Indirect Flight Muscles Research Articles

Nicotine increases lifespan and rescues olfactory and motor deficits in a Drosophila model of Parkinson's disease

Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.

Drosophila melanogaster model for Mycobacterium abscessus infection

Mycobacterium abscessus is a human pathogen that is responsible for a broad spectrum of tissue infections and disseminated infections in immunodeficient patients. This pathogen is one of the most resistant organisms to chemotherapeutic agents. Therefore, we tested the hypothesis that the fruit fly, Drosophila melanogaster, is a genetically tractable model host for M. abscessus. In this context, we infected D. melanogaster with M. abscessus. This M. abscessus infection results in dissemination in the fly body, followed by death, which is accompanied by severe indirect flight muscle and brain damage. Our data show that M. abscessus can grow and replicate in D. melanogaster w1118 and that it elicited a humoral immune response, especially of the Toll antimicrobial peptide pathway. To the best of our knowledge, this is the first report that mycobacteria induce the production of antimicrobial peptides in D. melanogaster.

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs.

The myosin converter region influences cross‐bridge stiffness and muscle kinetics

The converter has been proposed to be the myosin elastic element that sets cross‐bridge stiffness. According to most cross‐bridge models, the elastic element's stiffness should directly influence muscle fiber type force and power generation levels. We tested these hypotheses by using the natural variation in converter regions found in Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative exon versions. We expressed four non‐native versions in Drosophila indirect flight muscle (IFM) and found that two of the three versions, 11b and 11e (found in larval and adult body wall muscles) decreased IFM rigor stiffness by 63% and 43%, respectively. This supports the hypothesis that the converter is the critical elastic element of myosin. The converters also influenced IFM fiber and myosin kinetics. 11b, 11d, and 11e decreased the frequency at which maximum power was generated (fmax) by 28%, 40%, and 53%, respectively. All three decreased myosin attachment kinetics, however, 11d and 11e also increased detachment kinetics. Differences in force generation are minimal, likely due to myosin stiffness changes being offset by changes in myosin duty ratio. With little to no force differences, changes in muscle power almost directly correlated with fmax. All three lines displayed reduced wing beat frequency, 11 to 15% less than control lines, with flight performance better correlating with fiber fmax than cross‐bridge stiffness. Thus, the converter is critical for muscle fiber type diversity as it helps set myosin stiffness, cross‐bridge kinetics and muscle power generation. Supported by NIH grant R01 AR055611 to D.S.

A novel Mito‐Timer reporter gene for measurement of mitochondrial quantity and quality in vivo

Mitochondrial dysfunction plays an important role in many diseases, but there is no satisfactory method to assess mitochondrial quantity and quality in vivo. Here we engineered a novel pMito‐Timer reporter gene from the existing pTimer‐1 reporter gene. pMito‐Timer encodes a mitochondria‐targeted green fluorescent protein (GFP) when newly synthesized, which shifts irreversibly to red fluorescence (DsRed) when oxidized. Confocal microscopy confirmed that Mito‐Timer targets to mitochondria in cultured mouse myoblasts, Drosophila heart and indirect flight muscles, and adult mouse skeletal muscle. Ratiometric algorithm showed that conditions that cause mitochondrial stress lead to a significant fluorescence shift toward DsRed with a significant increase of DsRed puncta. Somatic gene transfer in adult skeletal muscle in mice showed that high‐fat diet induces the same changes, which could be completely ameliorated by exercise training. Exercise training also results in increases in mitochondrial volume that could be quantified. Therefore, pMito‐Timer can be used to report mitochondrial health under both normal and pathological conditions and will provide a valuable tool to multiple disciplines investigating the role of the mitochondria in disease.

Molecular characterization of the flightin gene in the wing-dimorphic planthopper, Nilaparvata lugens, and its evolution in Pancrustacea

Flightin was initially identified in Drosophila melanogaster. Previous work has shown that Drosophila flightin plays a key role in indirect flight muscle (IFM) function and has limited expression in the IFM. In this study, we demonstrated that flightin is conserved across the Pancrustacea species, including winged insects, non-winged insects, non-insect hexapods and several crustaceans. The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a long-distance migration insect with wing dimorphism, is the most destructive rice pest in Asia. We showed that flightin was one of the most differentially expressed genes in macropterous and brachypterous BPH adults. In female BPHs, flightin was expressed in the IFM of macropterous adults, no expression was detected in brachypterous ones; while in male BPHs, flightin was not only expressed in the IFM of macropterous adults, but also in the dorsal longitudinal muscle (DLM) in the basal two abdominal segments of both macropterous and brachypterous ones. RNAi and transmission electron microscopy results showed that flightin played key roles in maintaining IFM and male DLM structure, which drive wing movements in macropterous adults and the vibration of the male-specific tymbal, respectively. Using Daphnia magna as an example of a crustacean species, we observed that flightin was expressed in juvenile instars and adults, and was localized in the antenna muscles. These results illustrate the functional variations of flightin in insects and other arthropod species and provide clues as to how insects with flight apparatuses evolved from ancient pancrustaceans.

Drosophila type XV/XVIII collagen mutants manifest integrin mediated mitochondrial dysfunction, which is improved by cyclosporin A and losartan

Vertebrate collagen types XV and XVIII are broadly distributed basement membrane components, classified into a structurally distinct subgroup called “multiplexin collagens”. Mutations in mammalian multiplexins are identified in some degenerative diseases such as Knobloch syndrome 1 (KNO1) or skeletal/cardiac myopathies, however, these progressive properties have not been elucidated. Here we investigated Drosophila mutants of Multiplexin (Mp), the only orthologue of vertebrate collagen types XV and XVIII, to understand the pathogenesis of multiplexin-related diseases. The mp mutants exhibited morphological changes in cardiomyocytes and progressive dysfunction of the skeletal muscles, reminiscent phenotypes observed in Col15a1-null mice. Ultrastructural analysis revealed morphologically altered mitochondria in mutants’ indirect flight muscles (IFMs), resulting in severely attenuated ATP production and enhanced reactive oxygen species (ROS) production. In addition, mutants’ IFMs exhibited diminished βPS integrin clustering and abolished focal adhesion kinase (FAK) phosphorylation. Furthermore, mutants’ defective IFMs are improved by the administrations of cyclosporin A, an inhibitor against mitochondrial permeability transition pore (mPTP) opening or losartan, an angiotensin II type 1 receptor (AT1R) blocker. Thus, our results suggest that Mp modulates mPTP opening and AT1R activity through its binding to integrin and that lack of Mp causes unregulated mPTP opening and AT1R activity, leading to mitochondrial dysfunctions. Hence, our results provide new insights towards the roles of multiplexin collagens in mitochondrial homeostasis and may serve as pharmacological evidences for the potential use of cyclosporin A or losartan for the therapeutic strategies.

The Drosophila Z-disc Protein Z(210) Is an Adult Muscle Isoform of Zasp52, Which Is Required for Normal Myofibril Organization in Indirect Flight Muscles

The Z-disc is a critical anchoring point for thin filaments as they slide during muscle contraction. Therefore, identifying components of the Z-disc is critical for fully comprehending how myofibrils assemble and function. In the adult Drosophila musculature, the fibrillar indirect flight muscles accumulate a >200 kDa Z-disc protein termed Z(210), the identity of which has to date been unknown. Here, we use mass spectrometry and gene specific knockdown studies, to identify Z(210) as an adult isoform of the Z-disc protein Zasp52. The Zasp52 primary transcript is extensively alternatively spliced, and we describe its splicing pattern in the flight muscles, identifying a new Zasp52 isoform, which is the one recognized by the Z(210) antibody. We also demonstrate that Zasp52 is required for the association of α-actinin with the flight muscle Z-disc, and for normal sarcomere structure. These studies expand our knowledge of Zasp isoforms and their functions in muscle. Given the role of Zasp proteins in mammalian muscle development and disease, our results have relevance to mammalian muscle biology.

Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle

Over 190 mutations in the human skeletal muscle α-actin gene, ACTA1 cause congenital actin myopathies. We transgenically expressed six different mutant actins, G15R, I136M, D154N, V163L, V163M and D292V in Drosophila indirect flight muscles and investigated their effects in flies that express one wild type and one mutant actin copy. All the flies were flightless, and the IFMs showed incomplete Z-discs, disorganised actin filaments and 'zebra bodies'. No differences in levels of sarcomeric protein expression were observed, but tropomodulin staining was somewhat disrupted in D164N, V163L, G15R and V163M heterozygotes. A single copy of D292V mutant actin rescued the hypercontractile phenotypes caused by TnI and TnT mutants, suggesting that the D292V mutation interferes with thin filament regulation. Our results show that expression of actin mutations homologous to those in humans in the indirect flight muscles of Drosophila disrupt sarcomere organisation, with somewhat similar phenotypes to those observed in humans. Using Drosophila to study actin mutations may help aid our understanding of congential myopathies caused by actin mutations.

Drosophila Erect wing (Ewg) controls mitochondrial fusion during muscle growth and maintenance by regulation of the Opa1-like gene

Mitochondrial biogenesis and morphological changes are associated with tissue-specific functional demand, but the factors and pathways that regulate these processes have not been completely identified. A lack of mitochondrial fusion has been implicated in various developmental and pathological defects. The spatiotemporal regulation of mitochondrial fusion in a tissue such as muscle is not well understood. Here, we show in Drosophila indirect flight muscles (IFMs) that the nuclear-encoded mitochondrial inner membrane fusion gene, Opa1-like, is regulated in a spatiotemporal fashion by the transcription factor/co-activator Erect wing (Ewg). In IFMs null for Ewg, mitochondria undergo mitophagy and/or autophagy accompanied by reduced mitochondrial functioning and muscle degeneration. By following the dynamics of mitochondrial growth and shape in IFMs, we found that mitochondria grow extensively and fuse during late pupal development to form the large tubular mitochondria. Our evidence shows that Ewg expression during early IFM development is sufficient to upregulate Opa1-like, which itself is a requisite for both late pupal mitochondrial fusion and muscle maintenance. Concomitantly, by knocking down Opa1-like during early muscle development, we show that it is important for mitochondrial fusion, muscle differentiation and muscle organization. However, knocking down Opa1-like, after the expression window of Ewg did not cause mitochondrial or muscle defects. This study identifies a mechanism by which mitochondrial fusion is regulated spatiotemporally by Ewg through Opa1-like during IFM differentiation and growth.

Alleviation of Skeletal Muscle Defects Induced by Huntington's Disease-causing Amyloid by Modulating TOR Pathway in a Drosophila Model

Huntington's disease (HD) is caused by an expanded polyglutamine (Poly-Q) repeat in the Huntingtin (HTT) protein. Beyond the brain, htt gene is also expressed in skeletal muscle. Muscle atrophy is common in HD patients, likely from accumulation of toxic amyloid. To explore the mechanism of Poly-Q-induced skeletal muscle defects associated with HD-causing amyloid protein, we expressed mutant HTT with short (Httex1-PolyQ25) and expanded (Httex1-PolyQ72) Poly-Q exclusively in the Drosophila indirect flight muscle (IFM). Expression of Httex1-PolyQ-72 in the IFM resulted in severe defects in skeletal muscle function and ultrastructure. Progressive reduction in flight performance was observed in 3-day and 3-week old flies (a 20% and 90% decrease in flight index, respectively) compared to age matched Httex1-PolyQ-25 flies. Further, 3-week old flies expressing Httex1-PolyQ-72 showed amyloid aggregates, whereas expression of Httex1-PolyQ-25 was benign. At the ultrastructural level, the myofibrils of control IFM showed well-organized Z-bands and M-lines, however, IFM of 3-week old flies expressing Httex1-PolyQ-72 showed severe ultrastructural deterioration with broken Z-disks, M-lines and a loss of thick filaments. In addition to myofibril defects, abnormal vacuole-like structures of various sizes and shapes containing mitochondrial remnants were present. Mutant fibers also display rimmed vacuoles fused with additional membranes, suggesting the formation of type 2 autophagic vacuoles. To further explore the mechanism underlying the PolyQ-induced skeletal muscle defects, we treated flies expressing Httex1-PolyQ-72 with rapamycin, a known suppressor of mTOR. Rapamycin-treated flies expressing Httex1-PolyQ-72 showed significantly improved flight function (a 30% increase in flight index) and improved myofibril integrity (organized Z-bands and M-lines) compared to flies expressing Httex1-PolyQ-72 without rapamycin treatment. Thus, we have developed a novel Drosophila model to explore and suppress skeletal muscle defects linked with HD-causing amyloid by modulating the TOR pathway.

Characterization of the Intracellular Distribution of Adenine Nucleotide Translocase (ANT) in Drosophila Indirect Flight Muscles

Background: The high power output necessary for insect flight has driven the evolution of muscles with large myofibrils (primary energy consumers) and abundant mitochondria (primary energy suppliers). The intricate functional interrelationship between these two organelles remains largely unknown despite its fundamental importance in understanding insect flight bioenergetics. Unlike vertebrate muscle that relies on a phosphagen (creatine phosphate/creatine kinase) system to regulate high energy phosphate flux, insect flight muscle has been reported to lack mitochondrial arginine kinase (analogous to creatine kinase), a key enzyme that enables intracellular energy transport. Creatine kinase is known to interact with mitochondrial adenine nucleotide translocase (ANT) in the transfer of ADP and ATP into and out of the mitochondria. Results: Here, we use quantitative immunogold transmission electron microscopy to show that in Drosophila melanogaster indirect flight muscles (IFM), ANT is present in the mitochondria as well as throughout the myofibril. To confirm this unexpected result, we created a transgenic line that expresses a chimeric GFP-ANT protein and used an anti-GFP antibody to determine the intracellular distribution of the fusion protein in the IFM. Similar to results obtained with anti-ANT, the fusion GFP-ANT protein is detected in myofibrils and mitochondria. We confirmed the absence of arginine kinase from IFM mitochondria and show that its sarcomeric (i.e., intramyofibrillar) distribution is similar to that of ANT. Conclusions: These results raise the possibility that direct channeling of nucleotides between mitochondria and myofibrils is assisted by an ANT protein thereby circumventing the need for a phosphagen shuttle in the IFM. The myofibrillar ANT may represent a unique adaptation in the muscles that require efficient exchange of nucleotides between mitochondria and myofibrils.

Zasp PDZ Domain Proteins Cooperate in Z-Disc Formation and Myofibril Assembly

Zasp52 is a multi-domain protein, composed of an N-terminal PDZ domain, ZM motif, and four LIM domains, and is found predominantly in Drosophila muscle. It is part of the PDZ-LIM domain protein family, which comprises Enigma, ENH, ZASP, Alp, CLP36, RIL, and Mystique in vertebrates. In Drosophila embryos, Zasp52 was shown to co-localize with α-actinin at Z-discs and with integrins at myotendinous junctions. Mutations in Zasp52 cause first instar larval lethality with defects in Z-disc assembly and maintenance as well as muscle attachment. During Drosophila indirect flight muscle (IFM) development in the pupa, Zasp52 is present at very early stages of myofibril assembly. It localizes with α-actinin in rudimentary Z-bodies along the assembling myofibril, before other muscle proteins show any periodicity. RNA interference against the last exon encoding the most C-terminal LIM domain results in the depletion of all high molecular weight isoforms. This gives rise to viable but flightless adult animals. IFM sarcomeres show thin and interrupted Z-discs as well as distorted H-zones from early stages of myofibril assembly onwards. These findings suggest that Zasp52 is required for the establishment of normal Z-discs in the IFM and subsequent sarcomere stability after onset of contractility. A closely related PDZ domain containing protein, Zasp66, is also required for Z-disc formation and stability. Simultaneous knock down of Zasp52 and Zasp66 lead to more severe, synergistic myofibril defects, demonstrating that both proteins act together during Z-disc formation, likely because both biochemically bind α-actinin.

A Troponin-T Mutation Initiates Cardiac and Skeletal Myopathy due to Impaired Inhibition of Contraction in Drosophila Melanogaster

Muscle contraction results from a series of molecular events that involve transient interactions between myosin-containing thick and actin-containing thin filaments. The high affinity of myosin for actin suggests that without regulation, muscle would remain in a continuous state of contraction. Regulation of striated muscle contraction is primarily achieved by Ca2+-dependent modulation of myosin crossbridge cycling on actin by the thin filament (TF) troponin-tropomyosin complex. Alterations in various subunits of the complex trigger contractile dysregulation and myopathy. For example, point mutations located over a span of ten amino acids (130-39) of human cardiac troponin T (cTnT) are associated with distinct cardiomyopathic responses. The Drosophila up101 (E88K) mutation localizes to the end of this well-conserved region of TnT. Here we examined the consequences of the lesion on distinct muscle types from the fly. Cardiac performance and morphology were assessed using direct immersion DIC optics, high-speed video imaging and motion analysis. Relative to controls, up101 hearts displayed a phenotype reminiscent of human restrictive cardiomyopathy. End-diastolic and end-systolic dimensions and percent fractional shortening were significantly reduced. Furthermore systolic intervals were significantly prolonged. This suggests TF dysregulation initiates excessive periods of force production and diastolic dysfunction. To characterize the fundamental cause of cardiac remodeling, we isolated TFs from the indirect flight muscles (IFM). The constitutively expressed up101 mutation results in flightlessness due to severe IFM hypercontraction. Electron microscopy and three-dimensional reconstruction of IFM TFs revealed Ca+2-dependent tropomyosin movement, typical of control filaments, was not a general feature of the TnT mutants. The vast majority of Ca+2-free mutant TFs exhibited tropomyosin associated with successive actin monomers over actin subdomains 3 and 4, distal to known myosin binding sites. Thus, up101 TnT likely promotes TF dysinhibition and ubiquitous myopathic remodeling.

Myosin Stiffness and Cross-Bridge Kinetics are Influenced by Five Drosophila Alternative Converters

The converter has been proposed to be the myosin elastic element that sets cross-bridge stiffness. According to most cross-bridge models, the elastic element's stiffness should influence muscle fiber type force and power generation levels. We tested these hypotheses by exploiting the natural variation in converter regions found in Drosophila muscle types. In Drosophila, myosin isoforms are generated by alternative splicing from a single myosin heavy chain gene. The converter region is encoded by five alternative versions. We forced the four non-native versions to be expressed in Drosophila indirect flight muscle (IFM) and found that two of the three versions tested to date, 11b and 11e (found in larval and adult body wall muscles) significantly decreased IFM rigor stiffness by 63% and 43%, respectively. This supports the hypothesis that the converter is at least part of the elastic element of myosin. The converters influenced more than myosin stiffness, as all three converters slowed the super fast IFM fiber kinetics. 11b, 11d, and 11e decreased the frequency at which maximum power was generated (fmax) by 28%, 40%, and 53%, respectively. All three decreased apparent rate constant 2πb (set by myosin attachment and Pi release kinetics). However, 11d and 11e increased 2πc (set by detachment kinetics). Differences in force generation are minimal, likely due to myosin stiffness changes being offset by changes in duty ratio. With little to no force differences, power almost directly correlated with fmax, and the optimal percent ML amplitude for power inversely correlated with fmax. All three lines showed reduced wing beat frequency, with flight performance better correlating with fiber fmax than rigor stiffness. Thus, the converter is critical for muscle fiber type diversity as it helps set myosin stiffness, cross-bridge kinetics and power generation.

Exploration and Suppression of Tau-Induced Cardiac and Skeletal Muscle Defects in a Drosophila Model

Hyperphosphorylation and aggregation of the microtubule-associated protein tau into tangles occurs in several neurodegenerative diseases referred to as tauopathies. Several tau mutations, including tauR406W and tauV337M, have been associated with tau hyperphosphorylation and neurodegeneration. Accumulation of phosphorylated tau in cardiac and skeletal muscle biopsy samples suggests that striated muscle tissue generates tau-amyloid which causes the destruction/malfunctioning of myocytes. However, it is unknown how mutations in the tau gene lead to myopathies and moreover, there is no experimental model to understand tau-mediated striated muscle dysfunction. To test the effects of tau mutations on cardiac structure and function, we developed a novel Drosophila model that expresses pathological human tau in the heart using the UAS-Gal4 expression system with a cardiac specific driver. Cardiac physiology was assessed using high-speed video recording of heart tube contractile parameters from semi-intact heart preparations. Compared to control human tau (h-tau), expression of mutant tau (h-tauR406W and h-tauV337M) resulted in progressive cardiac dysfunction and ultrastructural abnormalities. Expression of h-tauR406W in 4 week-old hearts resulted in severe cardiac dilation, reduced contractility, increased arrythmia, and ultrastructural defects, including myofibrillar degeneration and mitochondrial elongation. Expression of h-tauV337M in 4 week-old hearts resulted in reduced contractility, increased arrythmia, and fragmented mitochondria. Interestingly, cardiac dilation and ultrastructural defects in 4 week-old h-tauR406W flies were suppressed by cardiac overexpression of DRP-1 (a regulator of mitochondrial biogenesis), or TRAP-1 (a mitochondrial chaperone). Additionally, expression of mutant tau (h-tau R406W and h-tau V337M) in indirect flight muscles using Actin88F driver resulted in reduced flight ability and ultrastructural defects, including myofibrillar disorganization, bulk Z-disk accumulation, and mitochondrial abnormalities. Our data demonstrate pathological consequences for tau mutations in striated muscle and a link between tau-induced myopathies and mitochondrial defects.

N-Terminal Truncation of Flightin Reduces Oscillatory Power Output without Affecting Cross-Bridge Kinetics in Drosophila Indirect Flight Muscles

The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive stiffness and exceptionally fast myosin kinetics, two attributes that enhance power output to sustain flight. Flightin is an IFM-specific, 20kDa myosin rod-binding protein required for normal thick filament stiffness, sarcomere integrity, and flight. Previously, we showed that a COOH-terminal truncation of flightin (flnDeltaC44) decreased myofilament lattice order and myosin kinetics, resulting in lower oscillatory power output and flightlessness. Here, we investigate the function of the flightin N-terminal 62 amino acids by creating transgenic Drosophila (flnDeltaN62) expressing a truncated flightin. flnDeltaN62 flies were flight impaired (flight index: 2.8±0.1 vs. 4.2±0.4 for flnDeltaN62 vs. fln+ rescued null control) despite having a normal wing-beat frequency (195±4 vs. 198±2 Hz for fln+). Mechanical analysis of skinned IFM fibers showed that the flightin N-terminal truncation reduced passive, active, and rigor stiffness without affecting cross-bridge kinetics (frequency of maximum power: 205±7 vs. 217±7 Hz for fln+). flnDeltaN62 fibers produced approximately half the isometric tension (passive: 0.9±0.1 vs. 1.7±0.3 kN/m2, active: 0.8±0.1 vs. 1.5±0.2 kN/m2, rigor: 1.1±0.2 vs. 3.1±0.4 kN/m2) and maximum oscillatory power output (38.0±4.6 vs. 89.5±9.6 W/m3) as fln+ fibers. Moreover, about 60% of the flnDeltaN62 fibers tore in rigor, demonstrating mechanical failure near isometric tension values that were sustained by fln+ fibers. Fourier transform analysis of cross-sectional electron micrographs revealed that the flightin N-terminal truncation compromised myofilament lattice crystallinity and reduced inter-thick filament spacing by 10% (44.1±1.3 vs. 49.7±0.4 nm). These results indicate that the flightin N-terminal region enhances myofilament lattice order and mechanical integrity, which in turn is required for effective force transmission, normal oscillatory power output, and flight.

An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight

Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers - 1% muscle length (ML) amplitude and 150 Hz oscillation frequency - were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance.

Defining Myosin Relay Domain Interactions with the Converter Domain and with the SH1-SH2 Helix Region and their Significance in Muscle Contraction

Interaction of the myosin relay domain with the converter domain and with the SH1-SH2 helix region appears to be vital for the power stroke and muscle contraction. To define involvement of specific residues in these interactions we used mutation and compensatory mutation approach. The mutant myosins were expressed transgenically in Drosophila indirect flight muscles, which allow assessment of myosin function in vitro and in vivo. We mutated residue R714 near the SH1-SH2 helix in the main body of myosin. We found that R714E myosin ATPase is reduced by ∼80% and that no in vitro motility is detected. R714E organisms show myofibril assembly defects that are exacerbated with age and these organisms cannot fly. Putative compensatory mutation E499R in the relay domain improves ATPase activity so that there is only ∼35% reduction compared to wild type. Further, R714E/E499R myosin moves actin filaments at ∼30% of wild-type velocity. Normal myofibril assembly is restored and myofibrils are more stable. We next studied mutation R759E in the converter domain. It depresses myosin ATPase activity, in vitro motility and flight ability. Although myofibrils assemble normally, they degenerate as flies age. We inserted a lysine mutation at putative interacting relay domain loop residue N509 in an attempt to suppress the R759E mutant phenotypes. N509K dramatically rescues muscle structure and function, with significant increases in ATPase rates and in vitro motility. These interactions are critical for normal ATPase activity, in vitro motility, myofibril assembly and/or stability and flight muscle function. Thus the specific residue contacts identified permit the myosin relay domain to communicate information between the nucleotide binding pocket and the converter domain and are essential for progress through the mechanochemical cycle that powers muscle contraction.

Native bees pollinate tomato flowers and increase fruit production

The tomato plant has a specific relationship with native pollinators because the form of its flowers is adapted to buzz pollination carried out by some pollen-gatherer bees that vibrate their indirect flight muscles to obtain that floral resource. The absence and the low density of these bees in tomato fields can lead to pollination deficits for crop. The aim of this study is to demonstrate that open tomato flowers, probably visited by native pollinator, have greater pollen load on their stigma than unvisited flowers. Another objective is to show that this great pollen load increases fruit production. We selected crops of the Italian tomato cultivar in areas of the State of Goiás, Brazil. Thirty seven plants of three crops each had one inflorescence bagged in the field. Bagged and non-bagged flowers had their stigmas collected and the amount of pollen on their surfaces was quantified. For the comparison of fruit production, we monitored bagged and not-bagged inflorescences and after 40 days, their fruits were counted, weighed, measured and had their seeds counted. The amount of pollen grains on the stigma of flowers available to pollinators was higher than that on the stigma of bagged flowers. On average, fruit production was larger in not-bagged inflorescences than in bagged inflorescences. In addition, not-bagged flowers produced heavier fruits than did bagged flowers. There was a significant difference in the number of seeds between treatments, with significantly more seeds in the non-bagged fruit. Our results show that native bees buzz-pollinate tomato flowers, increasing the pollen load on their stigma and consequently fruit production and quality.