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1
Loya, Amy K., Sarah K. Van Houten, Bernadette M. Glasheen i Douglas M. Swank. "Shortening deactivation: quantifying a critical component of cyclical muscle contraction". American Journal of Physiology-Cell Physiology 322, nr 4 (1.04.2022): C653—C665. http://dx.doi.org/10.1152/ajpcell.00281.2021.
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A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA’s complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. Although it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, we developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. Our results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions.
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Kreuz, A. J., A. Simcox i D. Maughan. "Alterations in flight muscle ultrastructure and function in Drosophila tropomyosin mutants." Journal of Cell Biology 135, nr 3 (1.11.1996): 673–87. http://dx.doi.org/10.1083/jcb.135.3.673.
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Drosophila indirect flight muscle (IFM) contains two different types of tropomyosin: a standard 284-amino acid muscle tropomyosin, Ifm-TmI, encoded by the TmI gene, and two > 400 amino acid tropomyosins, TnH-33 and TnH-34, encoded by TmII. The two IFM-specific TnH isoforms are unique tropomyosins with a COOH-terminal extension of approximately 200 residues which is hydrophobic and rich in prolines. Previous analysis of a hypomorphic TmI mutant, Ifm(3)3, demonstrated that Ifm-TmI is necessary for proper myofibrillar assembly, but no null TmI mutant or TmII mutant which affects the TnH isoforms have been reported. In the current report, we show that four flightless mutants (Warmke et al., 1989) are alleles of TmI, and characterize a deficiency which deletes both TmI and TmII. We find that haploidy of TmI causes myofibrillar disruptions and flightless behavior, but that haploidy of TmII causes neither. Single fiber mechanics demonstrates that power output is much lower in the TmI haploid line (32% of wild-type) than in the TmII haploid line (73% of wild-type). In myofibers nearly depleted of Ifm-TmI, net power output is virtually abolished (< 1% of wild-type) despite the presence of an organized fibrillar core (approximately 20% of wild-type). The results suggest Ifm-TmI (the standard tropomyosin) plays a key role in fiber structure, power production, and flight, with reduced Ifm-TmI expression producing corresponding changes of IFM structure and function. In contrast, reduced expression of the TnH isoforms has an unexpectedly mild effect on IFM structure and function.
3
Gu, Wenzhi, Qiufang Li, Meng Ding, Yurou Cao, Tongquan Wang, Shihu Zhang, Jiadong Feng, Hongyu Li i Lan Zheng. "Regular Exercise Rescues Heart Function Defects and Shortens the Lifespan of Drosophila Caused by dMnM Downregulation". International Journal of Environmental Research and Public Health 19, nr 24 (9.12.2022): 16554. http://dx.doi.org/10.3390/ijerph192416554.
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Although studies have shown that myomesin 2 (MYOM2) mutations can lead to hypertrophic cardiomyopathy (HCM), a common cardiovascular disease that has a serious impact on human life, the effect of MYOM2 on cardiac function and lifespan in humans is unknown. In this study, dMnM (MYOM2 homologs) knockdown in cardiomyocytes resulted in diastolic cardiac defects (diastolic dysfunction and arrhythmias) and increased cardiac oxidative stress. Furthermore, the knockdown of dMnM in indirect flight muscle (IFM) reduced climbing ability and shortened lifespan. However, regular exercise significantly ameliorated diastolic cardiac dysfunction, arrhythmias, and oxidative stress triggered by dMnM knockdown in cardiac myocytes and also reversed the reduction in climbing ability and shortening of lifespan caused by dMnM knockdown in Drosophila IFM. In conclusion, these results suggest that Drosophila cardiomyocyte dMnM knockdown leads to cardiac functional defects, while dMnM knockdown in IFM affects climbing ability and lifespan. Furthermore, regular exercise effectively upregulates cardiomyocyte dMnM expression levels and ameliorates cardiac functional defects caused by Drosophila cardiomyocyte dMnM knockdown by increasing cardiac antioxidant capacity. Importantly, regular exercise ameliorates the shortened lifespan caused by dMnM knockdown in IFM.
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Glasheen, Bernadette M., Catherine C. Eldred, Leah C. Sullivan, Cuiping Zhao, Michael K. Reedy, Robert J. Edwards i Douglas M. Swank. "Stretch activation properties of Drosophila and Lethocerus indirect flight muscle suggest similar calcium-dependent mechanisms". American Journal of Physiology-Cell Physiology 313, nr 6 (1.12.2017): C621—C631. http://dx.doi.org/10.1152/ajpcell.00110.2017.
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Muscle stretch activation (SA) is critical for optimal cardiac and insect indirect flight muscle (IFM) power generation. The SA mechanism has been investigated for decades with many theories proposed, but none proven. One reason for the slow progress could be that multiple SA mechanisms may have evolved in multiple species or muscle types. Laboratories studying IFM SA in the same or different species have reported differing SA functional properties which would, if true, suggest divergent mechanisms. However, these conflicting results might be due to different experimental methodologies. Thus, we directly compared SA characteristics of IFMs from two SA model systems, Drosophila and Lethocerus, using two different fiber bathing solutions. Compared with Drosophila IFM, Lethocerus IFM isometric tension is 10- or 17-fold higher and SA tension was 5- or 10-fold higher, depending on the bathing solution. However, the rate of SA tension generation was 9-fold faster for Drosophila IFM. The inverse differences between rate and tension in the two species causes maximum power output to be similar, where Drosophila power is optimized in the bathing solution that favors faster muscle kinetics and Lethocerus in the solution that favors greater tension generation. We found that isometric tension and SA tension increased with calcium concentration for both species in both solutions, reaching a maximum plateau around pCa 5.0. Our results favor a similar mechanism for both species, perhaps involving a troponin complex that does not fully calcium activate the thin filament thus leaving room for further tension generation by SA.
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Kulke, Michael, Ciprian Neagoe, Bernhard Kolmerer, Ave Minajeva, Horst Hinssen, Belinda Bullard i Wolfgang A. Linke. "Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle". Journal of Cell Biology 154, nr 5 (3.09.2001): 1045–58. http://dx.doi.org/10.1083/jcb.200104016.
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Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and α-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM–I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with μ-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.
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Zhao, Cuiping, i Douglas M. Swank. "The Drosophila indirect flight muscle myosin heavy chain isoform is insufficient to transform the jump muscle into a highly stretch-activated muscle type". American Journal of Physiology-Cell Physiology 312, nr 2 (1.02.2017): C111—C118. http://dx.doi.org/10.1152/ajpcell.00284.2016.
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Stretch activation (SA) is a delayed increase in force that enables high power and efficiency from a cyclically contracting muscle. SA exists in various degrees in almost all muscle types. In Drosophila, the indirect flight muscle (IFM) displays exceptionally high SA force production ( FSA), whereas the jump muscle produces only minimal FSA. We previously found that expressing an embryonic (EMB) myosin heavy chain (MHC) isoform in the jump muscle transforms it into a moderately SA muscle type and enables positive cyclical power generation. To investigate whether variation in MHC isoforms is sufficient to produce even higher FSA, we substituted the IFM MHC isoform (IFI) into the jump muscle. Surprisingly, we found that IFI only caused a 1.7-fold increase in FSA, less than half the increase previously observed with EMB, and only at a high Pi concentration, 16 mM. This IFI-induced FSA is much less than what occurs in IFM, relative to isometric tension, and did not enable positive cyclical power generation by the jump muscle. Both isometric tension and FSA of control fibers decreased with increasing Pi concentration. However, for IFI-expressing fibers, only isometric tension decreased. The rate of FSA generation was ~1.5-fold faster for IFI fibers than control fibers, and both rates were Pi dependent. We conclude that MHC isoforms can alter FSA and hence cyclical power generation but that isoforms can only endow a muscle type with moderate FSA. Highly SA muscle types, such as IFM, likely use a different or additional mechanism.
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QIU, Feng, Anne LAKEY, Bogos AGIANIAN, Amanda HUTCHINGS, Geoffrey W. BUTCHER, Siegfried LABEIT, Kevin LEONARD i Belinda BULLARD. "Troponin C in different insect muscle types: identification of two isoforms in Lethocerus, Drosophila and Anopheles that are specific to asynchronous flight muscle in the adult insect". Biochemical Journal 371, nr 3 (1.05.2003): 811–21. http://dx.doi.org/10.1042/bj20021814.
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The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca2+ concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca2+-binding subunit of troponin, troponin C (TnC), with a single Ca2+-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca2+-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca2+-binding sites. Ca2+ binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca2+-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca2+ binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.
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Brault, V., M. C. Reedy, U. Sauder, R. A. Kammerer, U. Aebi i C. Schoenenberger. "Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila". Journal of Cell Science 112, nr 21 (1.11.1999): 3627–39. http://dx.doi.org/10.1242/jcs.112.21.3627.
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The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5′ promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5′ and 3′ untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.
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Babu, Sajesh, i Nallur B. Ramachandra. "Screen for new mutations on the 2nd chromosome involved in indirect flight muscle development in Drosophila melanogaster". Genome 50, nr 4 (kwiecień 2007): 343–50. http://dx.doi.org/10.1139/g07-012.
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An extensive ethylmethanesulfonate mutagenesis of Drosophila melanogaster was undertaken to isolate the stronger alleles of 3 indirect flight-muscle mutations. We isolated 17 strong mutant lines, with nearly complete penetrance and expressivity, using direct screening under polarized light, from more than 1700 mutagenized chromosomes. On complementation, we found 11 of these 17 mutant lines to be alleles of 3 indirect flight-muscle mutations (Ifm(2)RU1, 3 noncomplementing lines; ifm(2)RU2, 6 alleles; ifm(2)RU3, 2 alleles) of the previously isolated 8 complementation groups (Ifm(2)RU1to ifm(2)RU8). In addition, we found 6 new complementation groups with strong defects in adult-muscle morphology; we named these ifm(2)RS1 to ifm(2)RS6. All mutant lines were mapped by meiotic recombination, and 5 of the 6 new complementation lines were mapped using chromosome deficiencies. ifm(2)RS1 maps to a region that harbors ifm(2)RU4 (a mutation that was isolated previously); however, theses are not alleles because each complements the other mutation, and the mutant-muscle phenotype is very different. We used direct screening under polarized light to find recessive mutations; although this method was labor intensive, it can be used to identify recessive genes involved in myogenesis, unlike screens for flightlessness or wing-position defects. This screen identifies regions on the second chromosome that harbor probable genes that are likely expressed in the mesoderm and are thought to be involved in myogenesis. This screen has generated valuable resources that will help us to understand the role of many molecular players involved in myogenesis.
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Reedy, Mary C., Belinda Bullard i Jim O. Vigoreaux. "Flightin Is Essential for Thick Filament Assembly and Sarcomere Stability in Drosophila Flight Muscles". Journal of Cell Biology 151, nr 7 (25.12.2000): 1483–500. http://dx.doi.org/10.1083/jcb.151.7.1483.
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Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a null mutation for flightin, fln0, that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln0 IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25–30% longer than in wild type. fln0 fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln0 sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.
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Hao, Yudong, Sanford I. Bernstein i Gerald H. Pollack. "Passive stiffness of Drosophila IFM myofibrils: a novel, high accuracy". Journal of Muscle Research and Cell Motility 25, nr 4-5 (lipiec 2004): 359–66. http://dx.doi.org/10.1007/s10974-004-0684-5.
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Tanner, Bertrand C. W., Mark S. Miller, Becky M. Miller, Panagiotis Lekkas, Thomas C. Irving, David W. Maughan i Jim O. Vigoreaux. "COOH-terminal truncation of flightin decreases myofilament lattice organization, cross-bridge binding, and power output in Drosophila indirect flight muscle". American Journal of Physiology-Cell Physiology 301, nr 2 (sierpień 2011): C383—C391. http://dx.doi.org/10.1152/ajpcell.00016.2011.
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The indirect flight muscle (IFM) of insects is characterized by a near crystalline myofilament lattice structure that likely evolved to achieve high power output. In Drosophila IFM, the myosin rod binding protein flightin plays a crucial role in thick filament organization and sarcomere integrity. Here we investigate the extent to which the COOH terminus of flightin contributes to IFM structure and mechanical performance using transgenic Drosophila expressing a truncated flightin lacking the 44 COOH-terminal amino acids ( flnΔ C44). Electron microscopy and X-ray diffraction measurements show decreased myofilament lattice order in the flnΔ C44 line compared with control, a transgenic flightin-null rescued line ( fln +). flnΔ C44 fibers produced roughly 1/3 the oscillatory work and power of fln +, with reduced frequencies of maximum work (123 Hz vs. 154 Hz) and power (139 Hz vs. 187 Hz) output, indicating slower myosin cycling kinetics. These reductions in work and power stem from a slower rate of cross-bridge recruitment and decreased cross-bridge binding in flnΔ C44 fibers, although the mean duration of cross-bridge attachment was not different between both lines. The decreases in lattice order and myosin kinetics resulted in flnΔ C44 flies being unable to beat their wings. These results indicate that the COOH terminus of flightin is necessary for normal myofilament lattice organization, thereby facilitating the cross-bridge binding required to achieve high power output for flight.
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Dhanyasi, Nagaraju, Dagan Segal, Eyal Shimoni, Vera Shinder, Ben-Zion Shilo, K. VijayRaghavan i Eyal D. Schejter. "Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles". Journal of Cell Biology 211, nr 1 (12.10.2015): 191–203. http://dx.doi.org/10.1083/jcb.201503005.
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Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.
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Fernandes, J., M. Bate i K. Vijayraghavan. "Development of the indirect flight muscles of Drosophila". Development 113, nr 1 (1.09.1991): 67–77. http://dx.doi.org/10.1242/dev.113.1.67.
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We have followed the pupal development of the indirect flight muscles (IFMs) of Drosophila melanogaster. At the onset of metamorphosis larval muscles start to histolyze, with the exception of a specific set of thoracic muscles. Myoblasts surround these persisting larval muscles and begin the formation of one group of adult indirect flight muscles, the dorsal longitudinal muscles. We show that the other group of indirect flight muscles, the dorsoventral muscles, develops simultaneously but without the use of larval templates. By morphological criteria and by patterns of specific gene expression, our experiments define events in IFM development.
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Warmke, J. W., A. J. Kreuz i S. Falkenthal. "Co-localization to chromosome bands 99E1-3 of the Drosophila melanogaster myosin light chain-2 gene and a haplo-insufficient locus that affects flight behavior." Genetics 122, nr 1 (1.05.1989): 139–51. http://dx.doi.org/10.1093/genetics/122.1.139.
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Abstract Using overlapping synthetic deficiencies, we find that a haplo-insufficient locus affecting flight behavior and the myosin light chain-2 gene co-map to the Drosophila melanogaster polytene chromosome interval 99D9-E1 to 99E2-3. From screening over 9000 EMS-treated chromosomes, we obtained alleles of two complementation groups that map to this same interval. One of these complementation groups lfm(3)99Eb, exhibits dominant flightless behavior; thus, flightless behavior of the deficiency is in all likelihood due to hemizygosity of this single locus. Rescue of flightless behavior by a duplication indicates that the single allele, E38, of the Ifm(3)99Eb complementation group is a hypomorph. Based upon its map position and a reduction in concentration of myosin light chain-2 mRNA in heterozygotes, we propose that Ifm(3)Eb(E38) is a mutant allele of the myosin light chain-2 gene. Our genetic analysis also resulted in the identification of four dominant flightless alleles of an unlinked locus, l(3)nc99Eb, that exhibits dominant lethal synergism with Ifm(3)99Eb.
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Brault, Véronique, Ursula Sauder, Mary C. Reedy, Ueli Aebi i Cora-Ann Schoenenberger. "Differential Epitope Tagging of Actin in TransformedDrosophila Produces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle". Molecular Biology of the Cell 10, nr 1 (styczeń 1999): 135–49. http://dx.doi.org/10.1091/mbc.10.1.135.
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We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.
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Standiford, David M., Wei Tao Sun, Mary Beth Davis i Charles P. Emerson. "Positive and Negative Intronic Regulatory Elements Control Muscle-Specific Alternative Exon Splicing of Drosophila Myosin Heavy Chain Transcripts". Genetics 157, nr 1 (1.01.2001): 259–71. http://dx.doi.org/10.1093/genetics/157.1.259.
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Abstract Alternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts is precisely regulated to ensure the expression of specific MHC isoforms required for the distinctive contractile activities of physiologically specialized muscles. We have used transgenic expression analysis in combination with mutagenesis to identify cis-regulatory sequences that are required for muscle-specific splicing of exon 11, which is encoded by five alternative exons that produce alternative “converter” domains in the MHC head. Here, we report the identification of three conserved intronic elements (CIE1, -2, and -3) that control splicing of exon 11e in the indirect flight muscle (IFM). Each of these CIE elements has a distinct function: CIE1 acts as a splice repressor, while CIE2 and CIE3 behave as splice enhancers. These CIE elements function in combination with a nonconsensus splice donor to direct IFM-specific splicing of exon 11e. An additional cis-regulatory element that is essential in coordinating the muscle-specific splicing of other alternative exon 11s is identified. Therefore, multiple interacting intronic and splice donor elements establish the muscle-specific splicing of alternative exon 11s.
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Tansey, T., J. R. Schultz, R. C. Miller i R. V. Storti. "Small differences in Drosophila tropomyosin expression have significant effects on muscle function". Molecular and Cellular Biology 11, nr 12 (grudzień 1991): 6337–42. http://dx.doi.org/10.1128/mcb.11.12.6337-6342.1991.
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The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.
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Tansey, T., J. R. Schultz, R. C. Miller i R. V. Storti. "Small differences in Drosophila tropomyosin expression have significant effects on muscle function." Molecular and Cellular Biology 11, nr 12 (grudzień 1991): 6337–42. http://dx.doi.org/10.1128/mcb.11.12.6337.
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The effects of promoter deletions on Drosophila tropomyosin I (TmI) gene expression have been determined by measuring TmI RNA levels in transformed flies. Decreases in RNA levels have been correlated with rescue of flightless and jumpless mutant phenotypes in Ifm(3)3 mutant transformed flies and changes in muscle ultrastructure. The results of this analysis have allowed us to identify a region responsible for 20% of maximal TmI expression, estimate threshold levels of TmI RNA required for indirect flight and jump muscle function, and obtain evidence suggesting that sarcomere length may be an important determinant of flight muscle function.
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Schultz, J. R., T. Tansey, L. Gremke i R. V. Storti. "A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression". Molecular and Cellular Biology 11, nr 4 (kwiecień 1991): 1901–11. http://dx.doi.org/10.1128/mcb.11.4.1901-1911.1991.
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The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.
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Schultz, J. R., T. Tansey, L. Gremke i R. V. Storti. "A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression." Molecular and Cellular Biology 11, nr 4 (kwiecień 1991): 1901–11. http://dx.doi.org/10.1128/mcb.11.4.1901.
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The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.
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Newhard, Christopher S., Sam Walcott i Douglas M. Swank. "The load dependence of muscle’s force-velocity curve is modulated by alternative myosin converter domains". American Journal of Physiology-Cell Physiology 316, nr 6 (1.06.2019): C844—C861. http://dx.doi.org/10.1152/ajpcell.00494.2018.
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The hyperbolic shape of the muscle force-velocity relationship (FVR) is characteristic of all muscle fiber types. The degree of curvature of the hyperbola varies between muscle fiber types and is thought to be set by force-dependent properties of different myosin isoforms. However, the structural elements in myosin and the mechanism that determines force dependence are unresolved. We tested our hypothesis that the myosin converter domain plays a critical role in the force-velocity relationship (FVR) mechanism. Drosophila contains a single myosin heavy chain gene with five converters encoded by alternative exons. We measured FVR properties of Drosophila jump muscle fibers from five transgenic lines each expressing a single converter. Consistent with our hypothesis, we observed up to 2.4-fold alterations in FVR curvature. Maximum shortening velocity ( v0) and optimal velocity for maximum power generation were also altered, but isometric tension and maximum power generation were unaltered. Converter 11a, normally found in the indirect flight muscle (IFM), imparted the highest FVR curvature and v0, whereas converter 11d, found in larval body wall muscle, imparted the most linear FVR and slowest v0. Jump distance strongly correlated with increasing FVR curvature and v0, meaning flies expressing the converter from the IFM jumped farther than flies expressing the native jump muscle converter. Fitting our data with Huxley’s two-state model and a biophysically based four-state model suggest a testable hypothesis that the converter sets muscle type FVR curvature by influencing the detachment rate of negatively strained myosin via changes in the force dependence of product release.
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Komlós, Marcell, Janka Szinyákovics, Gergő Falcsik, Tímea Sigmond, Bálint Jezsó, Tibor Vellai i Tibor Kovács. "The Small-Molecule Enhancers of Autophagy AUTEN-67 and -99 Delay Ageing in Drosophila Striated Muscle Cells". International Journal of Molecular Sciences 24, nr 9 (30.04.2023): 8100. http://dx.doi.org/10.3390/ijms24098100.
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Background: Autophagy (cellular self-degradation) plays a major role in maintaining the functional integrity (homeostasis) of essentially all eukaryotic cells. During the process, superfluous and damaged cellular constituents are delivered into the lysosomal compartment for enzymatic degradation. In humans, age-related defects in autophagy have been linked to the incidence of various age-associated degenerative pathologies (e.g., cancer, neurodegenerative diseases, diabetes, tissue atrophy and fibrosis, and immune deficiency) and accelerated ageing. Muscle mass decreases at detectable levels already in middle-aged patients, and this change can increase up to 30–50% at age 80. AUTEN-67 and -99, two small-molecule enhancers of autophagy with cytoprotective and anti-ageing effects have been previously identified and initially characterized. These compounds can increase the life span in wild-type and neurodegenerative model strains of the fruit fly Drosophila melanogaster. Methods: Adult flies were treated with these AUTEN molecules via feeding. Fluorescence and electron microscopy and Western blotting were used to assess the level of autophagy and cellular senescence. Flying tests were used to measure the locomotor ability of the treated animals at different ages. Results: In the current study, the effects of AUTEN-67 and -99 were observed on striated muscle cells using the Drosophila indirect flight muscle (IFM) as a model. The two molecules were capable of inducing autophagy in IFM cells, thereby lowering the accumulation of protein aggregates and damaged mitochondria, both characterizing muscle ageing. Furthermore, the two molecules significantly improved the flying ability of treated animals. Conclusions: AUTEN-67 and -99 decrease the rate at which striated muscle cells age. These results may have a significant medical relevance that could be further examined in mammalian models.
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Hastings, G. A., i C. P. Emerson. "Myosin functional domains encoded by alternative exons are expressed in specific thoracic muscles of Drosophila." Journal of Cell Biology 114, nr 2 (15.07.1991): 263–76. http://dx.doi.org/10.1083/jcb.114.2.263.
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The Drosophila 36B muscle myosin heavy chain (MHC) gene has five sets of alternatively spliced exons that encode functionally important domains of the MHC protein and provide a combinatorial potential for expression of as many as 480 MHC isoforms. In this study, in situ hybridization analysis has been used to examine the complexity and muscle specificity of MHC isoform expression in the fibrillar indirect flight muscle (IFM), the tubular direct flight muscles (DFM) and tubular tergal depressor of the trochanter muscle (TDT), and the visceral esophageal muscle in the adult thorax. Our results show that alternative splicing of the MHC gene transcripts is precisely regulated in these thoracic muscles, which express three MHC isoforms. Individual thoracic muscles each express transcripts of only one isoform, as detectable by in situ hybridization. An apparently novel fourth MHC isoform, with sequence homology to the rod but not to the head domain of the 36B MHC, is expressed in two direct flight muscles. These findings form a basis for transgenic experiments designed to analyze the muscle-specific functions of MHC domains encoded by alternative exons.
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Nongthomba, Upendra, Mark Cummins, Samantha Clark, Jim O. Vigoreaux i John C. Sparrow. "Suppression of Muscle Hypercontraction by Mutations in the Myosin Heavy Chain Gene of Drosophila melanogaster". Genetics 164, nr 1 (1.05.2003): 209–22. http://dx.doi.org/10.1093/genetics/164.1.209.
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Abstract The indirect flight muscles (IFM) of Drosophila melanogaster provide a good genetic system with which to investigate muscle function. Flight muscle contraction is regulated by both stretch and Ca2+-induced thin filament (actin + tropomyosin + troponin complex) activation. Some mutants in troponin-I (TnI) and troponin-T (TnT) genes cause a “hypercontraction” muscle phenotype, suggesting that this condition arises from defects in Ca2+ regulation and actomyosin-generated tension. We have tested the hypothesis that missense mutations of the myosin heavy chain gene, Mhc, which suppress the hypercontraction of the TnI mutant held-up2 (hdp2), do so by reducing actomyosin force production. Here we show that a “headless” Mhc transgenic fly construct that reduces the myosin head concentration in the muscle thick filaments acts as a dose-dependent suppressor of hypercontracting alleles of TnI, TnT, Mhc, and flightin genes. The data suggest that most, if not all, mutants causing hypercontraction require actomyosin-produced forces to do so. Whether all Mhc suppressors act simply by reducing the force production of the thick filament is discussed with respect to current models of myosin function and thin filament activation by the binding of calcium to the troponin complex.
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Menard, Lynda M., Neil B. Wood i Jim O. Vigoreaux. "Contiguity and Structural Impacts of a Non-Myosin Protein within the Thick Filament Myosin Layers". Biology 10, nr 7 (2.07.2021): 613. http://dx.doi.org/10.3390/biology10070613.
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Myosin dimers arranged in layers and interspersed with non-myosin densities have been described by cryo-EM 3D reconstruction of the thick filament in Lethocerus at 5.5 Å resolution. One of the non-myosin densities, denoted the ‘red density’, is hypothesized to be flightin, an LMM-binding protein essential to the structure and function of Drosophila indirect flight muscle (IFM). Here, we build upon the 3D reconstruction results specific to the red density and its engagement with the myosin coiled-coil rods that form the backbone of the thick filament. Each independent red density winds its way through the myosin dimers, such that it links four dimers in a layer and one dimer in a neighboring layer. This area in which three distinct interfaces within the myosin rod are contacted at once and the red density extends to the thick filament core is designated the “multiface”. Present within the multiface is a contact area inclusive of E1563 and R1568. Mutations in the corresponding Drosophila residues (E1554K and R1559H) are known to interfere with flightin accumulation and phosphorylation in Drosophila. We further examine the LMM area in direct apposition to the red density and identified potential binding residues spanning up to ten helical turns. We find that the red density is associated within an expanse of the myosin coiled-coil that is unwound by the third skip residue and the coiled-coil is re-oriented while in contact with the red density. These findings suggest a mechanism by which flightin induces ordered assembly of myosin dimers through its contacts with multiple myosin dimers and brings about reinforcement on the level of a single myosin dimer by stabilization of the myosin coiled-coil.
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Madan, Aditi, Divesh Thimmaiya, Ari Franco-Cea, Mohammed Aiyaz, Prabodh Kumar, John C. Sparrow i Upendra Nongthomba. "Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations". Gene 631 (październik 2017): 16–28. http://dx.doi.org/10.1016/j.gene.2017.07.061.
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Chun, M., i S. Falkenthal. "Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster." Journal of Cell Biology 107, nr 6 (1.12.1988): 2613–21. http://dx.doi.org/10.1083/jcb.107.6.2613.
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Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.
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Chakravorty, Samya, Bertrand C. W. Tanner, Veronica Lee Foelber, Hien Vu, Matthew Rosenthal, Teresa Ruiz i Jim O. Vigoreaux. "Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila". Proceedings of the Royal Society B: Biological Sciences 284, nr 1854 (3.05.2017): 20170431. http://dx.doi.org/10.1098/rspb.2017.0431.
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The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. We show that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain ( fln ΔN62 ) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, we show that fln ΔN62 males produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, we propose that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs.
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Cripps, R. M., E. Ball, M. Stark, A. Lawn i J. C. Sparrow. "Recovery of dominant, autosomal flightless mutants of Drosophila melanogaster and identification of a new gene required for normal muscle structure and function." Genetics 137, nr 1 (1.05.1994): 151–64. http://dx.doi.org/10.1093/genetics/137.1.151.
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Abstract To identify further mutations affecting muscle function and development in Drosophila melanogaster we recovered 22 autosomal dominant flightless mutations. From these we have isolated eight viable and lethal alleles of the muscle myosin heavy chain gene, and seven viable alleles of the indirect flight muscle (IFM)-specific Act88F actin gene. The Mhc mutations display a variety of phenotypic effects, ranging from reductions in myosin heavy chain content in the indirect flight muscles only, to reductions in the levels of this protein in other muscles. The Act88F mutations range from those which produce no stable actin and have severely abnormal myofibrillar structure, to those which accumulate apparently normal levels of actin in the flight muscles but which still have abnormal myofibrils and fly very poorly. We also recovered two recessive flightless mutants on the third chromosome. The remaining five dominant flightless mutations are all lethal alleles of a gene named lethal(3)Laker. The Laker alleles have been characterized and the gene located in polytene bands 62A10,B1-62B2,4. Laker is a previously unidentified locus which is haplo-insufficient for flight. In addition, adult wild-type heterozygotes and the lethal larval trans-heterozygotes show abnormalities of muscle structure indicating that the Laker gene product is an important component of muscle.
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Menard, Lynda M., Neil B. Wood i Jim O. Vigoreaux. "Secondary Structure of the Novel Myosin Binding Domain WYR and Implications within Myosin Structure". Biology 10, nr 7 (29.06.2021): 603. http://dx.doi.org/10.3390/biology10070603.
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Structural changes in the myosin II light meromyosin (LMM) that influence thick filament mechanical properties and muscle function are modulated by LMM-binding proteins. Flightin is an LMM-binding protein indispensable for the function of Drosophila indirect flight muscle (IFM). Flightin has a three-domain structure that includes WYR, a novel 52 aa domain conserved throughout Pancrustacea. In this study, we (i) test the hypothesis that WYR binds the LMM, (ii) characterize the secondary structure of WYR, and (iii) examine the structural impact WYR has on the LMM. Circular dichroism at 260–190 nm reveals a structural profile for WYR and supports an interaction between WYR and LMM. A WYR–LMM interaction is supported by co-sedimentation with a stoichiometry of ~2.4:1. The WYR–LMM interaction results in an overall increased coiled-coil content, while curtailing ɑ helical content. WYR is found to be composed of 15% turns, 31% antiparallel β, and 48% ‘other’ content. We propose a structural model of WYR consisting of an antiparallel β hairpin between Q92-K114 centered on an ASX or β turn around N102, with a G1 bulge at G117. The Drosophila LMM segment used, V1346-I1941, encompassing conserved skip residues 2-4, is found to possess a traditional helical profile but is interpreted as having <30% helical content by multiple methods of deconvolution. This low helicity may be affiliated with the dynamic behavior of the structure in solution or the inclusion of a known non-helical region in the C-terminus. Our results support the hypothesis that WYR binds the LMM and that this interaction brings about structural changes in the coiled-coil. These studies implicate flightin, via the WYR domain, for distinct shifts in LMM secondary structure that could influence the structural properties and stabilization of the thick filament, scaling to modulation of whole muscle function.
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Walls, Stanley M., Dale A. Chatfield, Karen Ocorr, Greg L. Harris i Rolf Bodmer. "Systemic and heart autonomous effects of sphingosine Δ4 desaturase deficiency in lipotoxic cardiac pathophysiology". Disease Models & Mechanisms 13, nr 8 (8.07.2020): dmm043083. http://dx.doi.org/10.1242/dmm.043083.
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ABSTRACTLipotoxic cardiomyopathy (LCM) is characterized by cardiac steatosis, including the accumulation of fatty acids, triglycerides and ceramides. Model systems have shown the inhibition of ceramide biosynthesis to antagonize obesity and improve insulin sensitivity. Sphingosine Δ4 desaturase (encoded by ifc in Drosophila melanogaster) enzymatically converts dihydroceramide into ceramide. Here, we examine ifc mutants to study the effects of desaturase deficiency on cardiac function in Drosophila. Interestingly, ifc mutants exhibited classic hallmarks of LCM: cardiac chamber dilation, contractile defects and loss of fractional shortening. This outcome was phenocopied in global ifc RNAi-mediated knockdown flies. Surprisingly, cardiac-specific ifc knockdown flies exhibited cardiac chamber restriction with no contractile defects, suggesting heart autonomous and systemic roles for ifc activity in cardiac function. Next, we demonstrated that ifc mutants exhibit suppressed Sphingosine kinase 1 (Sk1) expression. Ectopic overexpression of Sk1 was sufficient to prevent cardiac chamber dilation and loss of fractional shortening in ifc mutants. Partial rescue was also observed with cardiac- and fat-body-specific Sk1 overexpression. Finally, we showed that cardiac-specific expression of Drosophila inhibitor of apoptosis (dIAP) also prevented cardiac dysfunction in ifc mutants, suggesting a role for caspase activity in the observed cardiac pathology. Collectively, we show that spatial regulation of sphingosine Δ4 desaturase activity differentially affects cardiac function in heart autonomous and systemic mechanisms through tissue interplay.
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Bloemink, Marieke J., Karen H. Hsu, Michael A. Geeves i Sanford I. Bernstein. "Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain". Journal of Biological Chemistry 295, nr 42 (19.08.2020): 14522–35. http://dx.doi.org/10.1074/jbc.ra120.014684.
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We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3–encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3–encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.
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Wang, Yang, Girish C. Melkani, Jennifer A. Suggs, Anju Melkani, William A. Kronert, Anthony Cammarato i Sanford I. Bernstein. "Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness". Molecular Biology of the Cell 23, nr 11 (czerwiec 2012): 2057–65. http://dx.doi.org/10.1091/mbc.e12-02-0120.
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Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.
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Littlefield, Kimberly Palmiter, Douglas M. Swank, Becky M. Sanchez, Aileen F. Knowles, David M. Warshaw i Sanford I. Bernstein. "The converter domain modulates kinetic properties ofDrosophila myosin". American Journal of Physiology-Cell Physiology 284, nr 4 (1.04.2003): C1031—C1038. http://dx.doi.org/10.1152/ajpcell.00474.2002.
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Recently the converter domain, an integral part of the “mechanical element” common to all molecular motors, was proposed to modulate the kinetic properties of Drosophila chimeric myosin isoforms. Here we investigated the molecular basis of actin filament velocity ( V actin) changes previously observed with the chimeric EMB-IC and IFI-EC myosin proteins [the embryonic body wall muscle (EMB) and indirect flight muscle isoforms (IFI) with genetic substitution of the IFI and EMB converter domains, respectively]. In the laser trap assay the IFI and IFI-EC myosins generate the same unitary step displacement (IFI = 7.3 ± 1.0 nm, IFI-EC = 5.8 ± 0.9 nm; means ± SE). Thus converter-mediated differences in the kinetics of strong actin-myosin binding, rather than the mechanical capabilities of the protein, must account for the observed V actin values. Basal and actin-activated ATPase assays and skinned fiber mechanical experiments definitively support a role for the converter domain in modulating the kinetic properties of the myosin protein. We propose that the converter domain kinetically couples the Pi and ADP release steps that occur during the cross-bridge cycle.
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Moshrefi, Mandana, Kamal Ahmadi, Amin Purhematy, Maziar Jajarmi i yasin SarveAhrabi. "Detection of Antibacterial Properties of Musca domestica, Drosophila melanogaster, and Sarcophaga nodosa Using Resazurin as A Growth Indicator in Bacterial Cells". Infection Epidemiology and Microbiology 6, nr 3 (1.08.2020): 201–9. http://dx.doi.org/10.29252/iem.6.3.201.
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Lee, Pauline, Ngoc Ho, Terri Gelbart i Ernest Beutler. "Polymorphisms in the human homologue of the drosophila Indy (I'm not dead yet) gene". Mechanisms of Ageing and Development 124, nr 8-9 (sierpień 2003): 897–902. http://dx.doi.org/10.1016/s0047-6374(03)00149-0.
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Cormier, Sarah, Stéphanie Le Bras, Céline Souilhol, Sandrine Vandormael-Pournin, Béatrice Durand, Charles Babinet, Patricia Baldacci i Michel Cohen-Tannoudji. "The Murine Ortholog of Notchless, a Direct Regulator of the Notch Pathway in Drosophila melanogaster, Is Essential for Survival of Inner Cell Mass Cells". Molecular and Cellular Biology 26, nr 9 (1.05.2006): 3541–49. http://dx.doi.org/10.1128/mcb.26.9.3541-3549.2006.
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ABSTRACT Notch signaling is an evolutionarily conserved pathway involved in intercellular communication and is essential for proper cell fate choices. Numerous genes participate in the modulation of the Notch signaling pathway activity. Among them, Notchless (Nle) is a direct regulator of the Notch activity identified in Drosophila melanogaster. Here, we characterized the murine ortholog of Nle and demonstrated that it has conserved the ability to modulate Notch signaling. We also generated mice deficient for mouse Nle (mNle) and showed that its disruption resulted in embryonic lethality shortly after implantation. In late mNle −/− blastocysts, inner cell mass (ICM) cells died through a caspase 3-dependent apoptotic process. Most deficient embryos exhibited a delay in the temporal down-regulation of Oct4 expression in the trophectoderm (TE). However, mNle-deficient TE was able to induce decidual swelling in vivo and properly differentiated in vitro. Hence, our results indicate that mNle is mainly required in ICM cells, being instrumental for their survival, and raise the possibility that the death of mNle-deficient embryos might result from abnormal Notch signaling during the first steps of development.
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Zheng, Jolene, David Heber, Mingming Wang, Chenfei Gao, Steven B. Heymsfield, Roy J. Martin, Frank L. Greenway i in. "Pomegranate juice and extract extended lifespan and reduced intestinal fat deposition in Caenorhabditis elegans". International Journal for Vitamin and Nutrition Research 87, nr 3-4 (1.05.2017): 149–58. http://dx.doi.org/10.1024/0300-9831/a000570.
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Abstract. Pomegranate juice with a high content of polyphenols, pomegranate extract, ellagic acid, and urolithin A, have anti-oxidant and anti-obesity effects in humans. Pomegranate juice extends lifespan of Drosophila melanogaster. Caenorhabditis elegans (C. elegans) (n = 6) compared to the control group in each treatment, lifespan was increased by pomegranate juice in wild type (N2, 56 %, P < 0.001) and daf-16 mutant (daf-16(mgDf50)I) (18 %, P = 0.00012), by pomegranate extract in N2 (28 %, P = 0.00004) and in daf-16(mgDf50)I (10 %, P < 0.05), or by ellagic acid (11 %, P < 0.05). Pomegranate juice reduced intestinal fat deposition (IFD) in C. elegans (n = 10) N2 (–68 %, P = 0.0003) or in the daf-16(mgDf50)I (–33 %, P = 0.0034). The intestinal fat deposition was increased by pomegranate extract in N2 (137 %, P < 0.0138) and in daf-16(mgDf50)I (26 %, P = 0.0225), by ellagic acid in N2 (66 %, P < 0.0001) and in daf-16(mgDf50)I (74 %, P < 0.0001), or by urolithin A in N2 (57 %, P = 0.0039) and in daf-16(mgDf50)I (43 %, P = 0.0001). These effects were partially mediated by the daf-16 pathway. The data may offer insights to human aging and obesity due to homology with C. elegans.
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INOUE, Katsuhisa, Lina ZHUANG, Dennis M. MADDOX, Sylvia B. SMITH i Vadivel GANAPATHY. "Human sodium-coupled citrate transporter, the orthologue of Drosophila Indy, as a novel target for lithium action". Biochemical Journal 374, nr 1 (15.08.2003): 21–26. http://dx.doi.org/10.1042/bj20030827.
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NaCT (sodium-coupled citrate transporter) is an Na+-coupled citrate transporter identified recently in mammals that mediates the cellular uptake of citrate. It is expressed predominantly in the liver. NaCT is structurally and functionally related to the product of the Indy (I'm not dead yet) gene in Drosophila, the dysfunction of which leads to lifespan extension. Here, we show that NaCT mediates the utilization of extracellular citrate for fat synthesis in human liver cells, and that the process is stimulated by lithium. The transport function of NaCT is enhanced by lithium at concentrations found in humans treated with lithium for bipolar disorders. Valproate and carbamazepine, two other drugs that are used for the treatment of bipolar disorder, do not affect the function of NaCT. The stimulatory effect of Li+ is specific for human NaCT, since NaCTs from other animal species are either inhibited or unaffected by Li+. The data also suggest that two of the four Na+-binding sites in human NaCT may become occupied by Li+ to produce the stimulatory effect. The stimulation of NaCT in humans by lithium at therapeutically relevant concentrations has potential clinical implications. We also show here that a single base mutation in codon-500 (TTT→CTT) in the human NaCT gene, leading to the replacement of phenylalanine with leucine, stimulates the transport function and abolishes the stimulatory effect of lithium. This raises the possibility that genetic mutations in humans may lead to alterations in the constitutive activity of the transporter, with associated clinical consequences.
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Kopel, Jonathan J., Yangzom D. Bhutia, Sathish Sivaprakasam i Vadivel Ganapathy. "Consequences of NaCT/SLC13A5/mINDY deficiency: good versus evil, separated only by the blood–brain barrier". Biochemical Journal 478, nr 3 (5.02.2021): 463–86. http://dx.doi.org/10.1042/bcj20200877.
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NaCT/SLC13A5 is a Na+-coupled transporter for citrate in hepatocytes, neurons, and testes. It is also called mINDY (mammalian ortholog of ‘I'm Not Dead Yet’ in Drosophila). Deletion of Slc13a5 in mice leads to an advantageous phenotype, protecting against diet-induced obesity, and diabetes. In contrast, loss-of-function mutations in SLC13A5 in humans cause a severe disease, EIEE25/DEE25 (early infantile epileptic encephalopathy-25/developmental epileptic encephalopathy-25). The difference between mice and humans in the consequences of the transporter deficiency is intriguing but probably explainable by the species-specific differences in the functional features of the transporter. Mouse Slc13a5 is a low-capacity transporter, whereas human SLC13A5 is a high-capacity transporter, thus leading to quantitative differences in citrate entry into cells via the transporter. These findings raise doubts as to the utility of mouse models to evaluate NaCT biology in humans. NaCT-mediated citrate entry in the liver impacts fatty acid and cholesterol synthesis, fatty acid oxidation, glycolysis, and gluconeogenesis; in neurons, this process is essential for the synthesis of the neurotransmitters glutamate, GABA, and acetylcholine. Thus, SLC13A5 deficiency protects against obesity and diabetes based on what the transporter does in hepatocytes, but leads to severe brain deficits based on what the transporter does in neurons. These beneficial versus detrimental effects of SLC13A5 deficiency are separable only by the blood-brain barrier. Can we harness the beneficial effects of SLC13A5 deficiency without the detrimental effects? In theory, this should be feasible with selective inhibitors of NaCT, which work only in the liver and do not get across the blood-brain barrier.
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Knauf, Felix, Nilufar Mohebbi, Carsten Teichert, Diana Herold, Blanka Rogina, Stephen Helfand, Maik Gollasch, Friedrich C. Luft i Peter S. Aronson. "The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates". Biochemical Journal 397, nr 1 (14.06.2006): 25–29. http://dx.doi.org/10.1042/bj20060409.
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A longevity gene called Indy (for ‘I'm not dead yet’), with similarity to mammalian genes encoding sodium–dicarboxylate cotransporters, was identified in Drosophila melanogaster. Functional studies in Xenopus oocytes showed that INDY mediates the flux of dicarboxylates and citrate across the plasma membrane, but the specific transport mechanism mediated by INDY was not identified. To test whether INDY functions as an anion exchanger, we examined whether substrate efflux is stimulated by transportable substrates added to the external medium. Efflux of [14C]citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate–succinate exchange. The succinate-stimulated [14C]citrate efflux was sensitive to inhibition by DIDS (4,4′-di-isothiocyano-2,2′-disulphonic stilbene), as demonstrated previously for INDY-mediated succinate uptake. INDY-mediated efflux of [14C]citrate was also stimulated by external citrate and oxaloacetate, indicating citrate–citrate and citrate–oxaloacetate exchange. Similarly, efflux of [14C]succinate from INDY-expressing oocytes was stimulated by external citrate, α-oxoglutarate and fumarate, indicating succinate–citrate, succinate–α-oxoglutarate and succinate–fumarate exchange respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [14C]succinate uptake was markedly stimulated, confirming succinate–succinate and succinate–citrate exchange. Exchange of internal anion for external citrate was markedly pHo-dependent, consistent with the concept that citrate is co-transported with a proton. Anion exchange was sodium-independent. We conclude that INDY functions as an exchanger of dicarboxylate and tricarboxylate Krebs-cycle intermediates. The effect of decreasing INDY activity, as in the long-lived Indy mutants, may be to alter energy metabolism in a manner that favours lifespan extension.
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Shwartz, Arkadi, Nagaraju Dhanyasi, Eyal D. Schejter i Ben-Zion Shilo. "The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly". eLife 5 (12.10.2016). http://dx.doi.org/10.7554/elife.16540.
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Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. We have used formation of the Drosophila indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, we identify several distinct phases in the dynamic construction of thin-filament arrays. This sequence includes assembly of nascent arrays after an initial period of intensive microfilament synthesis, followed by array elongation, primarily from filament pointed-ends, radial growth of the arrays via recruitment of peripheral filaments and continuous barbed-end turnover. Using genetic approaches we have identified Fhos, the single Drosophila homolog of the FHOD sub-family of formins, as a primary and versatile mediator of IFM thin-filament organization. Localization of Fhos to the barbed-ends of the arrays, achieved via a novel N-terminal domain, appears to be a critical aspect of its sarcomeric roles.
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Gunderson, Jakob T., Ashley E. Peppriell, Ian N. Krout, Daria Vorojeikina i Matthew D. Rand. "Neuroligin-1 is a mediator of methylmercury neuromuscular toxicity". Toxicological Sciences, 21.09.2021. http://dx.doi.org/10.1093/toxsci/kfab114.
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Abstract Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. We investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, while a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg1 and nrx1 revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20 – 36-hour window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. We identified Nlg1 as a muscle-specific, NMJ structural component, that can mediate MeHg neuromuscular toxicity resulting from early life exposure.
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Lin, Min-Han, Madeline K. Jensen, Nathan D. Elrod, Kai-Lieh Huang, Kevin A. Welle, Eric J. Wagner i Liang Tong. "Inositol hexakisphosphate is required for Integrator function". Nature Communications 13, nr 1 (30.09.2022). http://dx.doi.org/10.1038/s41467-022-33506-3.
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AbstractIntegrator is a multi-subunit protein complex associated with RNA polymerase II (Pol II), with critical roles in noncoding RNA 3′-end processing and transcription attenuation of a broad collection of mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 Integrator cleavage module (ICM). Here we report a cryo-EM structure of the Drosophila ICM, at 2.74 Å resolution, revealing stable association of an inositol hexakisphosphate (IP6) molecule. The IP6 binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 Å away from the IntS11 active site and generally conserved in other ICMs. We also confirmed IP6 association with the same site in human ICM. IP6 binding is not detected in ICM samples harboring mutations in this binding site. Such mutations or disruption of IP6 biosynthesis significantly reduced Integrator function in snRNA 3′-end processing and mRNA transcription attenuation. Our structural and functional studies reveal that IP6 is required for Integrator function in Drosophila, humans, and likely other organisms.
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Naït-Saïdi, Rima, Aymeric Chartier, Emmanuelle Abgueguen, Philippe Guédat i Martine Simonelig. "The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response". Open Biology 13, nr 4 (kwiecień 2023). http://dx.doi.org/10.1098/rsob.230008.
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Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.