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.

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.

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.

Accomplishing Career Transitions 2019: facilitating success towards the professoriate.

The Minorities Affairs Committee of the American Society for Cell Biology through its Accomplishing Career Transitions (ACT) program aims to ease critical transitions for postdocs and junior faculty from underrepresented backgrounds in STEM or from minority-serving institutions as they work towards promotion and tenure at a wide range of academic institutions. The ACT program is a 2-year cohort-based professional and skills development program that kicks off with a summer workshop and continues with additional online training sessions on selected topics, forging the creation of a permanent mentoring community for the participants. In this BMC Proceedings Supplement, we highlight selected content from the first ACT summer workshop held in 2019 at the Rizzo Center in Chapel Hill, NC. The goal of this BMC Proceedings Supplement is to amplify impact of ACT programming in a way that transcends the ACT Fellow community to benefit an increased number of scientists.

Multi-institutional study of GRE scores as predictors of STEM PhD degree completion: GRE gets a low mark.

The process of selecting students likely to complete science, technology, engineering and mathematics (STEM) doctoral programs has not changed greatly over the last few decades and still relies heavily on Graduate Record Examination (GRE) scores in most U.S. universities. It has been long debated whether the GRE is an appropriate selection tool and whether overreliance on GRE scores may compromise admission of students historically underrepresented in STEM. Despite many concerns about the test, there are few studies examining the efficacy of the GRE in predicting PhD completion and even fewer examining this question in STEM fields. For the present study, we took advantage of a long-lived collaboration among institutions in the Northeast Alliance for Graduate Education and the Professoriate (NEAGEP) to gather comparable data on GRE scores and PhD completion for 1805 U.S./Permanent Resident STEM doctoral students in four state flagship institutions. We found that GRE Verbal (GRE V) and GRE Quantitative (GRE Q) scores were similar for women who completed STEM PhD degrees and those who left programs. Remarkably, GRE scores were significantly higher for men who left than counterparts who completed STEM PhD degrees. In fact, men in the lower quartiles of GRE V or Q scores finished degrees more often than those in the highest quartile. This pattern held for each of the four institutions in the study and for the cohort of male engineering students across institutions. GRE scores also failed to predict time to degree or to identify students who would leave during the first year of their programs. Our results suggests that GRE scores are not an effective tool for identifying students who will be successful in completing STEM doctoral programs. Considering the high cost of attrition from PhD programs and its impact on future leadership for the U.S. STEM workforce, we suggest that it is time to develop more effective and inclusive admissions strategies.

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.

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.

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.

The structural and functional coordination of glycolytic enzymes in muscle: evidence of a metabolon?

Metabolism sustains life through enzyme-catalyzed chemical reactions within the cells of all organisms. The coupling of catalytic function to the structural organization of enzymes contributes to the kinetic optimization important to tissue-specific and whole-body function. This coupling is of paramount importance in the role that muscle plays in the success of Animalia. The structure and function of glycolytic enzyme complexes in anaerobic metabolism have long been regarded as a major regulatory element necessary for muscle activity and whole-body homeostasis. While the details of this complex remain to be elucidated through in vivo studies, this review will touch on recent studies that suggest the existence of such a complex and its structure. A potential model for glycolytic complexes and related subcomplexes is introduced.

Mutations of the Drosophila myosin regulatory light chain affect courtship song and reduce reproductive success.

The Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. Here, we examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (i) Dmlc2(Δ2-46) (Ext), an N-terminal extension truncation; (ii) Dmlc2(S66A,S67A) (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2(Δ2-46;S66A,S67A) (Dual), expressing both mutations. Our results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics (Phos and Dual) have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2 (+) rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight.

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.

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.

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.

Amplification of orthologous genes using degenerate primers.

This chapter describes the use of degenerate primers for PCR amplification of orthologous DNA from related species. While several methods for designing degenerate primers have been described, an important consideration is to base the design on a short region of highly conserved amino acids. Here, we present the use of a degenerate primer design strategy called Consensus-degenerate hybrid oligonucleotide primer (CODEHOP). This strategy is very useful when there is low conservation in the multiple alignments of sequences across species. We demonstrate the use of CODEHOP to amplify the entire coding region of the flightin gene from multiple Drosophila species.

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.

Flightin is necessary for length determination, structural integrity, and large bending stiffness of insect flight muscle thick filaments.

Despite the fundamental role of thick filaments in muscle contraction, little is known about the mechanical behavior of these filaments and how myosin-associated proteins dictate differences between muscle types. In this study, we used atomic force microscopy to study the morphological and mechanical properties of fully hydrated native thick filaments isolated from indirect flight muscle (IFM) of normal and mutant Drosophila lacking flightin (fln(0)). IFM thick filaments from newly eclosed (0-1 h old) wild-type flies have a mean length of 3.04+/-0.05 microm. In contrast, IFM thick filaments from newly eclosed fln(0) flies are more variable in length and, on average, are significantly longer (3.90+/-1.33 microm) than wild-type filaments from flies of the same age. In the absence of flightin, thick filaments can attain lengths >300% of wild-type filaments, indicating that flightin is required for setting the proper filament length in vivo. Filaments lacking flightin are structurally compromised, and filament preparations from fully matured 3- to 5-day-old adult fln(0) IFM yielded fragments of variable length much shorter than 3.20+/-0.04 microm, the length obtained from wild-type flies of similar age. The persistence length, an index of bending stiffness, was calculated from measurements of filament end-to-end length and contour length. We show that the presence of flightin increases persistence length by more than 40% and that wild-type filaments increase in stiffness with age. These results indicate that flightin fulfills an essential role in defining the structural and mechanical properties of IFM thick filaments.

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.