Seven genes associated with the Pat phenotype (Paralyzed, Arrested elongation at Two-fold) are needed for early membrane-related steps in the assembly of the contractile apparatus of body wall muscle cells (Williams and Waterston, 1994). The products of two of these genes,
unc-52 and
pat-3, are known to code for the worm homologue of the basement membrane proteoglycan perlecan (Rogalski et al., 1993) and an integrin beta subunit, betaPAT-3 (Gettner et al., 1995), respectively. The integrins are a family of alpha-beta heterodimeric receptors for the extracellular matrix that function in cell adhesion, cytoskeletal organization, and signaling. To identify other related components we have cloned
pat-2, using a candidate gene approach. We rescued
pat-2 mutants with a predicted integrin alpha subunit gene identified by the Genome Sequencing Consortium on cosmid F54F2. The open reading frame of this gene contains single nucleotide substitutions corresponding to each of seven existing
pat-2 mutant alleles, confirming that it is
pat-2. cDNA and RT-PCR analysis predict a single 4.0 kb transcript with a trans-spliced SL1 leader. The transcript can code for a 1226 residue polypeptide, referred to as alphaPAT-2, that is homologous to previously described integrin alpha subunits, and is most similar to the vertebrate subfamily which binds ligands containing the RGD motif. Characteristic of integrin alpha subunits, the alphaPAT-2 extracellular domain has seven N-terminal repeat sequences. Repeats 4 through 7 contain a core sequence similar to the calcium binding EF hand consensus motif Dx(D/N)xD(D/G)xxD. Allele
st422 introduces an opal stop codon within the C-terminal part of the extracellular domain. Alleles
e2444 and
e2465 (both generously provided by Joel Rothman) alter a highly conserved gly residue in N-terminal repeat 3.
e2476 (also from Joel Rothman) and
st567 alter highly conserved gly residues 7 or 8 positions N-terminal to the cation binding motif of repeats 5 and 7, respectively.
st538 and
st543, which are slightly milder alleles that allow some delayed, very weak embryonic twitching movements (in contrast to absolute paralysis), alter a less well conserved gly residue 20 positions N-terminal to the cation binding motif of repeat 7. We stained
pat-2 mutant embryos at the 1-1/2 fold stage with anti-beta PAT-3 (MH25). In wild-type embryos betaPAT-3 is located at the muscle cell membrane where it is becoming organized into nascent dense body and M-line attachments. In contrast, in each of the
pat-2 mutants, betaPAT-3 is disorganized and is located within the same focal plane as the muscle cell nucleus. These results suggest that integrin dimer formation and/or maturation is abnormal. To determine the expression pattern of alphaPAT-2, we tagged it with Green Fluorescent Protein (GFP) by inserting a sequence coding for GFP just before the normal stop codon. The alphaPAT-2::GFP chimeric protein, expressed from heritable extrachromosomal arrays, rescues
pat-2 mutants and is located specifically at sites previously shown to contain betaPAT-3 in adults. Thus, the tagged protein is functional and appears to behave like a wild-type integrin alpha subunit. alphaPAT-2::GFP is located in body wall muscle cell dense bodies, M-lines, and attachment plaques. It is also present in vulval, uterine, anal depressor, and anal sphincter muscle attachments, as well as within the myoepithelial sheath cells of the gonad. In conclusion, our results establish that
pat-2 codes for an integrin alpha subunit expressed by muscle and myoepithelial cells and is critical for myofilament lattice assembly in body wall muscles. The alphaPAT-2::GFP chimera should be a useful tool for future analysis of alphaPAT-2 function.