Muscle LIM protein at 60A

Transcriptional Regulation

Drosophila MLP1 expression was examined in embryos ectopically expressing MEF2 in the epidermis and ventral midline cells. The ectopic expression of Drosophila MLP1 in ventral midline cells, but not in the epidermis, is detected starting at stage 13, when Drosophila MLP1 is normally activated in the mesoderm. Drosophila MLP1, which is not normally expressed in the dorsal vessel, is ectopically expressed in the dorsal vessel in embryos overexpressing MEF2 in the mesoderm and muscles. Drosophila MLP1 may be a target of MEF2-regulated gene expression in muscles. This is suggested by the fact that overexpression of MEF2 in the dorsal vessel induces the expression of Drosophila MLP1 (Lin, 1997).

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, have been characterized that show restricted expression in differentiating muscle lineages. To extend this analysis of Drosophila Mlps, the expression of Mlps have been characterized in mutant backgrounds that disrupt specific aspects of muscle development. A genetic requirement is found for the transcription factor dMEF2 in regulating Mlp expression, and dMEF2 can bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, the subcellular distribution of Mlp84B has been analyzed in detail. Immunofluorescent studies reveal the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also fail to perturb Mlp84B distribution. In conclusion, molecular epistasis analysis has been used to position Mlp function downstream of events involving mesoderm specification and patterning, concomitant with terminal muscle differentiation. Furthermore, these results are consistent with a structural role for Mlps as components of muscle cytoarchitecture (Stronach, 1999).

The Mlp60A gene exhibits three exons interrupted by two small introns, one in the 5' untranslated region and another in the coding region. The Mlp84B gene contains one noncoding and one coding exon separated by a single large intron. Analysis of noncoding DNA within and surrounding the Mlp genes reveals the presence of multiple A/T-rich sequences matching exactly the reported MEF2 target binding consensus sequence. The Mlp60A gene contains three potential dMEF2 binding sites; two of these sites are located in the region 5' to the start of gene, whereas the third is found 3' to the coding sequence. Six putative dMEF2 binding sites are found in the Mlp84B gene. Four of the six are clustered in the intron, another is located 3' to the coding region of the gene, and another is contained completely within the first exon (Stronach, 1999).

Knowledge of the subcellular distribution of a protein often contributes substantially to an understanding of its function. In embryonic somatic muscles, Mlp60A and Mlp84B are found in both the nuclear and cytoplasmic compartments, consistent with either a regulatory or structural role in differentiating muscle. When expressed in rat embryo fibroblast cells, Drosophila Mlps showed a specific association with the actin cytoskeleton. To determine the precise localization of Mlps within mature myofibrils at higher resolution, whole, third instar larval midguts were double labelled using antibodies directed against the Mlps and alpha-actinin, which marks Z-bands. Surrounding the midgut, elongated visceral mesodermal cells form a lattice of transverse and longitudinal fibers. Although these cells do not undergo myoblast fusion, they appear striated and display sarcomeric repeats. Within the midgut visceral mesoderm, alpha-actinin prominently localizes to Z-bands. Z-bands demarcate the ends of individual sarcomeres, where the barbed ends of actin thin filaments terminate. In the same tissue, Mlp84B distributes as a doublet that flanks each Z-band. As seen in merged images, Alpha-actinin and Mlp84B are localized in adjacent regions. Mlp84B extends away from the periphery of the Z-band, whereas alpha-actinin is clearly more restricted. The localization of Mlp84B to discrete sites within the muscle sarcomere provides evidence for a specific association of Mlp84B with the microfilament cytoskeleton in vivo. Although Western immunoblot analysis reveals that Mlp60A is present in isolated midgut preparations, the protein was not detected using immunofluorescent methods. It is unclear why Mlp60A protein was not observed in situ, but perhaps within the mature myofibril, Mlp60A is complexed with protein partners such that the epitopes recognized by the antibodies were masked (Stronach, 1999).

Protein Interactions

When either Drosophila MLP60A or MLP84B are expressed in mammalian cultured cells, each shows significant colocalization with actin bundles, illustrating that LIM-glycine repeats found in fly proteins share with their vertebrate relative the ability to associate with the actin cytoskeleton. This resulet suggests that MLP60A is a cytoskeletal protein acting to join different elements in the myosin cytoskeleton. MLP60A is found occasionally in cell nuclei but MLP84B is never observed in vertebrate cell nuclei (Stronach, 1996).

DEVELOPMENTAL BIOLOGY

Embryonic

The Drosophila MLP1 transcripts are present in visceral mesoderm and in a segmentally repeated pattern in somatic mesoderm of early stage 13 Drosophila embryos. No expression is detected in the endoderm, ectoderm or nervous system (Arber, 1994).

Like MLP60A, a second Drosophila muscle LIM protein, MLP84B, is detected late in development. Messenger RNAs for both proteins decline in larval development and elevate again during the larval to pupal transition. MLP60A transcripts persist in adults. The initial expression of both proteins is detected in growing syncytial myotubes visualized as segmentally repeated groups of cells positioned dorsally, laterally and ventrally within the embryo. Although mRNAs for both proteins are coexpressed in somatic muscles, their patterns of hybridization are distinct. Mlp60A mRNA appears to be distributed throughout mature myotubes, whereas Mlp84B mRNA is concentrated at the terminal portions of the myotubes near where they make attachments to the epidermis. Confocal microscopy was used to visualize the distribution of MLP proteins fluorescently labeled with anti-MLP antibody in parallel with an anti-muscle myosin antibody. The MLPs, although not enriched in muscle cell nuclei, do not show a significant nuclear exclusion as does myosin. MLP84B uniquely, becomes associated with the developing myotendinous junction, visualized as bright staining at the ends of myotubules. This enrichment is largely absence before stage 16. The redistribution of MLP84B to the ends of muscle fibers after 14 hours of development correlates with early signs of the development of functional myotendinous junctions, including somatic muscle attachments and visible muscle contractions. Both muscle LIM proteins appear to associate with linear cytoplasmic elements within the muscle cell syncytium, suggestive of the sarcomeric actin filament network (Stronach, 1996).

Both transcripts are also coexpressed in the visceral musculature surrounding the fore-, mid- and hindgut of stage 14 and older embryos. In addition to the presence in visceral mesoderm, both mRNAs are strongly expressed in pharyngeal muscle. In contrast with what was observed in the somatic musculature, polarized distribution of MLP84B transcripts in visceral or pharyngeal muscle was not observed (Stronach, 1996).

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date revised: 10 October 99

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