Differential expression of proteins in muscle and electric organ, a muscle derivative

Abstract

The electric organ of electric fish develops from a myogenic lineage. We have used immunohistochemistry and immunoblotting to determine which features of the muscle phenotype are retained and whether any new ones are expressed in mature electrocytes of the electric fish Sternopygus. The muscle-specific intermediate filament desmin was found throughout the electrocytes, and different desmin antibodies detected molecules with different subcellular distributions. Western blots confirm that these antibodies recognize a protein of MW = 53 kD, the molecular weight of desmin. Other muscle proteins were also present within electrocytes: Actin and sarcomeric alpha-actinin were found within the subsynaptic membrane beneath the plasmalemma of the electrocytes, and talin and acetylcholine receptors were detected both at the innervated posterior face and at the non-innervated anterior face. This was confirmed using rhodamine-conjugated alpha-bungarotoxin. Neither myosin heavy chain nor tropomyosin was present in electrocytes. Finally, we detected within electrocytes a type I acidic keratin that forms a filamentous meshwork within each cell. Immunoblots corroborate this result: A keratin-positive doublet of MW = 50 kD and 57 kD was found in both electrocytes and skin. Myosin, actin, talin, tropomyosin, desmin, alpha-actinin, and acetylcholine receptor, but not keratin, were all expressed in fish skeletal muscle fibers. Thus, electrocytes retain some muscle-specific proteins, do not express others, and in addition, express a non-muscle protein.

Mechanisms of muscle gene regulation in the electric organ ofSternopygus macrurus

“…The mature electrocyte phenotype is more than just a partially suppressed muscle because electrocytes also express some genes that muscle fibers do not, such as keratin and the Na(v)1.4a sodium channel (Patterson and Zakon, 1996;Unguez and Zakon, 1998b;Zakon et al, 2006). Identification of additional genes that are upregulated in electrocytes and not expressed in muscle fibers will provide a more comprehensive characterization of the electrocyte phenotype.…”

“…Ultrastructural studies show that electrocytes in S. macrurus are multinucleated like their muscle precursors but do not have sarcomeres or T-tubules ( Fig.1) (Unguez and Zakon, 1998a;Unguez and Zakon, 1998b). Consistent with results from ultrastructural studies, immunolabeling studies show that electrocytes express some muscle proteins including desmin, titin, contractile proteins α-actinin and α-actin, and endplate proteins dystrophin and acetylcholine receptors (AChRs), but do not express sarcomeric proteins like myosin heavy chains (MHCs), tropomyosin, and troponin-T (Figs1, 2) (Cuellar et al, 2006;Kim et al, 2008;Patterson and Zakon, 1996;Unguez and Zakon, 1998a). As the nervous system plays a major role in the maintenance and plasticity of muscle fibers in adult vertebrates, it is important to note that the EO of S. macrurus is innervated by a population of spinal motoneurons that exerts activation patterns (continuous rate of 50-200Hz) (Bennett, 1971;Mills et al, 1992) that are markedly different from those that activate muscle fibers (Bellemare et al, 1983; Coughlin and Rome, 1999).…”

“…In general, quantitative analysis showed similar levels of these muscle transcripts in EO and skeletal muscle, confirming previously published qualitative reports ( Fig.4) (Cuellar et al, 2006). Detection of transcripts for these contraction-associated genes in EO was unexpected given that protein expression studies using mammalian antibodies against tropomyosin, troponin-T, fast SERCA, and slow and fast isoforms of MHC failed to detect these proteins in mature electrocytes and EO lysate (Fig.2, Fig.4B) (Cuellar et al, 2006;Kim et al, 2004;Patterson and Zakon, 1996;Unguez and Zakon, 1998b).…”

“…In general, quantitative analysis showed similar levels of these muscle transcripts in EO and skeletal muscle, confirming previously published qualitative reports ( Fig.4) (Cuellar et al, 2006). Detection of transcripts for these contraction-associated genes in EO was unexpected given that protein expression studies using mammalian antibodies against tropomyosin, troponin-T, fast SERCA, and slow and fast isoforms of MHC failed to detect these proteins in mature electrocytes and EO lysate (Fig.2, Fig.4B) (Cuellar et al, 2006;Kim et al, 2004;Patterson and Zakon, 1996;Unguez and Zakon, 1998b).While detection of these transcripts suggested that the MRFdependent muscle activation in mammals is conserved in S. macrurus muscle fibers and electrocytes, the transcription of some muscle genes without apparent translation suggested that posttranscriptional events might be involved. In contrast to the above contraction-associated genes, expression profiles of metabolic genes and other muscle components such as α-actin, α-AChR and desmin show little evidence of post-transcriptional regulation as the relative abundance of transcripts and corresponding proteins or enzymatic activities are similar between skeletal muscle and EO (Fig.4).…”

Electric fish: new insights into conserved processes of adult tissue regeneration

“…Recently, we have also begun to address some aspects of the cellular and molecular mechanisms underlying the regeneration of complex tail structures such as skeletal muscle and the electric organ in this teleost fish. Tail regeneration in gymnotiforms has been most studied in S. macrurus (Fig.2) (Patterson and Zakon, 1993;Patterson and Zakon, 1996;Patterson and Zakon, 1997;Unguez and Zakon, 2002;Weber et al, 2012). Like all gymnotiforms studied to date, tail regeneration in S. macrurus begins by epidermal cells covering the wound followed by formation of a blastema (Fig.2).…”