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doi: 10.1242/10.1242/dev.00492

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Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes

Huntsman Cancer Institute and Department of Biology, University of Utah, Salt Lake City, UT 84112, USA

View larger version (80K): [in a new window]   Fig. 3. PINCH protein distribution in developing muscle and other tissues. (A) Dorsal view of a stage 16 embryo. PINCH protein can be seen in the developing dorsal vessel (DV; the two rows of immunoreactive cells at the midline) and the pharyngeal musculature (PM). Also note enrichment of PINCH at the muscle-attachment sites (MAS), where the somatic muscles attach to each other and to the epidermal tendon cells. (B) Optical section through a stage 16 embryo. PINCH protein is prominent in the gut, and in the pharyngeal musculature and the visceral muscle (VM) surrounding the gut. (C) Higher magnification of PINCH localization in the pharyngeal, somatic and cardiac muscle lineages. (D) Higher magnification of PINCH in the digestive tract. PINCH is prominent in both the visceral musculature surrounding the gut and the gut epithelial cells (EC).

View larger version (22K): [in a new window]   Fig. 1. Drosophila pinch is encoded by the steamer duck (stck) locus. (A) Protein sequence similarity between the LIM domains of human PINCH1 (LIMS1 – Human Gene Nomenclature Database) and Drosophila PINCH. (B) Northern blot analysis of staged RNA samples from different developmental timepoints. Embryonic stages are numbered and represent time collected after egg laying at 25°C. Larval samples (L) are from the three larval stages (instar); the third instar larval sample is represented twice to confirm the decrease in rp49 probe seen at that time of development (Borie et al., 1999). Pupal and adult samples are labeled P and A, respectively. RNA markers (not shown) indicated the size of the hybridizing band to be 1.4 kb. Northern blot quantitation is indicated below by the graph. (C) Sequence analysis of the stck alleles. The pinch transcription unit contains six exons, indicated by the blocks, with the initiating MET codon encoded by the second exon. Individual LIM domains are color coded. The pinch sequence in stck17 contains a 571 bp deletion encompassing nucleotides 2095-2664 (corresponding to 615-1066 of the published cDNA sequence; Accession Number AF078907). stck18 harbors a 2 bp deletion removing nucleotides 2309-2310 in the fifth exon (774-775 of the published cDNA sequence), resulting in a frame shift in the middle of the fourth LIM domain. (D) Western blot demonstrating reduction of PINCH protein in stck mutants. Each lane contains 10 µg of protein lysate from the following samples: stage 17 wild-type embryo (lane 1); stage 17 l(3)097 homozygous embryo (lane 2); stage17 l(3)097/stck17 embryo (lane 3); stage 17 l(3)097/stck17 embryo from stck17 germline clone (lane 4). The blot has been hybridized with the affinity-purified PINCH antiserum. The faint band present in lane 4 that migrates at 40 kDa is nonspecific immunoreactivity.

View larger version (147K): [in a new window]   Fig. 2. Loss of PINCH function disrupts muscle morphology and actin filament organization. (A,B) Lateral views of stage 16 embryos stained with an antibody against the Mlp84B protein to visualize the somatic muscles. The stck mutant (B) displays a disruption in muscle fiber morphology. Arrowheads in B indicate areas where the muscles have lost their attachment to the tendon matrix. Arrows in A,B indicate enrichment of Mlp84B at muscle-attachment sites. (C-G) Confocal micrographs of embryonic muscle from wild-type (C,E), stck18/l(3)097 (D), stck17/l(3)097 (F) and l(3)097 homozygote (G) embryos, labeled with fluorescent-phalloidin to visualize F-actin. (C,D) Muscle fibers from early stage 17 embryos. A set of lateral muscles from two segments is shown in each panel. Actin bundles are readily distinguished in the wild-type muscles because of the precise orientation of the actin filaments in each muscle. This arrangement is not maintained in the mutant muscles (D). (E-G) Late stage 17 embryos. Note that the defects exhibited by a stck17/l(3)097 embryo (F) are similar to those from the l(3)097 homozygote (G), when compared with a wild-type embryo (E). Equivalent regions are indicated by an asterisk in the mutant and wild type.

View larger version (139K): [in a new window]   Fig. 4. PINCH is dependent on integrins for its enrichment at muscle-attachment sites. (A,B) Optical section through a stage 16 embryo, showing localization of the indicated proteins at the muscle-attachment sites. (A) PINCH immunoreactivity. (B) ßPS integrin immunoreactivity. The merge of the boxed regions in the stained embryos is shown in the lower corner of the panel. (C,D) Optical sections near the lateral surface of stage 16 embryos stained for PINCH. (C) PINCH enrichment at muscle-attachment sites in wild-type muscle cells. (D) PINCH distribution in myospheroid mutant muscle cells. Note lack of enrichment at the muscle termini (arrows). (E,F) Lateral views of stage 16 myospheroid embryos. (E) PINCH distribution. (F) Pak distribution. Pak remains prominently enriched at muscle-attachment sites (arrows in F), while PINCH is diffuse. Arrowheads in E indicate background immunoreactivity against chordotonal organs present in the affinity-purified PINCH antiserum. (G,H) Ventral views of stage 16 embryos stained with a monoclonal antibody against ßPS integrin. (G) ßPS integrin distribution in a wild-type embryo. (H) ßPS integrin distribution in a stck18/l(3)097 embryo. ßPS integrin remains enriched at the muscle-attachment sites, indicating that functional PINCH is not required for integrin localization to the myotendinous junction.

View larger version (104K): [in a new window]   Fig. 5. PINCH and ILK co-precipitate and colocalize at integrin-rich sites in Drosophila embryos. (A-C) An optical section of a stage 16 embryo showing the localization of the indicated proteins. (A) PINCH immunoreactivity. (B) ILK::GFP. (C) Merged image of A,B. (D) Native immunoprecipitation of PINCH and associated proteins from Drosophila embryonic lysates. Lanes 1-4 represent different immunoprecipitation experiments, run out on an SDS-PAGE gel. The resulting blot was probed with the antisera indicated on the right-hand side. Immunoprecipitations with the PINCH preimmune serum (PI) serve as negative controls, while PINCH immunocomplexes were isolated with the affinity-purified PINCH antiserum (anti-Pin). (E,F) ILK::GFP distribution in a wild-type (E) and stck17 maternal/zygotic mutant (F) stage 16 embryo. The ILK fusion protein is still concentrated at the muscle-attachment sites in the stck mutant embryo (arrows in F).

View larger version (76K): [in a new window]   Fig. 6. PINCH function is essential for adhesion between wing epithelial sheets. (A) Production of stck17 homozygous clones in wing tissue leads to blister formation. (B-D) Confocal micrographs of a developing wing disc (45 hours after puparium formation). (B) ßPS integrin protein is enriched at the basal junctions between the two epithelial layers (arrow). (C) PINCH protein is also expressed in the developing wing, and displays a similar subcellular distribution (arrow). (D) Merge of B and C.