Elastic proteins in the flight muscle of Manduca sexta (original) (raw)

Localization of the Elastic Proteins in the Flight Muscle of Manduca sexta

International Journal of Molecular Sciences, 2020

The flight muscle of Manduca sexta (DLM1) is an emerging model system for biophysical studies of muscle contraction. Unlike the well-studied indirect flight muscle of Lethocerus and Drosophila, the DLM1 of Manduca is a synchronous muscle, as are the vertebrate cardiac and skeletal muscles. Very little has been published regarding the ultrastructure and protein composition of this muscle. Previous studies have demonstrated that DLM1 express two projectin isoform, two kettin isoforms, and two large Salimus (Sls) isoforms. Such large Sls isoforms have not been observed in the asynchronous flight muscles of Lethocerus and Drosophila. The spatial localization of these proteins was unknown. Here, immuno-localization was used to show that the N-termini of projectin and Salimus are inserted into the Z-band. Projectin spans across the I-band, and the C-terminus is attached to the thick filament in the A-band. The C-terminus of Sls was also located in the A-band. Using confocal microscopy and...

The molecular elasticity of the insect flight muscle proteins projectin and kettin

Proceedings of the National Academy of Sciences, 2006

Projectin and kettin are titin-like proteins mainly responsible for the high passive stiffness of insect indirect flight muscles, which is needed to generate oscillatory work during flight. Here we report the mechanical properties of kettin and projectin by single-molecule force spectroscopy. Force-extension and force-clamp curves obtained from Lethocerus projectin and Drosophila recombinant projectin or kettin fragments revealed that fibronectin type III domains in projectin are mechanically weaker (unfolding force, F u Ϸ 50-150 pN) than Ig-domains (Fu Ϸ 150-250 pN). Among Ig domains in Sls͞kettin, the domains near the N terminus are less stable than those near the C terminus. Projectin domains refolded very fast [85% at 15 s ؊1 (25°C)] and even under high forces (15-30 pN). Temperature affected the unfolding forces with a Q 10 of 1.3, whereas the refolding speed had a Q 10 of 2-3, probably reflecting the cooperative nature of the folding mechanism. High bending rigidities of projectin and kettin indicated that straightening the proteins requires low forces. Our results suggest that titin-like proteins in indirect flight muscles could function according to a folding-based-spring mechanism. force spectroscopy ͉ refolding ͉ single molecule ͉ titin Conflict of interest statement: No conflicts declared.

Mutations that affect flightin expression inDrosophilaalter the viscoelastic properties of flight muscle fibers

American Journal of Physiology-cell Physiology, 2004

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln 0 , a flightin null mutant, 2) Mhc 13 , a myosin rod point mutant with reduced levels of flightin, and 3) Mhc 6 , a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln 0 and Mhc 13 but not in Mhc 6. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln 0 and Mhc 13 but not in Mhc 6. Tension generation under isometric conditions was not impaired in fln 0. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over workproducing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles.

Flight muscle properties and aerodynamic performance of Drosophila expressing a flightin transgene

Journal of Experimental Biology, 2005

The myofibril is a multiprotein structure designed to produce and transmit contractile forces through the interaction of myosin-containing thick filaments and actin-containing thin filaments. In insect indirect flight muscles (IFM), these filaments are organized in a double hexagonal lattice and, as in other striated muscles, are stabilized laterally by structures at the M-line and Z-band. In particular, thick filaments are anchored at the center of the sarcomere through their association with unknown M-line proteins, and connected to the Z-band through projectin and kettin. Neither the composition of IFM thick filaments nor the nature of their assembly has been fully elucidated. In addition to myosin heavy chain (MHC) and its two associated (regulatory and essential) light chains, paramyosin/mini-paramyosin and flightin have been shown to be essential for normal thick filament structure and function (Arredondo et al., 2001; Liu et al., 2003; Reedy et al., 2000). Electron microscopy studies have provided insight into the precise and ordered manner by which the myofilament lattice of Drosophila IFM is assembled throughout development (Reedy and Beall, 1993; Vigoreaux and Swank, 2004). Genetic approaches have been instrumental in elucidating the role of myofibrillar proteins on sarcomere assembly and muscle structure stability (for reviews see: Bernstein et al., 1993; Cripps, 2004; Vigoreaux, 2001). In particular, Mhc gene mutants have provided insight into the role of MHC protein domains in flight muscle development and function (for reviews see: Miller and Bernstein, 2004; Swank et al., 2000). Analysis of flightin gene mutants also have shown that flightin plays an essential role in thick filament formation and

Flight muscle properties and aerodynamic performance ofDrosophilaexpressing aflightintransgene

The Journal of Experimental Biology, 2005

A Genetic Deficiency That Spans the Flightin Gene of Drosophila Melanogaster affects the Ultrastructure and Function of the Flight Muscles

The Journal of Experimental Biology, 1998

Striated muscle cells contain a highly ordered, threedimensional cytoskeletal network specialized for the generation and transmission of contractile forces. Myosincontaining thick filaments and actin-containing thin filaments are organized as interdigitating arrays that allow the cyclical interaction of myosin with actin, thus producing the force for work. The spatial arrangement of contractile myofilaments is dictated in part by two cross-linking structures, the M line and the Z band, whose variation in structure in part reflects tissue and/or fiber-specific differences in muscle properties (Squire, 1986; Schachat et al. 1985; Vigoreaux, 1994). The assembly and stability of the sarcomeric lattice results from diverse but poorly defined molecular interactions among structural proteins. Temporal scaffolding structures (e.g. sleeves of microtubules, Reedy and Beall, 1993) have been shown to participate in muscle fiber assembly but play no role in defining the structural stability of the actively contracting fiber. Contracting muscle cells undergo continuous changes in cell shape. A highly dynamic cytoskeleton well-suited to respond and adjust to internally derived tension and externally applied stress underlies these changes. Although the mechanism for generating contractile force has received a lot of attention, the ability of the muscle cell to contract and return to its original shape unscathed is often taken for granted. As a result, we know very little about the mechanism for preserving cellular integrity and of the molecular interactions that define the dynamic nature of the cytoskeleton. Genetic approaches have proved exceedingly useful for the analysis of muscle fiber assembly and muscle function (for reviews, see Epstein and Fischman, 1991; Bernstein et al. 1993). The indirect flight muscles (IFMs) of Drosophila melanogaster are well suited for genetic studies because the effects of a mutation can be studied in vivo as well as in situ using a combination of well-established functional, ultrastructural, mechanical and biochemical assays (Tohtong et al. 1995; Kreuz et al. 1996; Dickinson et al. 1997). Several mutations in contractile protein genes have been identified that appear to have little or no effect on myofibril assembly but which lead to rapid degeneration of the adult muscle. For example, Kronert et al. (1995) reported the characterization of three missense alleles of the myosin heavy chain gene that cause single amino acid changes in the light meromyosin region of the rod. None of the mutations appears to interfere with the normal assembly of myofibrils, but all three lead to sarcomeric degeneration as the adult ages (Kronert et al. 1995). An age-dependent degeneration of flight muscle has also been 2033

Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles

The Journal of Cell Biology, 1993

The indirect flight muscles of Drosophila are adapted for rapid oscillatory movements which depend on properties of the contractile apparatus itself. Flight muscles are stretch activated and the frequency of contraction in these muscles is independent of the rate of nerve impulses. Little is known about the molecular basis of these adaptations. We now report a novel protein that is found only in flight muscles and has, therefore, been named flightin. Although we detect only one gene (in polytene region 76D) for flightin, this protein has several isoforms (relative gel mobilities, 27-30 kD; pIs, 4.6-6.0). These isoforms appear to be created by posttranslational modifications. A subset of these isoforms is absent in newly emerged adults but appears when the adult develops the ability to fly. In intact muscles flightin is associated with the A band of the sarcomere, where evidence suggests it interacts with the myosin filaments. Computer database searches do not reveal extensive simila...

Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers

AJP: Cell Physiology, 2003

The Contributions of the Amino and Carboxy Terminal Domains of Flightin to the Biomechanical Properties of Drosophila Flight Muscle Thick Filaments

Biology, 2016

Flightin is a myosin binding protein present in Pancrustacea. In Drosophila, flightin is expressed in the indirect flight muscles (IFM), where it is required for the flexural rigidity, structural integrity, and length determination of thick filaments. Comparison of flightin sequences from multiple Drosophila species revealed a tripartite organization indicative of three functional domains subject to different evolutionary constraints. We use atomic force microscopy to investigate the functional roles of the N-terminal domain and the C-terminal domain that show different patterns of sequence conservation. Thick filaments containing a C-terminal domain truncated flightin (fln ∆C44) are significantly shorter (2.68˘0.06 µm; p < 0.005) than thick filaments containing a full length flightin (fln + ; 3.21˘0.05 µm) and thick filaments containing an N-terminal domain truncated flightin (fln ∆N62 ; 3.21˘0.06 µm). Persistence length was significantly reduced in fln ∆N62 (418˘72 µm; p < 0.005) compared to fln + (1386˘196µm) and fln ∆C44 (1128˘193 µm). Statistical polymer chain analysis revealed that the C-terminal domain fulfills a secondary role in thick filament bending propensity. Our results indicate that the flightin amino and carboxy terminal domains make distinct contributions to thick filament biomechanics. We propose these distinct roles arise from the interplay between natural selection and sexual selection given IFM's dual role in flight and courtship behaviors.

Mechanics and protein content of insect flight muscles

Journal of …, 1992

... 57 Printed in Great Britain © The Company of Biologists Limited 1992 MECHANICS AND PROTEIN CONTENT OF INSECT FLIGHT MUSCLES BY MICHELLE PECKHAM*, RICHARD CRIPPSt, DAVID WHITE Department of Biology, University of York, York, Y01 5DD ...

Elastic proteins in the flight muscle of Manduca sexta (original) (raw)

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