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In JoVE (2)
- Fluorescent Labeling of Drosophila Heart Structures
- Semi-automated Optical Heartbeat Analysis of Small Hearts
Other Publications (14)
- Biophysical Journal
- Journal of Muscle Research and Cell Motility
- Journal of Molecular Biology
- Journal of Molecular Biology
- Journal of Molecular Biology
- Molecular Biology of the Cell
- Cell
- Biochemical and Biophysical Research Communications
- Biophysical Journal
- Biochemical and Biophysical Research Communications
- Biophysical Journal
- PloS One
- Journal of Molecular Biology
- Journal of Cellular and Molecular Medicine
Articles by Anthony Cammarato in JoVE
JoVE General
Fluorescent Labeling of Drosophila Heart Structures
1Biology Department, San Diego State University, 2Development and Aging Program, NASCR Center, The Sanford Burnham Institute for Medical Research
Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.
JoVE General
Semi-automated Optical Heartbeat Analysis of Small Hearts
1Development and Aging Program, The Sanford Burnham Institute for Medical Research, 2Cardiac Electrophysiology Group, Dept. of Physiology, Anatomy and Genetics, The Sanford Burnham Institute for Medical Research, 3Biology Department and Heart Institute, San Diego State University
We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.
Other articles by Anthony Cammarato on PubMed
Drosophila Muscle Regulation Characterized by Electron Microscopy and Three-dimensional Reconstruction of Thin Filament Mutants
Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.
In Indirect Flight Muscles Drosophila Projectin Has a Short PEVK Domain, and Its NH2-terminus is Embedded at the Z-band
Insect indirect flight muscles (IFM) contain a third filament system made up of elastic connecting or C-filaments. The giant protein projectin is the main, if not the only, component of these structures. In this study we found that projectin is oriented within the IFM sarcomere with its NH2-terminus embedded in the Z-bands. We demonstrate that this protein has an elastic region that can be detected by the movement of specific epitopes following stretch. One possible elastic region is the PEVK-like domain located close to the NH2-terminus. The amino acid length of this region is short, and 52% of its residues are P, E, V or K. We propose a model in which projectin extends from the Z-band to the lateral borders of the A-band. The PEVK-like domain and a series of Ig domains spanning the intervening I-band may provide the elastic properties of projectin.
E93K Charge Reversal on Actin Perturbs Steric Regulation of Thin Filaments
Contraction in striated muscles is regulated by Ca2+-dependent movement of tropomyosin-troponin on thin filaments. Interactions of charged amino acid residues between the surfaces of tropomyosin and actin are believed to play an integral role in this steric mechanism by influencing the position of tropomyosin on the filaments. To investigate this possibility further, thin filaments were isolated from troponin-regulated, indirect flight muscles of Drosophila mutants that express actin with an amino acid charge reversal at residue 93 located at the interface between actin subdomains 1 and 2, in which a lysine residue is substituted for a glutamic acid. Electron microscopy and 3D helical reconstruction were employed to evaluate the structural effects of the mutation. In the absence of Ca2+, tropomyosin was in a position that blocked the myosin-binding sites on actin, as previously found with wild-type filaments. However, in the presence of Ca2+, tropomyosin position in the mutant filaments was much more variable than in the wild-type ones. In most cases (approximately 60%), tropomyosin remained in the blocking position despite the presence of Ca2+, failing to undergo a normal Ca2+-induced change in position. Thus, switching of a negative to a positive charge at position 93 on actin may stabilize negatively charged tropomyosin in the Ca2+-free state regardless of Ca2+ levels, an alteration that, in turn, is likely to interfere with steric regulation and consequently muscle activation. These results highlight the importance of actin's surface charges in determining the distribution of tropomyosin positions on thin filaments derived from troponin-regulated striated muscles.
AlphaB-crystallin Maintains Skeletal Muscle Myosin Enzymatic Activity and Prevents Its Aggregation Under Heat-shock Stress
Here, we provide functional and direct structural evidence that alphaB-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alphaB-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis, maintaining cytoskeletal integrity and sustaining muscle performance, since heat-shock temperatures can be produced during multiple stress conditions or vigorous exercise.
Alternative S2 Hinge Regions of the Myosin Rod Differentially Affect Muscle Function, Myofibril Dimensions and Myosin Tail Length
Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin "hinge" region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial "melting" of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was approximately 5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus, alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed.
Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles
Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.
A Global in Vivo Drosophila RNAi Screen Identifies NOT3 As a Conserved Regulator of Heart Function
Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.
Drosophila UNC-45 Prevents Heat-induced Aggregation of Skeletal Muscle Myosin and Facilitates Refolding of Citrate Synthase
UNC-45 belongs to the UCS (UNC-45, CRO1, She4p) domain protein family, whose members interact with various classes of myosin. Here we provide structural and biochemical evidence that Escherichia coli-expressed Drosophila UNC-45 (DUNC-45) maintains the integrity of several substrates during heat-induced stress in vitro. DUNC-45 displays chaperone function in suppressing aggregation of the muscle myosin heavy meromyosin fragment, the myosin S-1 motor domain, alpha-lactalbumin and citrate synthase. Biochemical evidence is supported by electron microscopy, which reveals the first structural evidence that DUNC-45 prevents inter- or intra-molecular aggregates of skeletal muscle heavy meromyosin caused by elevated temperatures. We also demonstrate for the first time that UNC-45 is able to refold a denatured substrate, urea-unfolded citrate synthase. Overall, this in vitro study provides insight into the fate of muscle myosin under stress conditions and suggests that UNC-45 protects and maintains the contractile machinery during in vivo stress.
Electron Microscopy and Persistence Length Analysis of Semi-rigid Smooth Muscle Tropomyosin Strands
The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.
Electron Microscopy and Three-dimensional Reconstruction of Native Thin Filaments Reveal Species-specific Differences in Regulatory Strand Densities
Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin-tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are approximately 20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin-tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands' length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.
In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium
Drosophila melanogaster is a genetically malleable organism with a short life span, making it a tractable system in which to study mechanical effects of genetic perturbation and aging on tissues, e.g., impaired heart function. However, Drosophila heart-tube studies can be hampered by its bilayered structure: a ventral muscle layer covers the contractile cardiomyocytes. Here we propose an atomic force microscopy-based analysis that uses a linearized-Hertz method to measure individual mechanical components of soft composite materials. The technique was verified using bilayered polydimethylsiloxane. We further demonstrated its biological utility via its ability to resolve stiffness changes due to RNA interference to reduce myofibrillar content or due to aging in Drosophila myocardial layers. This protocol provides a platform to assess the mechanics of soft biological composite systems and, to our knowledge, for the first time, permits direct measurement of how genetic perturbations, aging, and disease can impact cardiac function in situ.
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, Ca(2+)-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.
Structural Basis for Myopathic Defects Engendered by Alterations in the Myosin Rod
While mutations in the myosin subfragment 1 motor domain can directly disrupt the generation and transmission of force along myofibrils and lead to myopathy, the mechanism whereby mutations in the myosin rod influences mechanical function is less clear. Here, we used a combination of various imaging techniques and molecular dynamics simulations to test the hypothesis that perturbations in the myosin rod can disturb normal sarcomeric uniformity and, like motor domain lesions, would influence force production and propagation. We show that disrupting the rod can alter its nanomechanical properties and, in vivo, can drive asymmetric myofilament and sarcomere formation. Our imaging results indicate that myosin rod mutations likely disturb production and/or propagation of contractile force. This provides a unifying theory where common pathological cascades accompany both myosin motor and specific rod domain mutations. Finally, we suggest that sarcomeric inhomogeneity, caused by asymmetric thick filaments, could be a useful index of myopathic dysfunction.
Measuring Passive Myocardial Stiffness in Drosophila Melanogaster to Investigate Diastolic Dysfunction
Aging is marked by a decline in left ventricular diastolic function, which encompasses abnormalities in diastolic relaxation, chamber filling and/or passive myocardial stiffness. Genetic tractability and short life span make Drosophila melanogaster an ideal organism to study the effects of aging on heart function, including senescent-associated changes in gene expression and in passive myocardial stiffness. However, use of the Drosophila heart tube to probe deterioration of diastolic performance is subject to at least two challenges: the extent of genetic homology to mammals and the ability to resolve mechanical properties of the bilayered fly heart, which consists of a ventral muscle layer that covers the contractile cardiomyocytes. Here we argue for wide-spread use of Drosophila as a novel myocardial aging model by 1) describing diastolic dysfunction in flies, 2) discussing how critical pathways involved in dysfunction are conserved across species, and 3) demonstrating the advantage of an atomic force microscopy-based analysis method to measure stiffness of the multilayered Drosophila heart tube versus isolated myocytes from other model systems. By using powerful Drosophila genetic tools we aim to efficiently alter changes observed in factors that contribute to diastolic dysfunction to understand how one might improve diastolic performance at advanced ages in humans. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
