4 research outputs found
A Mighty Small Heart: The Cardiac Proteome of Adult Drosophila melanogaster
Drosophila melanogaster is emerging as a powerful model system
for the study of cardiac disease. Establishing peptide and protein maps of the
Drosophila heart is central to implementation of protein
network studies that will allow us to assess the hallmarks of
Drosophila heart pathogenesis and gauge the degree of
conservation with human disease mechanisms on a systems level. Using a
gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected
adult fly hearts. Contractile, cytostructural and mitochondrial proteins were
most abundant consistent with electron micrographs of the
Drosophila cardiac tube. Functional/Ontological enrichment
analysis further showed that proteins involved in glycolysis,
Ca2+-binding, redox, and G-protein signaling, among other
processes, are also over-represented. Comparison with a mouse heart proteome
revealed conservation at the level of molecular function, biological processes
and cellular components. The subsisting peptidome encompassed 5169 distinct
heart-associated peptides, of which 1293 (25%) had not been identified in
a recent Drosophila peptide compendium. PeptideClassifier
analysis was further used to map peptides to specific gene-models. 1872 peptides
provide valuable information about protein isoform groups whereas a further 3112
uniquely identify specific protein isoforms and may be used as a
heart-associated peptide resource for quantitative proteomic approaches based on
multiple-reaction monitoring. In summary, identification of
excitation-contraction protein landmarks, orthologues of proteins associated
with cardiovascular defects, and conservation of protein ontologies, provides
testimony to the heart-like character of the Drosophila cardiac
tube and to the utility of proteomics as a complement to the power of genetics
in this growing model of human heart disease
Fluorescent Labeling of Drosophila Heart Structures
The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of α-actinin in larval hearts. Additionally, we perform labeling of F-actin and α-actinin in myosin-GFP expressing adult flies and of α-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system