the myotactin cDNAs is attainable through Genbank. Two cDNA sequences were submitted: Form A includes exon 19A but excludes exons 19B, 24, and 25 (accession number AF148954) and form B includes exons 19B, 24, and 25 but excluded 19A (accession number AF148953). The two forms reflect the differences observed between cDNA pC-cc214 and pC-cc307.

Figure 4

Figure 4

Multiple messages are transcribed from the myotactin gene, the smallest being ∼15 kb. 20 μg of total RNA from mixed stage worms was run on a 0.7% agarose gel in the presence of formaldehyde and then transferred to nitrocellulose. A 32P-labeled DNA fragment corresponding to the sequence of pC-cc1A was used to probe the Northern blot. Molecular weight markers are indicated at the left, and were determined by either hybridization of the same blot with a twitchin probe (22 kb; 7) or a dynein heavy chain probe (14 kb; 35) or by the position of the 28S (3.5 kb) and 18S (1.75 kb) rRNAs of C. elegans.

Figure 5

Figure 5

Alignment of the 32 putative FNIII repeats. With the exception of repeats 8 and 27, the repeats have scores over 15 when analyzed by the fn3 HMM program (see Materials and Methods).

Figure 6

Figure 6

Embryonic expression of the myotactin gene assayed by in situ hybridization. Mixed stage embryos were fixed and incubated with anti-sense myotactin (A, B, D, and E) or myosin (C and F) single stranded DNA probes labeled with digoxigenin. Probes were visualized using an alkaline-phosphatase-conjugated anti-digoxigenin antibody. Anterior is toward the top and in D–F dorsal is to the right. Arrowheads designate dorsal hypodermal cells and arrows designate ventral hypodermal cells. (A) Dorsal and (B) mid-focal plane images of the same <290-min embryo. The anti-sense myotactin probe is detected on the dorsal surface of the embryo (A) and in cells at the lateral edges (B) of the embryo. These cells are the dorsal and ventral hypodermal cells, respectively. (C) Mid-focal plane of a 300-min embryo. The myosin probe is detected in the bodywall muscle cells giving a different pattern from that of the myotactin probe (compare to B). (D) Lateral view of a 320-min embryo and (E) lateral view of a comma stage embryo (390 min). Anti-sense myotactin probe is detected at the dorsal (dorsal hypodermal cells) and ventral (ventral hypodermal cells) edges of the embryo. (F) Lateral view of a 320-min embryo. The myosin probe is detected in bodywall muscle cells. The positive cells are away from the dorsal and ventral edges of the embryo. Embryos hybridized with either myosin or myotactin sense probes showed no staining.

Figure 7

Figure 7

Muscle cells detach in let-805(st456) mutant embryos. Immunofluorescence micrographs of embryos fixed and labeled with monoclonal antibodies MH46 and MH27 (B and D), or a polyclonal antibody against myosin (A, C, E, and F). MH27 recognizes the boundaries between hypodermal cells and is used to visualize the outline of the embryos. The staining pattern appears as a grid on the surface of the embryo (B and D, small arrowheads). Dorsal is to the left in each case, and large and small arrows mark the posterior and anterior ends of the embryos, respectively. (A–D) Lateral views of a wild-type (A and B) or a let-805(st456) homozygous (C and D) embryo at the 1.5-fold stage. Myosin positive cells (A and C) of one dorsal quadrant (large arrowheads) are seen. In both cases the muscle quadrant extends from the anterior to the posterior end of the embryo (the posterior of the quadrant in A is out of the focal plane). Myotactin is localized in the hypodermis adjacent to muscle cells in the wild-type embryo (B, large arrowheads). No myotactin staining is detected in the mutant embryo (D). (E and F) Lateral view of let-805(st456) homozygotes stained for myosin. Note the muscle quadrants do not extend to the anterior or posterior of either embryo. C, E, and F are images of increasingly older animals showing an increase in severity of the muscle cell detachment over time.

Figure 8

Figure 8

Wild-type and myotactin mutant embryos stained for intermediate filaments. Mixed stage embryos were fixed for immunofluorescence and labeled with MH4 (intermediate filament protein) and MH27. MH27 marks the boundaries between hypodermal cells and appears as a grid pattern on the embryos in B and D. (A and B) Dorsal view of a wild-type (A) and a st456 homozygous (B) embryo. In both embryos, the MH4-dependent signal is concentrated in the regions of the hypodermis adjacent to muscle. (C) Dorsal view of a st456 homozygote older than the one shown in B. MH4-dependent fluorescence is seen all through the dorsal hypodermis. (D) Lateral view of an embryo at a similar stage to the one shown in C. The MH4-dependent staining extends to the boundaries between the dorsal or ventral hypodermis and the seam hypodermis. The fluorescence pattern seen in C and D is one of circumferentially oriented bands. Arrowheads mark the boundaries between dorsal or ventral and seam hypodermis.

Figure 9

Figure 9

Myotactin organizes in response to muscle cells. (A) 310-min wild-type embryo stained with MH27 to illustrate the MH27 staining pattern. (B) An ∼1.25-fold (∼400 min) wild-type embryo stained with MH27 and MH46. At this stage the MH46 staining extends all the way to the anterior (top) and posterior (not seen in this focal plane) of the embryo. (C and D) Embryo ablated for MS.ap and MS.pp at the 28 cell stage, allowed to develop to the 1.25-fold stage, and then fixed and stained for myosin (D) and with MH46 and MH27 (C). (E and F) Embryo ablated for C.ap and allowed to develop to about the twofold stage and then fixed and stained for myosin (F) and with MH46 and MH27 (E). (B) Arrows indicate the portion of the staining due to MH46. (C–F) Arrows mark the position where the truncated muscle quadrants end, and show the corresponding gap in the organization of myotactin where hypodermal cells have failed to contact muscle.

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References

    1. Albertson D.G., Thomson J.N. The pharynx of Caenorhabditis elegans . Phil. Trans. R. Soc. Lond. Ser. B. Biol. 1976;275:299–325. - PubMed
    1. Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. Gapped BLAST and PSI-BLASTa new generation of protein database search programs. Nucleic Acid Res. 1997;25:3389–3402. - PMC - PubMed
    1. Avery L., Horvitz H.R. A cell that dies during wild-type C. elegans development can function as a neuron in a ced-3 mutant. Cell. 1987;51:1071–1078. - PMC - PubMed
    1. Barstead R.J., Waterston R.H. The basal component of the nematode dense body is vinculin. J. Biol. Chem. 1989;264:10177–10185. - PubMed
    1. Barstead R.J., Waterston R.H. Vinculin is essential for muscle function in the nematode. J. Cell Biol. 1991;114:715–724. - PMC - PubMed

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