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First published online 29 May 2007
doi: 10.1242/jcs.000695

Journal of Cell Science 120, 2066-2077 (2007)
Published by The Company of Biologists 2007
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The Drosophila muscle LIM protein, Mlp84B, cooperates with D-titin to maintain muscle structural integrity

Kathleen A. Clark1,2,*, Jennifer M. Bland2 and Mary C. Beckerle1,2,3

1 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
2 Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
3 Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA


Figure 1
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  		Fig. 1. Molecular genetic analysis of the mlp84B locus. (A) Identification of deficiencies that remove mlp84B. DNA from several deficiency lines in the 84B,C region was digested with EcoRI, and probed with the mlp84B cDNA. The second blot contains DNA from deficiency lines that harbor a balancer chromosome with a restriction fragment length polymorphism in the mlp84B region (TM3*). Loss of the larger EcoRI band (10 kb) indicates a deletion of mlp84B. (B) Southern blot analysis of the mlp84BP8 allele showing deletion of the mlp84B transcription unit. The arrangement of the HindIII fragments relative to the mlp84B transcription unit is shown to the right [arrow indicates band contributed by the Df(3R)dsx2M chromosome]. (C) Molecular map of the 84C1,2 region. Deficiencies used to generate mlp84B-null animals are shown above the map, and the deletion in mlp84BP8 is also shown. The mlp84B transcription unit is shown in detail below the map, with the position of the P-element in mlp84BP20 indicated. (D) Western blot of protein lysates from third-instar larvae demonstrating loss of the mlp84B gene product in mlp84B mutants. 10 µg of protein was loaded per lane. Genotypes for the larvae are indicated on the right; 4B2 is the mlp84B transgene, and 2X4B2 indicates two copies of the transgene. The open arrow indicates a weak non-specific band detected by this antiserum.

 

Figure 2
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  		Fig. 2. Loss of Mlp84B affects muscle-driven morphogenetic processes. (A) Pupal lethality associated with loss of mlp84B and rescue with the mlp84B transgene. (B) Axial ratios for pupae of indicated genotype. [*P>10–5, compared with two-copy rescue line (4B2 Scx2/4B2 dsx2M).] Data are displayed as a distribution of data points for each pupae, bounded by the upper quartile and lower quartile, with the median value indicated as a line in the box. Error bars show the upper and lower limits of the data, and any outliers are indicated as a circle. (C) Brightfield images of wild-type and Df(3R)dsx2M/Df(3R)Scx2 pupae (mlp84B–/–); bar, 200 µm. Anterior is to left. Arrow indicates air pocket that failed to translocate. Arrowhead shows failure in spiracle eversion. (D) Scanning electron microscopy image of wild-type and Df(3R)dsx2M/Df(3R)Scx2 pupae; bar, 200 µm. Arrowhead shows failure in spiracle eversion.

 

Figure 3
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  		Fig. 3. Loss of Mlp84B does not grossly disrupt muscle structure. (A,A') Wild-type third-instar muscle fillets labeled with actin (A) or {alpha}-actinin (A'). Bars, 200 µm (A); 20 µm (A'). (B,B') Fillets from Df(3R)dsx2M/Df(3R)Scx2 transheterozygous third-instar labeled with actin (B) or {alpha}-actinin (B'). (C) Ultrastructural analysis of flight muscle from wild-type (upper panel) and Mlp84B-deficient (lower panel) flies; bar, 10 µm. Double-headed arrows in each panel delineate one sarcomere, bounded by two Z-discs (electron-dense lines), with the M-lines in the middle (lighter line).

 

Figure 4
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  		Fig. 4. Mlp84B does not have an essential nuclear function. (A-C) Two muscle fibers from transgenic third-instar larvae expressing either an (A) Mlp84B::GFP, (B) Mlp84B::GFP::NES or (C) Mlp84B::GFP::NLS fusion protein. Muscle nuclei in A and B are highlighted by red circles. Note the lack of detectable nuclear GFP signal in B relative to A and the strong nuclear signal in C. The three micrographs were recorded using the same confocal settings. (D) Quantification of rescue of the mlp84BP8 mutants by the three Mlp84B fusion proteins. The Mlp84B::GFP::NLS does not provide any appreciable rescue of pupal lethality in the mlp84BP8 mutants.

 

Figure 5
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  		Fig. 5. Mlp84B and D-titin colocalize at the Z-disc and have similar mutant phenotypes. (A) Distribution of Mlp84B and {alpha}-actinin at the Z-disc of somatic body wall muscle; bar, 10 µm. Enlargements of two Z-bands are shown below the larger micrograph. (B) Diagram of the relative positions of Mlp84B, actin, {alpha}-actinin and D-titin in the Z-disc. (C) Colocalization of Mlp84B and D-titin in somatic muscle Z-disc; bar, 10 µm. Enlargements of two Z-bands are shown below the larger micrograph. (D) Representative pupal cases; bar, 200 µm. (E) Average axial ratios for pupae of indicated genotype (*P>10–6, comparing mutant to w1118).

 

Figure 6
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  		Fig. 6. Enhancement of mlp84B mutant phenotypes by reduction in D-titin activity. (A) Enhancement of pupal lethality at 18°C displayed by mlp84B P-element alleles and sls mutants. Numbers given are the percentage of the number of mutant adults divided by the expected number for the cross. (B) Average axial ratios for pupae of indicated genotype. (*P>10–6, compared with the appropriate P-element allele alone.) (C) Representative pupae; bar, 200 µm. (D-H) Muscle fillets of third-instar larvae stained with Alexa Fluor-488 phalloidin. (D) Wild type; bar, 200 µm. (E) mlp84BP8/Df(3R)dsx2M. (F) slsj1D7mlp84BP8/Df(3R)dsx2M; inset highlights torn muscle fibers. (G) slsg05/slse4. (H) slsj1D7/+. (I) Late-stage embryos labeled with D-titin antiserum to highlight muscle fiber morphology. (J) Single muscle fibers from third-instar larvae stained with Alexa Fluor-488 phalloidin: wild type (upper panel), mlp84BP8/Df(3R)dsx2M (middle panel), slsj1D7mlp84BP8/Df(3R)dsx2M (lower panel); bar, 20 µm.

 

Figure 7
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  		Fig. 7. Loss of Mlp84B does not affect D-titin protein localization or levels. (A) Muscle fibers from wild type (upper panel) or mlp84BP8 (lower panel) labeled with the N-terminal D-titin antibody; bar, 20 µm. (B) Western blot of protein lysates from wild-type and mlp84BP8 adults. Each lane contains protein from one-quarter of a thorax. An anti-LaminC antibody serves as a loading control (lower panel).

 

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© The Company of Biologists Ltd 2007