BIOLOGICAL OVERVIEW

The rat muscle LIM protein (MLP) was isolated in an attempt to identify genes expressed in muscles and in the formation on neuromuscular synapses. A subtractive library approach was used to isolate cDNAs induced in rat skeletal muscle 7 days after denervation. In this procedure, messenger RNAs from denervated rat skeletal muscle are copied into a complementary DNA form. Sequences appearing in normal (non-denervated) muscle are removed after hybridization of cDNA from denervated muscle with normal muscle cDNA, leaving only sequences expressed upon denervation. One of the cDNAs isolated by this approach codes for MLP, a novel LIM finger protein (Arber, 1994).

From there on, it was easy to jump from rat to chick to fly. Complementary RNA probes against rat MLP strongly hybridize with similar sized mRNAs found in chick and Drosophila. The Drosophila MLP60A transcripts are present in visceral mesoderm and in a segmentally repeated pattern in the somatic mesoderm of early stage 13 embryos. No expression is detected in the endoderm, the ectoderm or the 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. 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. In other words, the distribution of MLP proteins were analyzed by staining with antibody that reacts specifically with them. 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 prior to 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).

Rat MLP is detected as a nuclear antigen in cultured embryonic muscle tubes. MLP protein is first detected during myotube formation, and its expression is particularly elevated during muscle maturation. Later on, in maturing myotubes and muscle fibers, the protein also accumulates in the cytosol (Arber, 1994). The two Drosophila proteins, along with the rat and chick MLPs, define a subclass of LIM-only proteins having unique dual subcellular localization in the nucleus and along actin-based filaments in the cytosol. The associatiation of MLPs with actin suggests that MLPs are constituents of the actin based cytoskeleton (Arber, 1996).

To explore the possibility that mammalian MLP plays a role in myogenic differentiation, overexpression and antisense experiments were carried out in myogenic cell lines. While overexpression of MLP in differentiating myoblasts promotes myogenic differentiation, suppression of mRNA expression prevents this process. These results strongly suggest that the presence of MLP is essential for the expression of the myogenic phenotype in differentiating myotubes (Arber, 1994).

In contrast to its differentiation-promoting abilities, MLP does does not promote myogenic determination. Myogenic determination can be carried out by expression of myogenic HLH proteins or MyoD family members (MDFs) including MyoD, myogenin, Myf-5 and MRF-4. Overproduction of MDFs in several nonmyogenic cell lines leads to the expression of muscle-specific genes upon transfer to differentiation-promoting conditions (low serum). For example, one MLP expressing cell line is particularly responsive to MDFs, but this cell line shows no myogenic differentiation when transferred to a low serum in vitro environment. Early differentiation events preceding exit from the cell cycle apparently do not depend on MLP. Nevertheless, a drastic reduction of mitotic activity is found in MLP expressing cells when they are transferred to low serum medium. The ability of MLP to potentiate myogenic processes is obvious when cells are treated with growth factors that prevent myogenic differentiation. Significant inhibition of activation of muscle-specific genes takes place when cells are treated with transforming growth factor ß (TBFß). Overexpression of MLP has a marked effect on the inhibition of differentiation by TBFß. These findings are consistent with the conclusion that MLP efficiently potentiates myogenic differentiation and suggests that it may regulate processes controlling muscle-specific gene expression (Arber, 1994).

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).

Substantially different LIM finger proteins are able to mimic rat MLP in promoting differentiation of myogenic cells. Constructs of LIM domains of LIM1 and LIM2 (proteins involved in motor neuron differentation in mammalian cells) were prepared and expressed in cultured myogenic precursors. Both LIM1 and LIM2 LIM-only proteins are able to promote myogenic differentiation. In addition, the LIM fingers of rhombotin-1 and CRP, two other LIM domain proteins, also promote myogenesis. These studies strongly implicate LIM-only proteins in key regulatory interactions involving onset of myogenic differentiation (Arber, 1994).

Future research should reveal whether MLPs function as nuclear regulators of transcription, or whether their function is confined to the cytoplasm. MLP might function as a shuttle of information between the cytoplasm and nucleus, perhaps sending information to the nucleus concerning the developmental state of muscle. If this is shown to be the case, then MLP may well resemble many other Drosophila shuttle proteins, for example: Numb, the cytoplasmic domain of the receptor Notch, Suppressor of Hairless, and Armadillo.

GENE STRUCTURE

Bases in 5' UTR - 59

Bases in 3' UTR - 84

PROTEIN STRUCTURE

Amino Acids - 92

Structural Domains

The abbreviation LIM derives from a common protein motif shared by the homeodomain proteins Lin11, Isl-1 and Mec-3, Islet and Apterous. Each LIM finger binds two zinc ions. Each LIM domain coordinates two atoms of zinc in a tetrahedral fashion via the conserved cysteine and histidine residues of the LIM consensus. The majority of LIM finger proteins have homeodomains. Exceptions include Cys-rich protein (CRP), the single LIM finger Cys-rich intestinal protein, the T cell oncogene rhombotin-1, the focal adhesion protein zyxin, and a novel protein kinase. Rat MLP has two LIM fingers, each followed by a Gly-rich and hydrophobic residue-rich region. MLP is a conserved protein: complementary RNA probes against rat MLP strongly hybridize with an mRNA of similar size in chick and Drosophila. Chick MLP is 93% identical to rat MLP. Drosophila MLP has only one LIM Gly-rich motif. Drosophila MLP60A aligns best to the first LIM Gly-rich domain of rat MLP. Like vertebrate CRPs, the LIM domain of MLP60A exhibits the sequence CX2CX17HX2CX2CX2CX17CX2C. The potential nuclear targeting signal is retained with one conserved lysine to arginine substitution. The end of the Drosophila MLP coding sequence corresponds to the transition to the linker sequence that connects the two LIM Gly-rich domains of rat MLP (Arber, 1994 and Stronach, 1996).

The LIM domain is a cysteine-rich domain composed of 2 special zinc fingers joined by a 2-amino acid spacer. Some proteins are made up of only LIM domains, while others contain a variety of different functional domains. LIM proteins form a diverse group that includes transcription factors and cytoskeletal proteins. The primary role of LIM domains appears to be in protein-protein interaction, through the formation of dimers with identical or different LIM domains or by binding distinct proteins. In LIM homeodomain proteins, LIM domains seem to function as negative regulatory domains. LIM homeodomain proteins are involved in the control of cell lineage determination and the regulation of differentiation, and LIM-only proteins may have similar roles. LIM-only proteins are also implicated in the control of cell proliferation since several genes encoding such proteins are associated with oncogenic chromosome translocations. In analyzing sequence relationships among various LIM domains it is suggested that they may be arranged into 5 groups that appear to correlate with the structural and functional properties of the proteins containing these domains. All N-terminal LIM domains (LIM1) are segregated into cluster A, whereas all LIM2 domains of the same proteins constitute cluster B. This relationship suggests that the putative duplication leading to the LIM A and B domains is ancient, preceding their association with different structural motifs (e.g., homeodomains, kinases). Furthermore, the sequence relationships between the LIM domains (LIM1 and LIM2) in the same protein may be conserved by functional constraints based on cooperation between LIMA and B domains. In contrast, the two type C LIM (another LIM domain cluster) domains of some of the LIM-only proteins like CRP are more similar to one another, implying the possibility of a more recent duplication. Cluster D is a rather divergent set of LIM domains that includes the cytoskeletal proteins Zyxin and Paxillin. The closest homologs of Apterous, for both LIM1 and LIM2 domains, are human and rat LH2. The Islet LIM1 and LIM2 domains define Islet as a cohesive subfamily of LIM proteins (Dawid, 1995).

date revised: 9 APR 97 

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