A novel muscle LIM-only protein is generated from the paxillin gene locus in Drosophila

Abstract

Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musculature, largely utilizing the Paxillin LIM domains.

Fig. 1. Structure and expression of Drosophila DPxn37 and PDLP. (A) Intron–exon organization of DPxn37 and PDLP. Exons are indicated as thick bars, intronic and intergenic sequences as thin lines. Purple bars indicate the 5′- and 3′-untranslated regions. (B) A developmental northern blot analysis of poly(A)⁺ RNA from embryonic (numbered as hours of development at 25°C), larval, pupal or adult stages. RNAs were hybridized with ³²P-labeled DPxn37 cDNA. Numbers to the left refer to the size of the RNA markers. (C) Amino acid alignment of the third LIM domains of DPxn37 and PDLP. (D) Protein expression. S2 cells were transfected with empty vector, (myc)₆-tagged DPxn37 and PDLP plasmids. Ten micrograms of cell protein from each transfection of S2 cells, or from animals of the embryonic, the first larval or the adult stage, were resolved by SDS–PAGE and immunostained sequentially with affinity-purified polyclonal antibodies against DPxn37 (upper panel) and PDLP (lower panel). (E and F) Schematic representation of the DPxn37 and human Paxillin-superfamily proteins (E), and PDLP and Lepidopteran DALP (F). Numbers on the right depict amino acid identity of pairwise sequence comparisons of each human Paxillin-superfamily protein with DPxn37 (E), and of PDLP with DALP (F), regarding the entire coding region or the LIM1–LIM4 domains. The genetics computer program BLAST2 was used. Major tyrosine phosphorylation sites in human Paxillin and putative phosphorylation sites in DPxn37 are also shown in (E).

Fig. 2. Drosophila DPxn37 and PDLP gene expression in embryos. Embryos were hybridized with antisense DPxn37 (A) or PDLP (B) probes to reveal their gene expressions. Different stages of embryos were used as indicated, and are viewed laterally unless otherwise indicated. Several frames are enlarged, showing DPxn37 expression at the somatic muscle junction sites (indicated by arrowheads) and PDLP expression in the somatic muscles. Controls included stage 16 embryos hybridized with sense DPxn37 (C) or PDLP (D) probes. In all frames, embryos are oriented with anterior to the left.

Fig. 3. Protein expression of DPxn37 and PDLP at embryonic myotendinous junctions. Stage 16 embryos were stained with antibodies against DPxn37, PDLP, phosphotyrosine (pY) and αPS2 integrin, as indicated. The horizontal section (A) and lateral surface views (B–D) are presented showing prominent expression of DPxn37 at the center of the myotendinous junctions, well colocalized with phosphotyrosine signals (A and B), and showing PDLP expression in the somatic muscles with particularly strong staining near the segmentary boundaries (C and D). The right column shows merged views of the left and middle images.

Fig. 4. Association of PDLP with actin bundles. EGFP- or DsRed-tagged DPxn37, and EGFP-tagged PDLP were ectopically expressed in NIH 3T3 fibroblasts, as indicated, and visualized by detecting the autofluorescence from the tags, together with endogenous Paxillin (A) and F-actin (B). The focus was adjusted near the bottom of cells, to detect intracellular proteins at 0.5–2.0 µm above the surface of the glass chamber plates. The right column represents the merging of the left images. Bar, 10 µm. Note that PDLP only marginally overlapped with DPxn37 at the edges of actin stress fibers. HA- or Myc-tagged PDLP showed essentially the same subcellular localization as that of EGFP-PDLP (data not shown).