In mutant animals, the distance between apical hypodermal surface and the basal cuticle layer is consistently greater than in wild-type animals, including regions where muscles appear to remain attached to the body wall (compare Figs. at these sites. Thus, MUA-3 appears to be a protein that links the IF cytoskeleton of nematode epithelia to the cuticle at sites of mechanical stress. and adhesion complexes between muscle and cuticle, or required for their developmental regulation, mutations that showed abnormal tissue fragility in response to mechanical stress were isolated (Plenefisch et al., 2000). Here we show that one of these genes, result in the separation of hypodermis from cuticle. The MUA-3 protein is shown to be a novel transmembrane protein that localizes to hypodermal hemidesmosomes at the sites of skeletal muscle contact and to other epithelial sites where stress-resistant cuticular adhesion is required. Finally, we show that MUA-3 colocalizes with cytoplasmic IFs in the hypodermis, suggesting that it may physically link IFs to the cuticle. Results is required for postembryonic adhesion of the hypodermis to the cuticle Mutations in result in the detachment of the skeletal muscles from the body wall (Figs. 1 and ?and2 ;2 ; Plenefisch et al., 2000). Alleles range in severity from virtually complete muscle detachment in early larvae to partial detachment in late larvae and adults. Detachment typically initiates at a single site and progresses to include most or all of the affected muscle. Initial detachment usually occurs at the anterior tip of the worm or in the region of rectum, sites that may experience greater stress on attachment structures during normal use than elsewhere along the body wall. Eight of the alleles show detachment as early as the L1 stage. The allele shows a later onset (L2 or L3), but a similar pattern of phenotypic progression (Plenefisch et al., 2000). is the only viable allele. About 30% of adult homozygotes show limited muscle detachment in the region of the head or the rectum (Fig. 1, CCG). In animals, apparent separation between hypodermis and cuticle is seen at sites of muscle detachment. Interestingly, 7% of and 1% of Mouse monoclonal to HLA-DR.HLA-DR a human class II antigen of the major histocompatibility complex(MHC),is a transmembrane glycoprotein composed of an alpha chain (36 kDa) and a beta subunit(27kDa) expressed primarily on antigen presenting cells:B cells, monocytes, macrophages and thymic epithelial cells. HLA-DR is also expressed on activated T cells. This molecule plays a major role in cellular interaction during antigen presentation L1 animals show abnormal bulges in the head, a phenotype previously reported for animals with abnormal hypodermal cell positioning and enclosure (Costa et al., 1998). Open in a separate window Figure 1. DIC and polarized light micrographs of animals. (A) DIC micrograph of a animal showing typical curled posture. (B) Same animal as in A visualized by polarized light, a muscle band that has detached from the ventral body wall is visible as a bright birefringent band (arrows) that has collapsed dorsally. (C) DIC micrograph of homozygote showing localized separation of tissues from the cuticle at tail (arrowhead). (D) Same as C under polarized light showing separation of ventral body wall muscles from tip of tail. (E) DIC micrograph of homozygote showing localized separation of tissues from cuticle in head region (arrowheads). (F) Same as C under polarized light showing rearward retraction of the body wall muscles from the area of tissue separation. (G and H) Enlargements of area of tissue separation, two different focal planes of same animal as in E. Note retracted muscles (arrowhead in G) about the region of separation. The large blister labeled a appears to due to separation of the apical hypodermal membrane from the cuticle. Bars: (ACF) 100 m; (G and H) 10 m. Open in a separate window Figure 2. AMG-510 is required for attachment between the apical hypodermal surface and cuticle. (A and B) TEM micrographs of adult wild-type and body wall in intact muscle quadrants. Body wall muscle is indicated by m, hypodermis h, and the basal layer of the cuticle by AMG-510 bc. AMG-510 In the mutant an obviously substantial gap (asterisk) between apical hypodermis and cuticle can be observed. (C) TEM micrograph of mutant body wall in region of muscle detachment. Large gaps indicated by (asterisk) are observed between apical hypodermis and basal cuticle. Hypodermis (h) remains tightly apposed to muscle (m) in the detachment region. A portion of the hypodermis (region between arrows) has become decompressed in the detachment region, whereas immediately under the gaps it is still compressed. Bars, 0.5 m. Transmission electron microscopy (TEM) observations of detachment zones in adult animals demonstrated conclusively that is required for apical hypodermal attachment to the cuticle (Fig. 2). In mutant AMG-510 animals, the distance between apical hypodermal surface and the basal cuticle layer is consistently greater than in wild-type animals, including regions where muscles appear to remain attached to the body AMG-510 wall (compare Figs. 2 A and 3 B). No difference.