Anna Starzinski-Powitz

The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

Decrease in acetylcholine-receptor content of human myotube cultures mediated by monoclonal antibodies to α, β and γ subunits

One of the two main causes of acetylcholine‐receptor loss in myasthenia gravis is antigenic modulation, i.e. accelerated internalization and degradation rate by antibody‐crosslinking. This phenomenon has been studied only in animal tissues. Therefore, we tested antigenic modulation of the acetylcholine receptor on human embryonic myotubes in culture. Several monoclonal antibodies to the α, β and γ subunits of the receptor reduced its concentration, in some cases down to one‐third of the control. Some of these antibodies only form complexes of one antibody with two receptor molecules; consequently such small complexes are sufficient to accelerate internalization of the human acetylcholine receptor. This technique might be proved valuable for clinical screening of sera from myasthenie patients.

Description of Putative ribosomal RNAs with low abundance, developmental regulation, and the identifier sequence

Three RNA species (5, 2, 0.15 kb) characterized by the repetitive identifier (ID) sequence, expressed constitutively, and at low abundance have been identified in rat L6 muscle cells by hybridization to cDNA pL6-411. Comigration of these three RNAs with 28, 18, and 5.8 S ribosomal RNAs (rRNAs) has suggested the possibility that pL6-411 RNAs are related to ribosomes or ribosome-like structures. Subsequent experiments showed that pL6-411-related RNAs could indeed be found in ribosome-like particles which were indistinguishable from ribosomes when separated on sucrose gradients under native (low salt, isolation of intact ribosomes) or denaturing conditions (detergent, high salt, isolation of ribosome subunits). Furthermore, we demonstrate that pL6-411-related RNAs are cytoplasmic in L6 cells, may be transcribed in nucleoli, and, based on their nucleotide sequence, have the potential of inter- and intramolecular hybridization. Expression of pL6-411 RNAs was also shown in adult as well as in fetal rat tissues after Day 14 of gestation. These above findings provide supportive evidence for the hypothesis that pL6-411 5- and 2-kb RNAs could exist in a subset of ribosomes. These ribosome-like pL6-411 particles nevertheless differ from ribosomes in that their associated RNAs have different nucleotide sequences, are of lower abundance, and are up-regulated later in development than rRNAs. We discuss our results in the context of a postulated ribosome subset containing RNAs other than rRNAs. These ribosome-like particles might be involved in the translational control of ID-positive mRNAs.

In Situ Formats

Soon after the method of in situ hybridization (ISH) had been published (Pardue and Gall, 1969), reports appeared showing that it could also be used with great success in the study of virus-infected systems (Orth et al., 1970; Geukens and May, 1974). Now it was possible to study the biology of viruses and the mechanisms of viral infections in detail and to both improve diagnosis and form the basis of prognosis of viral diseases.

Pathogenicity of Myasthenic Sera and Monoclonal Antibodies Studied by the Use of Human Muscle Cells

Cross-linking of acetylcholine receptors (AChRs) by antibodies results in acceleration of AChR internalization rate. This phenomenon is called antigenic modulation of AChR and is thought to be one of the two main causes of AChR loss in myasthenia gravis (MG). Until recently antigenic modulation had been studied only on animal tissues (e.g., refs. 1 and 2), where only a small fraction of human anti-AChR antibodies bind. We present here the effect of monoclonal antibodies (mAbs) and MG sera on human embryonic AChR internalization rate, and we compare the capacity of the sera with the severity of the patients disease.