live-imaging-and-analysis-of-muscle-contractions-in-drosophila-embryo

Live Imaging and Analysis of Muscle Contractions in <em>Drosophila</em> Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Texas A&M University

Recent studies highlighted an emerging possibility of using Drosophila as a model system for investigating the mechanisms of human congenital muscular dystrophies, called dystroglycanopathies, resulting from the abnormal glycosylation of alpha-dystroglycan. Several of these diseases are associated with defects in O-mannosylation, one of the most prominent types of alpha-dystroglycan glycosylation mediated by two protein O-mannosyltransferases. Drosophila appears to possess homologs of all essential components of the mammalian dystroglycan-mediated pathway; however, the glycosylation of Drosophila Dystroglycan (DG) has not yet been explored. In this study, we characterized the glycosylation of Drosophila DG using a combination of glycosidase treatments, lectin blots, trypsin digestion, and mass spectrometry analyses. Our results demonstrated that DG extracellular domain is O-mannosylated in vivo. We found that the concurrent in vivo activity of the two Drosophila protein O-mannosyltransferases, Rotated Abdomen and Twisted, is required for O-mannosylation of DG. While our experiments unambiguously determined some O-mannose sites far outside of the mucin-type domain of DG, they also provided evidence that DG bears a significant amount of O-mannosylation within its central region including the mucin-type domain, and that O-mannose can compete with O-GalNAc glycosylation of DG. We found that Rotated Abdomen and Twisted could potentiate in vivo the dominant-negative effect of DG extracellular domain expression on crossvein development, which suggests that O-mannosylation can modulate the ligand-binding activity of DG. Taken together these results demonstrated that O-mannosylation of Dystroglycan is an evolutionarily ancient mechanism conserved between Drosophila and humans, suggesting that Drosophila can be a suitable model system for studying molecular and genetic mechanisms underlying human dystroglycanopathies.

Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted.

Protein O-mannosylation has a profound effect on the development and physiology of mammalian organisms. Mutations in genes affecting O-mannosyl glycan biosynthesis result in congenital muscular dystrophies. The main pathological mechanism triggered by O-mannosylation defects is a compromised interaction of cells with the extracellular matrix due to abnormal glycosylation of alpha-dystroglycan. Hypoglycosylation of alpha-dystroglycan impairs its ligand-binding activity and results in muscle degeneration and failure of neuronal migration. Recent experiments revealed the existence of compensatory mechanisms that could ameliorate defects of O-mannosylation. However, these mechanisms remain poorly understood. O-mannosylation and dystroglycan pathway genes show remarkable evolutionary conservation in a wide range of metazoans. Mutations and downregulation of these genes in zebrafish and Drosophila result in muscle defects and degeneration, also causing neurological phenotypes, which suggests that O-mannosylation has similar functions in mammals and lower animals. Thus, future studies in genetically tractable model organisms, such as zebrafish and Drosophila, should help to reveal molecular and genetic mechanisms of mammalian O-mannosylation and its role in the regulation of dystroglycan function.

Protein O-mannosylation in animal development and physiology: from human disorders to Drosophila phenotypes.

Genetic defects in protein O-mannosyltransferase 1 (POMT1) and POMT2 underlie severe muscular dystrophies.  genes are evolutionarily conserved in metazoan organisms. In , both male and female  mutants show a clockwise rotation of adult abdominal segments, suggesting a chirality of underlying pathogenic mechanisms. Here we described and analyzed a similar phenotype in  mutant embryos that shows left-handed body torsion. Our experiments demonstrated that coordinated muscle contraction waves are associated with asymmetric embryo rolling, unveiling a new chirality marker in  development. Using genetic and live-imaging approaches, we revealed that the torsion phenotype results from differential rolling and aberrant patterning of peristaltic waves of muscle contractions. Our results demonstrated that peripheral sensory neurons are required for normal contractions that prevent the accumulation of torsion. We found that  mutants show abnormal axonal connections of sensory neurons.  transgenic expression limited to sensory neurons significantly rescued the torsion phenotype, axonal connectivity defects, and abnormal contractions in  mutant embryos. Together, our data suggested that protein O-mannosylation is required for normal sensory feedback to control coordinated muscle contractions and body posture. This mechanism may shed light on analogous functions of  genes in mammals and help to elucidate the etiology of neurological defects in muscular dystrophies. Protein O-mannosyltransferases (POMTs) are evolutionarily conserved in metazoans. Mutations in  cause severe muscular dystrophies associated with pronounced neurological defects. However, neurological functions of POMTs remain poorly understood. We demonstrated that  mutations in  result in abnormal muscle contractions and cause embryo torsion. Our experiments uncovered a chirality of embryo movements and a unique -dependent mechanism that maintains symmetry of a developing system affected by chiral forces. Furthermore,  were found to be required for proper axon connectivity of sensory neurons, suggesting that O-mannosylation regulates the sensory feedback controlling muscle contractions. This novel POMT function in the peripheral nervous system may shed light on analogous functions in mammals and help to elucidate pathomechanisms of neurological abnormalities in muscular dystrophies.

Article	Year
Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted. Glycobiology\| PubMed ID: 19969597	2010
Protein O-mannosylation in animal development and physiology: from human disorders to Drosophila phenotypes. Seminars in cell & developmental biology\| PubMed ID: 20362685	2010
Protein O-Mannosyltransferases Affect Sensory Axon Wiring and Dynamic Chirality of Body Posture in the Embryo. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 29167399	2018

Naosuke Nakamura

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