Abstract
We have determined the molecular defect of the Drosophila melanogaster myosin heavy chain (MHC) mutation Mhc and the mutation's effect on indirect flight muscle, jump muscle, and larval intersegmental muscle. We show that the Mhc1 mutation is essentially a null allele which results in the dominant-flightless and recessive-lethal phenotypes associated with this mutant (Mogami, K., P. T. O'Donnell, S. I. Bernstein, T. R. F. Wright, C. P. Emerson, Jr. 1986. Proc. Natl. Acad. Sci. USA. 83:1393-1397). The mutation is a 101-bp deletion in the MHC gene which removes most of exon 5 and the intron that precedes it. S1 nuclease mapping indicates that mutant transcripts follow two alternative processing pathways. Both pathways result in the production of mature transcripts with altered reading frames, apparently yielding unstable, truncated MHC proteins. Interestingly, the preferred splicing pathway uses the more distal of two available splice donor sites. We present the first ultrastrutural characterization of a completely MHC- null muscle and show that it lacks any discernable thick filaments. Sarcomeres in these muscles are completely disorganized suggesting that thick filaments play a critical role in sarcomere assembly. To understand why the Mhc1 mutation severely disrupts indirect flight muscle and jump muscle function in heterozygotes, but does not seriously affect the function of other muscle types, we examined the muscle ultrastructure of Mhc1/+ heterozygotes. We find that these organisms have a nearly 50% reduction in the number of thick filaments in indirect flight muscle, jump muscle, and larval intersegmental muscle. In addition, aberrantly shaped thick filaments are common in the jump muscle and larval intersegmental muscle. We suggest that the differential sensitivity of muscle function to the Mhc1 mutation is a consequence of the unique myofilament arrays in each of these muscles. The highly variable myofilament array of larval intersegmental muscle makes its function relatively insensitive to changes in thick filament number and morphology. Conversely, the rigid double hexagonal lattice of the indirect flight muscle, and the organized lattice of the jump muscle cannot be perturbed without interfering with the specialized and evolutionarily more complex functions they perform.
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- Bernstein S. I., Hansen C. J., Becker K. D., Wassenberg D. R., 2nd, Roche E. S., Donady J. J., Emerson C. P., Jr Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain. Mol Cell Biol. 1986 Jul;6(7):2511–2519. doi: 10.1128/mcb.6.7.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bernstein S. I., Mogami K., Donady J. J., Emerson C. P., Jr Drosophila muscle myosin heavy chain encoded by a single gene in a cluster of muscle mutations. 1983 Mar 31-Apr 6Nature. 302(5907):393–397. doi: 10.1038/302393a0. [DOI] [PubMed] [Google Scholar]
- Bullard B., Luke B., Winkelman L. The paramyosin of insect flight muscle. J Mol Biol. 1973 Apr 5;75(2):359–367. doi: 10.1016/0022-2836(73)90026-0. [DOI] [PubMed] [Google Scholar]
- Dibb N. J., Brown D. M., Karn J., Moerman D. G., Bolten S. L., Waterston R. H. Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans. J Mol Biol. 1985 Jun 25;183(4):543–551. doi: 10.1016/0022-2836(85)90170-6. [DOI] [PubMed] [Google Scholar]
- Epstein H. F., Miller D. M., 3rd, Ortiz I., Berliner G. C. Myosin and paramyosin are organized about a newly identified core structure. J Cell Biol. 1985 Mar;100(3):904–915. doi: 10.1083/jcb.100.3.904. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Falkenthal S., Parker V. P., Mattox W. W., Davidson N. Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains. Mol Cell Biol. 1984 May;4(5):956–965. doi: 10.1128/mcb.4.5.956. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garamvölgyi N. The arrangement of the myofilaments in the insect flight muscle. I. J Ultrastruct Res. 1965 Dec;13(5):409–424. doi: 10.1016/s0022-5320(65)90004-3. [DOI] [PubMed] [Google Scholar]
- Garamvölgyi N. The arrangement of the myofilaments in the insect flight muscle. II. J Ultrastruct Res. 1965 Dec;13(5):425–434. doi: 10.1016/s0022-5320(65)90005-5. [DOI] [PubMed] [Google Scholar]
- Garcea R. L., Schachat F., Epstein H. F. Coordinate synthesis of two myosins in wild-type and mutant nematode muscle during larval development. Cell. 1978 Oct;15(2):421–428. doi: 10.1016/0092-8674(78)90011-9. [DOI] [PubMed] [Google Scholar]
- Hozumi T., Muhlrad A. Reactive lysyl of myosin subfragment 1: location on the 27K fragment and labeling properties. Biochemistry. 1981 May 12;20(10):2945–2950. doi: 10.1021/bi00513a035. [DOI] [PubMed] [Google Scholar]
- Karlik C. C., Coutu M. D., Fyrberg E. A. A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles. Cell. 1984 Oct;38(3):711–719. doi: 10.1016/0092-8674(84)90266-6. [DOI] [PubMed] [Google Scholar]
- Karlik C. C., Fyrberg E. A. An insertion within a variably spliced Drosophila tropomyosin gene blocks accumulation of only one encoded isoform. Cell. 1985 May;41(1):57–66. doi: 10.1016/0092-8674(85)90061-3. [DOI] [PubMed] [Google Scholar]
- Labbé J. P., Mornet D., Roseau G., Kassab R. Cross-linking of F-actin to skeletal muscle myosin subfragment 1 with bis(imido esters): further evidence for the interaction of myosin-head heavy chain with an actin dimer. Biochemistry. 1982 Dec 21;21(26):6897–6902. doi: 10.1021/bi00269a042. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Landel C. P., Krause M., Waterston R. H., Hirsh D. DNA rearrangements of the actin gene cluster in Caenorhabditis elegans accompany reversion of three muscle mutants. J Mol Biol. 1984 Dec 15;180(3):497–513. doi: 10.1016/0022-2836(84)90024-x. [DOI] [PubMed] [Google Scholar]
- MacLeod A. R., Waterston R. H., Brenner S. An internal deletion mutant of a myosin heavy chain in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5336–5340. doi: 10.1073/pnas.74.12.5336. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacLeod A. R., Waterston R. H., Fishpool R. M., Brenner S. Identification of the structural gene for a myosin heavy-chain in Caenorhabditis elegans. J Mol Biol. 1977 Jul;114(1):133–140. doi: 10.1016/0022-2836(77)90287-x. [DOI] [PubMed] [Google Scholar]
- Mackenzie J. M., Jr, Garcea R. L., Zengel J. M., Epstein H. F. Muscle development in Caenorhabditis elegans: mutants exhibiting retarded sarcomere construction. Cell. 1978 Nov;15(3):751–762. doi: 10.1016/0092-8674(78)90261-1. [DOI] [PubMed] [Google Scholar]
- Mahaffey J. W., Coutu M. D., Fyrberg E. A., Inwood W. The flightless Drosophila mutant raised has two distinct genetic lesions affecting accumulation of myofibrillar proteins in flight muscles. Cell. 1985 Jan;40(1):101–110. doi: 10.1016/0092-8674(85)90313-7. [DOI] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
- McLachlan A. D. Analysis of gene duplication repeats in the myosin rod. J Mol Biol. 1983 Sep 5;169(1):15–30. doi: 10.1016/s0022-2836(83)80173-9. [DOI] [PubMed] [Google Scholar]
- McLachlan A. D., Karn J. Periodic charge distributions in the myosin rod amino acid sequence match cross-bridge spacings in muscle. Nature. 1982 Sep 16;299(5880):226–231. doi: 10.1038/299226a0. [DOI] [PubMed] [Google Scholar]
- McLachlan A. D., Karn J. Periodic features in the amino acid sequence of nematode myosin rod. J Mol Biol. 1983 Mar 15;164(4):605–626. doi: 10.1016/0022-2836(83)90053-0. [DOI] [PubMed] [Google Scholar]
- Mogami K., Hotta Y. Isolation of Drosophila flightless mutants which affect myofibrillar proteins of indirect flight muscle. Mol Gen Genet. 1981;183(3):409–417. doi: 10.1007/BF00268758. [DOI] [PubMed] [Google Scholar]
- Mogami K., O'Donnell P. T., Bernstein S. I., Wright T. R., Emerson C. P., Jr Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1393–1397. doi: 10.1073/pnas.83.5.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nachtigall W., Wilson D. M. Neuro-muscular control of dipteran flight. J Exp Biol. 1967 Aug;47(1):77–97. doi: 10.1242/jeb.47.1.77. [DOI] [PubMed] [Google Scholar]
- Parker V. P., Falkenthal S., Davidson N. Characterization of the myosin light-chain-2 gene of Drosophila melanogaster. Mol Cell Biol. 1985 Nov;5(11):3058–3068. doi: 10.1128/mcb.5.11.3058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parry D. A. Structure of rabbit skeletal myosin. Analysis of the amino acid sequences of two fragments from the rod region. J Mol Biol. 1981 Dec 5;153(2):459–464. doi: 10.1016/0022-2836(81)90290-4. [DOI] [PubMed] [Google Scholar]
- Pringle J. W. The contractile mechanism of insect fibrillar muscle. Prog Biophys Mol Biol. 1967;17:1–60. doi: 10.1016/0079-6107(67)90003-x. [DOI] [PubMed] [Google Scholar]
- Reed R., Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. doi: 10.1016/0092-8674(86)90343-0. [DOI] [PubMed] [Google Scholar]
- Rozek C. E., Davidson N. Differential processing of RNA transcribed from the single-copy Drosophila myosin heavy chain gene produces four mRNAs that encode two polypeptides. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2128–2132. doi: 10.1073/pnas.83.7.2128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rozek C. E., Davidson N. Drosophila has one myosin heavy-chain gene with three developmentally regulated transcripts. Cell. 1983 Jan;32(1):23–34. doi: 10.1016/0092-8674(83)90493-2. [DOI] [PubMed] [Google Scholar]
- SHAFIQ S. A. Electron microscopic studies on the indirect flight muscles of Drosophila melanogaster. I. Structure of the myofibrils. J Cell Biol. 1963 May;17:351–362. doi: 10.1083/jcb.17.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
- SHAFIQ S. A. Electron microscopic studies on the indirect flight muscles of Drosophila melanogaster. II. Differentiation of myofibrils. J Cell Biol. 1963 May;17:363–373. doi: 10.1083/jcb.17.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spurr A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969 Jan;26(1):31–43. doi: 10.1016/s0022-5320(69)90033-1. [DOI] [PubMed] [Google Scholar]
- Sutoh K. Identification of myosin-binding sites on the actin sequence. Biochemistry. 1982 Jul 20;21(15):3654–3661. doi: 10.1021/bi00258a020. [DOI] [PubMed] [Google Scholar]
- Szilagyi L., Balint M., Sreter F. A., Gergely J. Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin. Biochem Biophys Res Commun. 1979 Apr 13;87(3):936–945. doi: 10.1016/0006-291x(79)92047-3. [DOI] [PubMed] [Google Scholar]
- Thorson J., White D. C. Distributed representations for actin-myosin interaction in the oscillatory contraction of muscle. Biophys J. 1969 Mar;9(3):360–390. doi: 10.1016/S0006-3495(69)86392-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toselli P. A., Pepe F. A. The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. I. Muscle structure at molting. J Cell Biol. 1968 May;37(2):445–461. doi: 10.1083/jcb.37.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toselli P. A., Pepe F. A. The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. II. Muscle changes in preparation for molting. J Cell Biol. 1968 May;37(2):462–481. doi: 10.1083/jcb.37.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wassenberg D. R., 2nd, Kronert W. A., O'Donnell P. T., Bernstein S. I. Analysis of the 5' end of the Drosophila muscle myosin heavy chain gene. Alternatively spliced transcripts initiate at a single site and intron locations are conserved compared to myosin genes of other organisms. J Biol Chem. 1987 Aug 5;262(22):10741–10747. [PubMed] [Google Scholar]
- Waterston R. H., Fishpool R. M., Brenner S. Mutants affecting paramyosin in Caenorhabditis elegans. J Mol Biol. 1977 Dec 15;117(3):679–697. doi: 10.1016/0022-2836(77)90064-x. [DOI] [PubMed] [Google Scholar]
- Waterston R. H., Hirsh D., Lane T. R. Dominant mutations affecting muscle structure in Caenorhabditis elegans that map near the actin gene cluster. J Mol Biol. 1984 Dec 15;180(3):473–496. doi: 10.1016/0022-2836(84)90023-8. [DOI] [PubMed] [Google Scholar]
- Waterston R. H., Smith K. C., Moerman D. G. Genetic fine structure analysis of the myosin heavy chain gene unc-54 of Caenorhabditis elegans. J Mol Biol. 1982 Jun 15;158(1):1–15. doi: 10.1016/0022-2836(82)90447-8. [DOI] [PubMed] [Google Scholar]
- Waterston R. H., Thomson J. N., Brenner S. Mutants with altered muscle structure of Caenorhabditis elegans. Dev Biol. 1980 Jun 15;77(2):271–302. doi: 10.1016/0012-1606(80)90475-3. [DOI] [PubMed] [Google Scholar]
- Weeds A. G. Light chains of myosin. Nature. 1969 Sep 27;223(5213):1362–1364. doi: 10.1038/2231362a0. [DOI] [PubMed] [Google Scholar]
- Weeds A. G., Lowey S. Substructure of the myosin molecule. II. The light chains of myosin. J Mol Biol. 1971 Nov 14;61(3):701–725. doi: 10.1016/0022-2836(71)90074-x. [DOI] [PubMed] [Google Scholar]