Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila.

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 (flnΔN62 ) 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 flnΔN62 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.

Diffusion coefficients of endogenous cytosolic proteins from rabbit skinned muscle fibers.

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10(-10) cm(2) s(-1)) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10(-7) cm(2) s(-1) (parvalbumin) to 0.20 × 10(-7) cm(2) s(-1) (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.

An evolutionary analysis of flightin reveals a conserved motif unique and widespread in Pancrustacea.

Flightin is a thick filament protein that in Drosophila melanogaster is uniquely expressed in the asynchronous, indirect flight muscles (IFM). Flightin is required for the structure and function of the IFM and is indispensable for flight in Drosophila. Given the importance of flight acquisition in the evolutionary history of insects, here we study the phylogeny and distribution of flightin. Flightin was identified in 69 species of hexapods in classes Collembola (springtails), Protura, Diplura, and insect orders Thysanura (silverfish), Dictyoptera (roaches), Orthoptera (grasshoppers), Pthiraptera (lice), Hemiptera (true bugs), Coleoptera (beetles), Neuroptera (green lacewing), Hymenoptera (bees, ants, and wasps), Lepidoptera (moths), and Diptera (flies and mosquitoes). Flightin was also found in 14 species of crustaceans in orders Anostraca (water flea), Cladocera (brine shrimp), Isopoda (pill bugs), Amphipoda (scuds, sideswimmers), and Decapoda (lobsters, crabs, and shrimps). Flightin was not identified in representatives of chelicerates, myriapods, or any species outside Pancrustacea (Tetraconata, sensu Dohle). Alignment of amino acid sequences revealed a conserved region of 52 amino acids, referred herein as WYR, that is bound by strictly conserved tryptophan (W) and arginine (R) and an intervening sequence with a high content of tyrosines (Y). This motif has no homologs in GenBank or PROSITE and is unique to flightin and paraflightin, a putative flightin paralog identified in decapods. A third motif of unclear affinities to pancrustacean WYR was observed in chelicerates. Phylogenetic analysis of amino acid sequences of the conserved motif suggests that paraflightin originated before the divergence of amphipods, isopods, and decapods. We conclude that flightin originated de novo in the ancestor of Pancrustacea > 500 MYA, well before the divergence of insects (~400 MYA) and the origin of flight (~325 MYA), and that its IFM-specific function in Drosophila is a more recent adaptation. Furthermore, we propose that WYR represents a novel myosin coiled-coil binding motif.

Courtship song analysis of Drosophila muscle mutants.

As part of the mating ritual, males of Drosophila species produce species-specific courtship songs through wing vibrations generated by the thoracic musculature. While previous studies have shown that indirect flight muscles (IFM) are neurally activated during courtship song production, the precise role of these muscles in song production has not been investigated. Fortunately, IFM mutants abound in Drosophila melanogaster and studies spanning several decades have shed light on the role of muscle proteins in IFM-powered flight. Analysis of courtship songs in these mutants offers the opportunity to uncover the role of the IFM in a behavior distinct than flight and subject to different evolutionary selection regimes. Here, we describe protocols for the recording and analysis of courtship behavior and mating song of D. melanogaster muscle transgenic and mutant strains. To record faint acoustic signal of courtship songs, an insulated mating compartment was used inside a recording device (INSECTAVOX) equipped with a modified electret microphone, a low-noise power supply, and noise filters. Songs recorded in the INSECTAVOX are digitized using Goldwave, whose several features enable extraction of critical song parameters, including carrier frequencies for pulse song and sine song. We demonstrate the utility of this approach by showing that deletion of the N-terminal region of the myosin regulatory light chain, a mutation known to decrease wing beat frequency and flight power, affects courtship song parameters.

Regulatory light chain phosphorylation and N-terminal extension increase cross-bridge binding and power output in Drosophila at in vivo myofilament lattice spacing.

The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2(+)), truncated N-terminal extension (Dmlc2(Δ2-46)), disrupted myosin light chain kinase phosphorylation sites (Dmlc2(S66A,S67A)), and dual mutant (Dmlc2(Δ2-46; S66A,S67A)). The N-terminal extension truncation and phosphorylation sites disruption mutations decreased oscillatory power output and the frequency of maximum power output in maximally Ca(2+)-activated fibers compressed to near in vivo inter-thick filament spacing, with the phosphorylation sites disruption mutation having a larger affect. The diminished power output parameters with the N-terminal extension truncation and phosphorylation sites disruption mutations were due to the reduction of the number of strongly-bound cross-bridges and rate of myosin force production, with the larger parameter reductions in the phosphorylation sites disruption mutation additionally related to reduced myosin attachment time. The phosphorylation and N-terminal extension-dependent boost in cross-bridge kinetics corroborates previous structural data, which indicate these RLC attributes play a complementary role in moving and orienting myosin heads toward actin target sites, thereby increasing fiber and whole fly power generation.

COOH-terminal truncation of flightin decreases myofilament lattice organization, cross-bridge binding, and power output in Drosophila indirect flight muscle.

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 (fln(ΔC44)). Electron microscopy and X-ray diffraction measurements show decreased myofilament lattice order in the fln(ΔC44) line compared with control, a transgenic flightin-null rescued line (fln(+)). fln(ΔC44) 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 fln(ΔC44) 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 fln(ΔC44) 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.

Obtaining a faculty position in STEM at a research-intensive institution.

Progressing from postdoctoral training to a STEM faculty appointment at a Research Intensive Institution (RII) is a daunting transition, and may be especially challenging to those who have followed a less-than-conventional path or whose peers have lost interest in academic careers. This article describes how to prepare for and progress through the application process for institutions in the USA, which takes approximately 1 year, including what to expect at each step and recommendations for a successful transition. The odds of success for any individual application are low, making good preparation and careful planning the more important, as does managing expectations to avoid becoming discouraged early in the process. The rewards of landing the faculty appointment at an institution that matches your professional and personal needs and for which you are best suited more than exceeds the effort required to attain it.

Secondary Structure of the Novel Myosin Binding Domain WYR and Implications within Myosin Structure.

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 ß, and 48% 'other' content. We propose a structural model of WYR consisting of an antiparallel ß hairpin between Q92-K114 centered on an ASX or ß 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.

Contiguity and Structural Impacts of a Non-Myosin Protein within the Thick Filament Myosin Layers.

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.

Comparative biomechanics of thick filaments and thin filaments with functional consequences for muscle contraction.

The scaffold of striated muscle is predominantly comprised of myosin and actin polymers known as thick filaments and thin filaments, respectively. The roles these filaments play in muscle contraction are well known, but the extent to which variations in filament mechanical properties influence muscle function is not fully understood. Here we review information on the material properties of thick filaments, thin filaments, and their primary constituents; we also discuss ways in which mechanical properties of filaments impact muscle performance.

Cardiac myosin binding protein-C is essential for thick-filament stability and flexural rigidity.

Using atomic force microscopy, we examined the contribution of cardiac myosin binding protein-C (cMyBP-C) to thick-filament length and flexural rigidity. Native thick filaments were isolated from the hearts of transgenic mice bearing a truncation mutation of cMyBP-C (t/t) that results in no detectable cMyBP-C and from age-matched wild-type controls (+/+). Atomic force microscopy images of these filaments were evaluated with an automated analysis algorithm that identified filament position and shape. The t/t thick-filament length (1.48 +/- 0.02 microm) was significantly (P < 0.01) shorter than +/+ (1.56 +/- 0.02 microm). This 5%-shorter thick-filament length in the t/t was reflected in 4% significantly shorter sarcomere lengths of relaxed isolated cardiomyocytes of the t/t (1.97 +/- 0.01 microm) compared to +/+ (2.05 +/- 0.01 microm). To determine if cMyBP-C contributes to the mechanical properties of thick filaments, we used statistical polymer chain mechanics to calculate a per-filament-specific persistence length, an index of flexural rigidity directly proportional to Young's modulus. Thick-filament-specific persistence length in the t/t (373 +/- 62 microm) was significantly lower than in +/+ (639 +/- 101 microm). Accordingly, Young's modulus of t/t thick filaments was approximately 60% of +/+. These results provide what we consider a new understanding for the critical role of cMyBP-C in defining normal cardiac output by sustaining force and muscle stiffness.

Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle.

X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2(Delta2-46)) or disruption of the phosphorylation sites by substituting alanines (Dmlc2(S66A, S67A)) decreased the equatorial intensity ratio (I(20)/I(10)), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2(S66A, S67A)), but not N-terminal extension truncation (Dmlc2(Delta2-46)), decreased the 14.5nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2(S66A, S67A)) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2(Delta2-46; S66A, S67A)), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a "tether" to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

Aging enhances indirect flight muscle fiber performance yet decreases flight ability in Drosophila.

We investigated the effects of aging on Drosophila melanogaster indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance.

Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.

The identification and quantification of specific phosphorylation sites within a protein by mass spectrometry has proved challenging when measured from peptides after protein digestion because each peptide has a unique ionization efficiency that alters with modification, such as phosphorylation, and because phosphorylation can alter cleavage by trypsin, shifting peptide distribution. In addition, some phosphorylated peptides generated by tryptic digest are small and hydrophilic and, thus, are not retained well on commonly used C18 columns. We have developed a novel C-terminal peptide (2)H-labeling derivatization strategy and a mass balance approach to quantify phosphorylation. We illustrate the application of our method using electrospray ionization liquid chromatography-mass spectrometry by quantifying phosphorylation of troponin I with protein kinase A and protein kinase C. The method also improves the retention and elution of hydrophilic peptides. The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage. Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.

Female Drosophila melanogaster respond to song-amplitude modulations.

Males in numerous animal species use mating songs to attract females and intimidate competitors. We demonstrate that modulations in song amplitude are behaviourally relevant in the fruit fly Drosophila We show that Drosophilamelanogaster females prefer amplitude modulations that are typical of melanogaster song over other modulations, which suggests that amplitude modulations are processed auditorily by D. melanogaster Our work demonstrates that receivers can decode messages in amplitude modulations, complementing the recent finding that male flies actively control song amplitude. To describe amplitude modulations, we propose the concept of song amplitude structure (SAS) and discuss similarities and differences to amplitude modulation with distance (AMD).This article has an associated First Person interview with the first author of the paper.

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

Flightin is a myosin binding protein present in Pancrustacea. In Drosophila, flightin is expressed in the indirect flight muscles (IFM), where it is required for the flexural rigidity, structural integrity, and length determination of thick filaments. Comparison of flightin sequences from multiple Drosophila species revealed a tripartite organization indicative of three functional domains subject to different evolutionary constraints. We use atomic force microscopy to investigate the functional roles of the N-terminal domain and the C-terminal domain that show different patterns of sequence conservation. Thick filaments containing a C-terminal domain truncated flightin (fln(ΔC44)) are significantly shorter (2.68 ± 0.06 µm; p < 0.005) than thick filaments containing a full length flightin (fln⁺; 3.21 ± 0.05 µm) and thick filaments containing an N-terminal domain truncated flightin (fln(ΔN62); 3.21 ± 0.06 µm). Persistence length was significantly reduced in fln(ΔN62) (418 ± 72 µm; p < 0.005) compared to fln⁺ (1386 ± 196µm) and fln(ΔC44)(1128 ± 193 µm). Statistical polymer chain analysis revealed that the C-terminal domain fulfills a secondary role in thick filament bending propensity. Our results indicate that the flightin amino and carboxy terminal domains make distinct contributions to thick filament biomechanics. We propose these distinct roles arise from the interplay between natural selection and sexual selection given IFM's dual role in flight and courtship behaviors.

Intrinsic disorder and multiple phosphorylations constrain the evolution of the flightin N-terminal region.

Flightin is a myosin binding phosphoprotein that originated in the ancestor to Pancrustacea ~500 MYA. In Drosophila melanogaster, flightin is essential for length determination and flexural rigidity of thick filaments. Here, we show that among 12 Drosophila species, the N-terminal region is characterized by low sequence conservation, low pI, a cluster of phosphorylation sites, and a high propensity to intrinsic disorder (ID) that is augmented by phosphorylation. Using mass spectrometry, we identified eight phosphorylation sites within a 29 amino acid segment in the N-terminal region of D. melanogaster flightin. We show that phosphorylation of D. melanogaster flightin is modulated during flight and, through a comparative analysis to orthologs from other Drosophila species, we found phosphorylation sites that remain invariant, sites that retain the charge character, and sites that are clade-specific. While the number of predicted phosphorylation sites differs across species, we uncovered a conserved pattern that relates the number of phosphorylation sites to pI and ID. Extending the analysis to orthologs of other insects, we found additional conserved features in flightin despite the near absence of sequence identity. Collectively, our results demonstrate that structural constraints demarcate the evolution of the highly variable N-terminal region.

Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster.

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 Ca(2+)-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 Ca(2+) 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-up(2) (hdp(2)), 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.

Mass processing--an improved technique for protein identification with mass spectrometry data.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis following tryptic digestion of polyacrylamide gel pieces is a common technique used to identify proteins. This approach is rapid, sensitive, and user friendly, and is becoming widely available to scientists in a variety of biological fields. Here we introduce a simple and effective strategy called "mass processing" where the list of masses generated from a mass spectrometer undergoes two stages of data reduction before identification. Mass processing improves the ability to identify in-gel tryptic-digested proteins by reducing the number of nonsample masses submitted to protein identification database search engines. Our results demonstrate that mass processing improves the statistical score and rank of putative protein identifications, especially with low-quantity samples, thus increasing the ability to confidently identify proteins with mass spectrometry data.

Flightin is a myosin rod binding protein.

The assembly of striated muscle myosin into thick filaments of precise and regular length requires the assistance of accessory proteins. Drosophila indirect flight muscle (IFM) contain flightin, a 20-kDa protein that has been shown to be essential for flight, for maintenance of sarcomeric integrity in active muscle, and informative in length determination of thick filaments during IFM development. Additionally, a point mutation in the myosin rod (Mhc(13)) negates flightin accumulation in the IFM in vivo. The manner in which flightin interacts with thick filaments is not known. Here, two different solid-state binding assays demonstrate that flightin binds to myosin and to a recombinant fragment of the myosin rod that include the COOH-terminal 600 amino acids (zone 19 to tail piece). The interaction of flightin and myosin is abolished by the single amino acid substitution in Mhc(13) at position 1e of zone 27 of the rod (residue 1554). The molar ratio of flightin to myosin is approx 1 : 1 to 1 : 2. Thus, the instability of thick filaments seen in vivo in the absence of flightin suggests that the flightin myosin interaction is critical for maintaining sarcomere integrity in active muscle.