Abstract
Projectin is a ca. 900 kDa protein that is a member of the titin protein superfamily. In skeletal muscle titins are involved in the longitudinal reinforcement of the sarcomere by connecting the Z-band to the M-line. In insect indirect flight muscle (IFM), projectin is believed to form the connecting filaments that link the Z-band to the thick filaments and is responsible for the high relaxed stiffness found in this muscle type. The Drosophila mutant bent D (bt D) has been shown to have a breakpoint close to the carboxy-terminal kinase domain of the projectin sequence. Homozygotes for bt D are embryonic lethal but heterozygotes (bt D/+) are viable. Here we show that bt D/+ flies have normal flight ability and a slightly elevated wing beat frequency (bt D/+ 223 ± 13 Hz; +/+203 ± 5 Hz, mean ± SD; P < 0.01). Electron microscopy of bt D/+ IFM show normal ultrastructure but skinned fiber mechanics show reduced stretch activation and oscillatory work. Although bt D/+ IFM power output was at wild-type levels, maximum power was achieved at a higher frequency of applied length perturbation (bt D/+ 151 ± 6 Hz; +/+ 102 ± 14 Hz; P < 0.01). Results were interpreted in the context of a viscoelastic model of the sarcomere and indicate altered cross-bridge kinetics of the power-producing step. These results show that the bt D mutation reduces oscillatory work in a way consistent with the proposed role of the connecting filaments in the stretch activation response of IFM.
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Abbott RH and Cage PE (1984) A possible mechanism of length activation in insect fibrillar flight muscle. J Muscle Res Cell Motil 5: 387-397.
Ayme-Southgate A, Southgate R, Saide J, Benian GM and Pardue ML (1995) Both Synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains. J Cell Biol. 128: 393-403.
Benian GM, Kiff JE, Neckelman N, Moerman DG and Waterson RH (1989) Sequence of an unusually large protein implicated in regulation of myosin activity in C. elegans. Nature 342: 45-50.
Bernstein SI, O'Donnell PT and Cripps RM (1993) Molecular genetic analysis of muscle development, structure, and function in Drosophila. Int Rev Cytol 143: 63-152.
Daley J, Southgate R and Ayme-Southgate A (1998) Structure of the Drosophila projectin protein: isoforms and implication for projectin filament assembly. J Mol Biol 279: 201-210.
Dibb NJ, Brown DM, Karn J, Moerman DG, Bolten SL and Waterston RH (1985) Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans. Mol Biol 183: 543-551.
Dickinson MH, Hyatt CJ, Lehmann F, Moore JR, Reedy MC, Simcox A, Tohtong R, Vigoreaux JO, Yamashita H and Maughan DW (1997) Phosphorylation-dependent power output of transgenic flies: an integrated study. Biophys J 73: 3122-3134.
Gautel M (1996) The super repeats of titin/connectin and their interactions: glimpses at sarcomeric assembly. Adv Biophys 33: 27-37.
Granzier HLM and Wang K (1993) Interplay between passive tension and strong and weak binding crossbridges in insect flight muscle: a functional dissection by gelsolin-mediated thin filament removal. J Gen Physiol 101: 235-270.
Hierhorst J, Kobe B, Feil SC, Parker MW, Benian GM, Weiss KR and Kemp BE (1996) Ca2./S100 regulation of giant protein kinases. Nature 380: 636-639.
Hierhorst J, Probst WC, Kohanski RA, Buku A and Weiss KR (1995a) Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase. Eur J Biochem 233: 426-431.
Hierhorst J, Probst WC, Vilim FS, Buku A and Weiss KR (1995b) Autophosphorylation of molluscan twitchin kinase and interaction of its kinase domain with calcium calmodulin. Eur J Biol Chem 269: 21086-21093.
Horowitz R and Podolsky RJ (1987) The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments. J Cell Biol 105: 2217-2223.
Hu SH, Parker MW, Lei JY, Wilce MC, Benian GM and Kemp BE (1994) Insights into autoregulation from the crystal structure of twitchin kinase. Nature 369: 581-584.
Huxley A (1974) Muscular contraction. J Physiol (Lond). 243: 1-43.
Hyatt CJ and Maughan DW (1994) Fourier analysis of wing beat signals: assessing the effects of genetic alterations of flight muscle structure in Diptera. Biophys J 67: 1149-1154.
Kammer AE and Heinrich B (1978) Insect flight metabolism. Adv Insect Physiol 13: 133-228.
Kawai M and Brandt PW (1980) Sinusoidal analysis: a high resolution method for correlating biochemical reactions with physiological processes in activated skeletal muscle of rabbit, frog and crayfish. J Muscle Res Cell Motil 1: 279-303.
Kobe B, Hierhorst J, Feil SC, Parker MW, Benian GM, Weiss KR and Kemp BE (1996) Giant protein kinases: domain interactions and structural basis of autoregulation. EMBO J 15: 6810-6821.
Kolsch B, Ziegler C and Beinbrech G (1995) Length determination of synthetic thick filaments by cooperation of two myosin-associated proteins, paramyosin and projectin. Naturwissenschaften 82: 239-241.
Lindsley DL and Zimm GG (1992) The Genome of Drosophila melanogaster, Academic Press, New York.
Machado C, Sunkel CE and Andrew DJ (1998) Human autoantibodies reveal titin as a chromosomal protein. J Cell Biol 141: 321-333.
Maroto M, Vinos J, Marco R and Cervera M (1992) Autophos-phorylating protein kinase activity in titin-like arthropod projectin. J Mol Biol 224: 287-291.
Messerli JM and Perriard JC (1995) Three-dimensional analysis and visualization of myofibrillogenesis in adult cardiomyocytes by confocal microscopy. Microsc Res Tech 30: 521-530.
Moerman DG, Plurad S, Waterston RH and Baillie DL (1982) Mutations in the unc-54 myosin heavy chain gene of Caenorhab-ditis elegans that alter contractility but not muscle structure. Cell 29: 773-781.
Noguchi J, Yanagisawa M, Imamura M, Kasuya Y, Sakurai T, Tanaka T and Masaki T (1992) Complete primary structure and tissue expression of chicken pectoralis M-protein J Biol Chem 267: 20302-20310.
Pringle JWS (1949) The excitation and contraction of the flight muscles of insects. J Physiol 108: 226-232.
Pringle JWS (1978) Stretch activation of muscle: function and mechanism. Proc R Soc Lond B 201: 107-130.
Probst WC, Cropper EC, Hierhorst J, Hooper SL, Jaffe H, Vilim F, Beuhausen S, Kupferman I and Weiss KR (1994) cAMP-depen-dent phosphorylation of Aplysia twitchin may mediate modulation of muscle contractions by neuropeptide cotransmitters. Proc Natl Acad Sci USA 91: 8487-8491.
Reedy MCand Beall C(1993) Ultrastructure of developing flight muscle in Drosophila. I. Assembly of myofibrils. Dev Biol 160: 443-465.
Reedy MC, Reedy MK, Leonard KR and Bullard B (1994) Gold/Fab immunoelectron microscopy localization of TnH and TnT in Lethocerus flight muscle. J Mol Biol 239: 52-67.
Royuela M, Fraile B, Paz De Miguel M, Cervera M and Paniagua R (1996) Immunohistochemical study and western blotting analysis of titin-like proteins in the striated muscle of Drosophila melano-gaster and in the striated and smooth muscle of the oligochaete Eisenia foetida. Mic Res Tech 35: 349-356.
Saide JS, Chin-Bow S, Hogan-Sheldon J, Busquets-Turner L, Vigor-eaux JO, Valgeirsdottir K and Pardue ML (1989) Characterization of components of Z-bands in the fibrillar ¯ight muscle of Drosophila melanogaster. J Cell Biol 109: 2157-2167.
Siegman MJ, Mooers SU, Li C, Narayan S, Trinkle-Mulcahy L, Watabe S, Hartshorne DJ and Butler TM (1997) Phosphorylation of a high molecular weight (600 kDa) protein regulates catch in invertebrate smooth muscle. J Muscle Res Cell Motil 18: 655-670.
Steiger GJ (1977) Stretch activation and tension transients in cardiac, skeletal and insect flight muscle. In: Tregear RT (ed.) Insect Flight Muscle (pp. 533-547.) North-Holland Publishing, Amsterdam.
Thorson and White (1983) Role of crossbridge distortion in the small signal dynamics of insect and rabbit striated muscle. J Physiol 343: 59-84.
Tohtong R, Rodriguez D, Maughan DM and Simcox A (1997) Analysis of cDNAs encoding Drosophila melanogaster myosin light chain kinase. J Muscle Res Cell Motil 18: 43-56.
Tohtong R, Yamashita H, Graham M, Haeberle J, Simcox A and Maughan D (1995) Impairment of muscle function caused by mutations of the phoshorylation sites in myosin regulatory light chain. Nature 374: 650-653.
Trinick J (1994) Titin and nebulin: protein rulers in muscle? Trends Biochem Sci 19: 405-408.
Trinick J (1996) Interaction of titin/connectin with the thick filament. Adv Biophys 33: 81-90.
Vigoreaux JO, Saide JS and Pardue ML (1991) Structurally distinct Drosophila striated muscles utilize distinct variants of Z-band associated proteins. J Muscle Res Cell Motil 12: 340-354.
Vigoreaux JO and Perry L (1994) Multiple isoelectric variants of flightin in Drosophila stretch-activated muscles are generated by temporally regulated phosphorylations. J Muscle Res Cell Motil 15: 607-616.
Vigoreaux JO, Hernandez C, Moore J, Ayer G and Maughan DW (1998) The effect of a genetic deficiency that spans the Drosophila flightin gene on the ultrastructure and function of the flight muscles. J Exp Biol 201: 2033-2044.
Vinkemeier U, Obermann W, Weber K and Furst DO (1993) The globular head domain of titin extends into the center of the sarcomeric M band. cDNA cloning, epitope mapping and immunoelectron microscopy of two titin-associated proteins. J Cell Sci 106 (Pt 1): 319-330.
Weitkamp B, Jurk K and Beinbrech G (1998) Projectin thin filament interaction and modulation of the sensitivity of the actomyosin ATPase to calcium by projectin kinase. J Biol Chem 273: 19802-19808.
White DCS (1983) The elasticity of relaxed insect flight muscle. J Physiol 343: 31-57.
Wray JS (1979) Filament geometry and the activation of insect flight muscle. Nature 280: 325-326.
Zhao Y and Kawai M (1993) The effect of lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers. II. Elementary steps affected by the spacing change. Biophys J 64: 197-210.
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Moore, J.R., Vigoreaux, J.O. & Maughan, D.W. The Drosophila projectin mutant, bent D, has reduced stretch activation and altered indirect flight muscle kinetics. J Muscle Res Cell Motil 20, 797–806 (1999). https://doi.org/10.1023/A:1005607818302
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DOI: https://doi.org/10.1023/A:1005607818302