Indirect Flight Muscle (IFM) expression profiling of actin and myosin nulls in Drosophila melanogaster
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE70252 | GEO | 2015/06/25
SECONDARY ACCESSION(S): PRJNA288041
REPOSITORIES: GEO
Similar Datasets
Project description:Expression profiling of IFMs from 1-2 day old adult male flies of 3 genotypes: Canton-S, IFM-specific actin null (Act88FKM88) and IFM-specific myosin null (Mhc7). Results provide insight into how muscles respond to the absence of major scaffold proteins, and whether these transcriptional responses are filament-specific or generic to the tissue. RNA was extracted from the IFMs of 1-2 day old adult male flies of 3 genotypes (Canton-S, Act88FKM88, Mhc7), labeled, and hybridized on Affymetrix GeneChip Drosophila Genome 2.0 microarrays. 9 microarray experiments from 3 independent biological replicates of each genotype. Data was analyzed using RMA method, and significance was tested using SAM version 3.02, with cut-off parameters of 10% FDR and minimum 2-fold change in expression levels.
2015-06-25 | E-GEOD-70252 | biostudies-arrayexpress
Project description:Building on a previous study where we examined changes in gene expression and splicing after knockdown of Bruno 1 (Bru1, Aret) in indirect flight muscle, we have now generated a CRISPR allele bru1[M3] that results in a phenotypic null. Here we examine changes in gene expression and alternative splicing in bru1[M3] mutant IFM dissected from 1 d adult flies.
2022-06-02 | GSE205092 | GEO
Project description:The goal of the study was to investigate the mechanistic basis for Time-restricted feeding (TRF) improvement in skeletal muscle by assessing transcriptomic data of wild type (WT), WT under High-fat diet (HFD), and genetic obesity linked mutant sphingosine kinase 2 (Sk2) under ad libitum feeding (ALF) and time-restricted feeding (TRF) conditions. Next generation sequencing was used to assess the changes along a diurnal cycle in the transcriptome of Drosophila indirect flight muscle (IFM) tissue at 3-week of age under ad libitum feeding (ALF) or time-restricted feeding (TRF).
2023-01-19 | GSE205334 | GEO
Project description:Structural interactions between the myosin converter and relay domains have been proposed to be critical for the myosin power stroke and muscle power generation. We tested this hypothesis by mutating converter residue 759, which interacts with relay residues I508, N509, and D511, to glutamate (R759E) and determined the effect on Drosophila indirect flight muscle mechanical performance. Work loop analysis of mutant R759E indirect flight muscle fibers revealed a 58% and 31% reduction in maximum power generation (P(WL)) and the frequency at which maximum power (f(WL)) is generated, respectively, compared to control fibers at 15 °C. Small amplitude sinusoidal analysis revealed a 30%, 36%, and 32% reduction in mutant elastic modulus, viscous modulus, and mechanical rate constant 2πb, respectively. From these results, we infer that the mutation reduces rates of transitions through work-producing cross-bridge states and/or force generation during strongly bound states. The reductions in muscle power output, stiffness, and kinetics were physiologically relevant, as mutant wing beat frequency and flight index decreased about 10% and 45% compared to control flies at both 15 °C and 25 °C. Thus, interactions between the relay loop and converter domain are critical for lever-arm and catalytic domain coordination, high muscle power generation, and optimal Drosophila flight performance.
| S-EPMC3164139 | biostudies-literature
Project description:LC-MS/MS analysis of peptide digests of myofibrils from wild type and Dmlc2(Delta 2-46; S66A, S67A) mutant Drosophila flies. Myofibrils and thick filament samples were solubilized in 150 ul 0.1% RapiGest SF Surfactant (Waters Corporation; 50 C, 1 hour). Proteins were reduced by addition of 0.75 ul of 1M dithiothreitol and heating (100 C, 10 min). Cysteines were alkylated by addition of 22.5 ul of 100 mM iodoacetamide in 50 mM ammonium bicarbonate and incubation in the dark (22C, 30 min). Proteins were digested to peptides by addition of 25 ul of 50 mM ammonium bicarbonate containing 5 ug of trypsin (Promega) and incubating (37 C, 18 hours). The samples were dried down in speed vacuum device. Trypsin was deactivated and RapiGest was cleaved by addition of 100 ul of 7% formic acid in 50 mM ammonium bicarbonate and heating (37 C, 1 hour). The resultant peptides were dried down. RapiGest was cleaved again by addition of 100 ul of 0.1% trifluoroacetic acid and heating (37 C, 1 hour). The resultant peptides were dried down and reconstituted a final time in 60 ul of 0.1% trifluoroacetic acid. The samples were centrifuged at 18,800 RCF for 5 minutes (Thermo, Sorvall Legend Micro 21R) to pellet the surfactant. The top 57 ul of solution was transferred into a mass spectrometry (MS) analysis vial.
A 20 ul aliquot of each sample was injected onto an Acquity UPLC HSS T3 column (100 A, 1.8 um, 1 x 150 mm) (Waters Corporation) attached to a UltiMate 3000 ultra-high pressure liquid chromatography (UHPLC) system (Dionex). The peptides were separated and the UHPLC effluent was directly infused into a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (MS) through an electrospray ionization source (Thermo Fisher Scientific). Data were collected in data-dependent MS/MS mode with the top five most abundant ions being selected for fragmentation.
2020-06-24 | MSV000085627 | MassIVE
Project description:Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.
| S-EPMC3308726 | biostudies-literature
Project description:BackgroundIsometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.MethodologyWe used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.ConclusionWe present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.
| S-EPMC2936580 | biostudies-literature
Project description:Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein’s amino terminal (N’)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C delta C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N’-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N’-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.
2021-03-23 | GSE169340 | GEO
Project description:We are investigating the influence of the converter and relay domains on elementary rate constants of the actomyosin cross-bridge cycle. The converter and relay domains vary between Drosophila myosin heavy chain isoforms due to alternative mRNA splicing. Previously, we found that separate insertions of embryonic myosin isoform (EMB) versions of these domains into the indirect flight muscle (IFM) myosin isoform (IFI) both decreased Drosophila IFM power and slowed muscle kinetics. To determine cross-bridge mechanisms behind the changes, we employed sinusoidal analysis while varying phosphate and MgATP concentrations in skinned Drosophila IFM fibers. Based on a six-state cross-bridge model, the EMB converter decreased myosin rate constants associated with actin attachment and work production, k(4), but increased rates related to cross-bridge detachment and work absorption, k(2). In contrast, the EMB relay domain had little influence on kinetics, because only k(4) decreased. The main alteration was mechanical, in that work production amplitude decreased. That both domains decreased k(4) supports the hypothesis that these domains are critical to lever-arm-mediated force generation. Neither domain significantly influenced MgATP affinity. Our modeling suggests the converter domain is responsible for the difference in rate-limiting cross-bridge steps between EMB and IFI myosin--i.e., a myosin isomerization associated with MgADP release for EMB and Pi release for IFI.
| S-EPMC2931743 | biostudies-literature
Project description:In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between cytoplasm and nuclei, and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific transcription and splicing of various sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.
2014-12-23 | GSE63707 | GEO