Muscle Myosin Research Articles

A simple and rapid preparation of smooth muscle myosin 2 for the electron microscopic analysis.

There has been an increase in the demand for purified protein as a result of recent developments in the structural biology of myosin 2. Although promising, current practices in myosin purification are usually time-consuming and cumbersome. The reported increased actin to myosin ratio in smooth muscles adds to the complexity of the purification process. Present study outlines a streamlined approach to isolate smooth muscle myosin 2 molecules from actomyosin suspension of chicken gizzard tissues. The procedure entails treating actomyosin for a brief period with actin-binding peptide phalloidin, followed by co-sedimentation and short column size exclusion chromatography. Typical myosin molecule with heavy and light chains and approximately 95% purity was examined using gel electrophoresis. Negative staining electron microscopy and image processing showed intact 10S myosin 2 molecules, proving that phalloidin is effective at eliminating majority of actin in the form of F-actin without dramatic alteration in the structure of myosin. The entire purification discussed here can be completed in a few hours, and further analysis can be done the same day. Thus, by offering quick and fresh supplies of native myosin molecules suited for structural research, specially cryo-electron microscopy, this innovative approach can be adapted to get around the drawbacks of time-intensive myosin purifying processes.

Aging related decreases in NM myosin expression and contractility in a resistance vessel.

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging.

Moderate dietary restriction delays the onset of age-associated sarcopenia in Caenorhabditis elegans due to reduced myosin UNC-54 degradation

Sarcopenia, a gradual decrease in skeletal muscle mass and strength, is a major component of frailty in the elderly, with age, (lack of) exercise and diet found to be the major risk factors. The nematode Caenorhabditis elegans is an important model of sarcopenia. Although many studies describe loss of muscle function in ageing C. elegans, surprisingly few report on the loss of muscle mass. Here, in order to quantify loss of muscle mass under various dietary restriction (DR) conditions, we used an internal GFP standard to determine levels of the major body wall muscle myosin (UNC-54) in transgenic unc-54::gfp worms over their lifespan. Myosin density linearly increased during the first week of adulthood and there was no significant effect of DR. In contrast, an exponential decrease in myosin density was seen during the second week of adulthood, with reduced rates of myosin loss for mild and medium DR compared to control. UNC-54 turnover rates, previously determined using pulse-labelling methods, correspond well with the t1/2 value found here for UNC-54-GFP using fluorescence (control t1/2 = 12.0 days), independently validating our approach. These data indicate that sarcopenia is delayed in worms under mild and medium DR due to a reduced rate of myosin UNC-54 degradation, thereby maintaining protein homeostasis.

Network pharmacology combined with an animal model to reveal the material basis and mechanism of Amomum villosum in alleviating constipation in mice

Constipation is a prevalent gastrointestinal disorder, with its prevalence showing an annual upward trend. There are many factors involved in the occurrence of constipation, such as abnormal smooth muscle contraction and disorders of gastrointestinal hormone secretion. Amomum villosum (A. villosum) has been proven to be effective in improving digestive system diseases, but there is no report on improving constipation. Therefore, we used network pharmacology prediction combined with animal experiments to explore the key active components of A. villosum and their pharmacological mechanisms. The results of network pharmacological prediction showed that β-sitosterol was the key laxative compound of A. villosum, which may play a laxative role by activating the adrenoceptor alpha 1 A-myosin light chain (ADRA1A-MLC) pathway. Further animal experiments showed that β-sitosterol could significantly shorten the time to first black stool; increase faecal weight, faecal number, and faecal water content; and promote gastrointestinal motility. β-sitosterol may promote intestinal motility by upregulating the expression of ADRA1A and myosin light chain 9 (Myl9) mRNA and protein in the colon, thereby activating the ADRA1A-MLC signalling pathway. In addition, it is possible to improve constipation symptoms by regulating serum neurotransmitters and gastrointestinal motility-related factors, such as the serum content of 5-hydroxytryptamine (5-HT) and acetylcholinesterase (AchE) and the mRNA expression of 5-hydroxytryptamine receptor 4 (5-HT4), stem cell factor (SCF), stem cell factor receptor (c-Kit) and smooth muscle myosin light chain kinase (smMLCK) in the colon. These results lay a foundation for the application of A. villosum and β-sitosterol in constipation.

Selective fast fiber damage after leg press exercise leading to failure: a pole vaulter case report

The aim of this study was to investigate, in a trained pole vaulter (PV) and in an endurance-trained physical education student (PE), the effect of a leg press exercise leading to failure (LPF) on changes in serum activity of muscle enzymes and serum concentration of fast (FM) and slow (SM) myosin isoforms, while simultaneously examining mechanical output components as indicators of performance and fatigue developed throughout exercise. A case report study design based on an observational comparison of response between two dichotomous participants, PV and PE, was used. Differences between the participants’ exercise outputs were examined by unpaired t-test or Mann-Whitney test and serum levels of muscle enzymes and myosin isoforms were analyzed at baseline and 24 and 48 hours after LPF. Exercise output analyses showed that the PV’s average fatigue index was significantly higher (P = 0.004). Moreover, during the first six sets, the concentric average power exerted by the PV was significantly (P < 0.01) higher (range: 14% to 35%) than that of the PE. The PV only showed acute mild increases of serum creatine kinase (CK) and FM 24 hours after exercise. In contrast, the PE presented persistent serum rises of several muscle enzymes and SM until 48 h after exercise. The PV’s exercise output revealed an explosive (power-oriented) profile leading to selective mild damage of fast fibers. In contrast, the PE exercise output showed a fatigue-resistant profile, which induced greater muscle enzyme activity and SM serum concentration, suggesting a higher extent of slow fiber damage. Keywords: muscle damage, creatine kinase, myosin isoforms, power output.

Discovery of the first unconventional myosin: Acanthamoeba myosin-I.

Having characterized actin from Acanthamoeba castellanii (Weihing and Korn, Biochemistry, 1971, 10, 590-600) and knowing that myosin had been isolated from the slime mold Physarum (Hatano and Tazawa, Biochim. Biophys. Acta, 1968, 154, 507-519; Adelman and Taylor, Biochemistry, 1969, 8, 4976-4988), we set out in 1969 to find myosin in Acanthamoeba. We used K-EDTA-ATPase activity to assay myosin, because it is a unique feature of muscle myosins. After slightly less than 3years, we purified a K-EDTA ATPase that interacted with actin. Actin filaments stimulated the Mg-ATPase activity of the crude enzyme, but this was lost with further purification. Recombining fractions from the column where this activity was lost revealed a "cofactor" that allowed actin filaments to stimulate the Mg-ATPase of the purified enzyme. The small size of the heavy chain and physical properties of the purified myosin were unprecedented, so many were skeptical, assuming that our myosin was a proteolytic fragment of a larger myosin similar to muscle or Physarum myosin. Subsequently our laboratories confirmed that Acanthamoeba myosin-I is a novel unconventional myosin that interacts with membrane lipids (Adams and Pollard, Nature, 1989, 340 (6234), 565-568) and that the cofactor is a myosin heavy chain kinase (Maruta and Korn, J. Biol. Chem., 1977, 252, 8329-8332). Phylogenetic analysis (Odronitz and Kollmar, Genome Biology, 2007, 8, R196) later established that class I myosin was the first myosin to appear during the evolution of eukaryotes.

Front Cover Image

ON THE FRONT COVER: Confocal image of the central quail myotube expressing mNeonGreen fluorescent protein linked to muscle myosin light chain 2 (green) and stained with phalloidin (red) reveals the premyofibrils, nascent and mature myofibrils extending in order from the extending edge during myofibrillogenesis.Credit: Jushuo Wang, Department of Cell and Developmental Biology, SUNY Upstate Medical University.image

Structure of the Drosophila melanogaster Flight Muscle Myosin Filament at 4.7 Å Resolution Reveals New Details of Non-Myosin Proteins.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long α-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. Here we report on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. Our results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which our results can define strategies for functional testing.

The influence of biological sex in human skeletal muscle transcriptome during ageing.

Sex is a crucial biological variable, and influence of biological sex on the change of gene expression in ageing skeletal muscle has not yet been fully revealed. In this study, the mRNA expression profiles were obtained from the Gene Expression Omnibus database. Key genes were identified by differential expression analysis and weighted gene co-expression network analysis. The gene set enrichment analysis software and Molecular Signatures Database were used for functional and enrichment analysis. A protein-protein interaction network was constructed using STRING and visualized in Cytoscape. The results were compared between female and male subgroups. Differentially expressed genes and enriched pathways in different sex subgroups shared only limited similarities. The pathways enriched in the female subgroup were more similar to the pathways enriched in the older groups without taking sex difference into consideration. The pathways enriched in the female subgroup were more similar to the pathways enriched in the older groups without taking sex difference into consideration. The muscle myosin filament pathways were downregulated in the both aged female and male samples whereas transforming growth factor beta pathway and extracellular matrix-related pathways were upregulated. With muscle ageing, the metabolism-related pathways, protein synthesis and degradation pathways, results of predicted immune cell infiltration, and gene cluster associated with slow-type myofibers drastically different between the female and male subgroups. This finding may indicate that changes in muscle type with ageing may differ between the sexes in vastus lateralis muscle.

PB0592 Evaluation of the Role of Skeletal Muscle Myosin as a Cofactor for Factor XI Activation of Factor XII

PB0592 Evaluation of the Role of Skeletal Muscle Myosin as a Cofactor for Factor XI Activation of Factor XII

The term “classic invasive lobular carcinoma” of the breast defines breast malignancies of vastly different nature

PurposeTo describe in detail the special features of a previously unappreciated “classic invasive lobular carcinoma” which is confined to the terminal ductal lobular units (TDLUs) and differs considerably from the extensive classic invasive lobular carcinoma, and to suggest specific terminology. MethodAll invasive breast cancer cases without associated microcalcifications diagnosed in our Institution with the histopathologic diagnosis of classic invasive lobular carcinoma during the years 1996–2019 (n = 560) formed the basis of this study. The cases were prospectively classified according to their imaging biomarkers (mammographic features) and followed up to Dec 31, 2021, to determine long-term patient outcome. An additional 2600 invasive breast cancer cases (diagnosed other than invasive lobular carcinoma) without associated microcalcifications served as a reference group. Detailed histopathologic analysis used large format (10x8 cm) thin section technique and staining methods including hematoxylin-eosin (H&E), E-cadherin, cytokeratin CK 5/6, a transmembrane glycoprotein (CD44) and anti-actin or anti-smooth muscle myosin heavy chain. ResultsThe imaging biomarkers differentiated two separate disease subgroups, having the same histopathologic diagnosis, classic invasive lobular carcinoma. One of these has the imaging biomarker of extensive architectural distortion with no central tumour mass, occupies the extralobular mesenchyme and has a long-term survival of 56%. The other subgroup forms stellate or circular non-calcified tumour masses usually smaller than 20 mm, which appear to arise in the intralobular mesenchyme, and has a significantly better long-term survival of 84%. ConclusionsThere is a striking difference between the subgross histopathology and the mammographic appearance (imaging biomarkers) of two breast malignancies having the same histopathologic diagnosis, “classic invasive lobular carcinoma”. The large difference in the long-term outcome of these two tumour types is even more striking. Using the same specific term, “classic invasive lobular carcinoma”, to describe these two separate entities can adversely affect management decisions.

A-Band assembly in avian skeletal muscles observed with super-resolution microscopy.

Myofibrils in vertebrate skeletal muscle are organized in aligned arrays of filaments formed from multiple protein components. Despite considerable information describing individual proteins, how they assemble de novo into mature myofibrils is still a challenge. Studies in our lab of sarcomeric protein localization during myofibril assembly led us to propose a three-step progression: premyofibrils to nascent myofibrils, culminating in mature myofibrils. Premyofibrils, forming at the spreading edges of muscle cells, are composed of minisarcomeres containing small bands of non-muscle myosin II filaments alternating with muscle-specific α-actinin Z-Bodies attached to barbed ends of actin filaments, establishing bipolar F-actin arrangements in sarcomeres. Assembly of nascent myofibrils occurs with addition of muscle-specific myosin II, F-actin, titin, and the alignment of Z-Bodies in adjacent fibrils to form beaded Z-Bands. Muscle-specific myosin II filaments in nascent myofibrils appear in an overlapping arrangement when viewed with wide-field and confocal microscopes. In mature myofibrils, non-muscle myosin II is absent, and M-Band proteins localize to the muscle myosin II filaments, aiding their alignment by cross-linking them into A-Bands. Super-resolution microscopy (SIM and STED) revealed muscle myosin II in mini-A-Bands in nascent myofibrils. In contrast to previous reports that vertebrate muscle myosin thick filaments form at their final 1.6 μm lengths, mini-A-Bands are first detected at a length of about 0.4 μm, and gradually increase four-fold in length to 1.6 μm in mature myofibrils. These new discoveries in avian skeletal muscle cells share a common characteristic with invertebrate muscles where some A-Bands can grow to lengths reaching 25 μm.

Immunohistochemical Staining Characteristics of Low-Grade Invasive Ductal Carcinoma Using the ADH5 Cocktail (CK5/14, P63, and CK7/18): A Potential Interpretative Pitfall.

Background: In our practice, the antibody cocktail ADH5 (CK5/14, p63, and CK7/18) helps with diagnostic challenges, such as identifying microinvasion and foci of invasive carcinoma, differentiating atypical ductal hyperplasia from hyperplasia of the usual type, and distinguishing basal phenotypes in triple-negative carcinomas. However, the ADH5 cocktail does have pitfalls and caveats. Methods: We describe our experience with the ADH5 cocktail of antibodies in breast pathology. Institutional knowledge and a literature search form our data sources. Results: We analyzed 44 cases. Four out of a total of 44 cases (9.1%)-two tubular carcinomas and two low-grade invasive breast carcinomas of no special type (ductal) with tubular features-showed an expected pattern of staining for ADH5 with a loss of brown (P63, CK5/14) staining around invasive glands and diffuse red (CK7/18) expression. Forty out of 44 (90.9%) cases showed an unexpected staining pattern (mixture of cytoplasmic brown and red). All 44 cases (100%) showed negative myoepithelial staining around invasive foci when separately stained for P63 and SMMH (Smooth Muscle Myosin Heavy). Conclusions: The unexpected staining pattern of ADH5 in low-grade invasive ductal carcinomas can be challenging to interpret in these lesions with low-grade cytology. The occurrence can cause confusion among users who employ multiplex stains, and it is important for users to be aware of this potential pitfall.

P90RSK2, a new MLCK mediates contractility in myosin light chain kinase null smooth muscle.

Introduction: Phosphorylation of smooth muscle (SM) myosin regulatory light chain (RLC20) is a critical switch leading to SM contraction. The canonical view held that only the short isoform of myosin light chain kinase (MLCK1) catalyzed this reaction. It is now accepted that auxiliary kinases may contribute to vascular SM tone and contractility. We have previously reported that p90 ribosomal S6 kinase (RSK2) functions as such a kinase, in parallel with MLCK1, contributing ∼25% of the maximal myogenic force in resistance arteries. Thus, RSK2 may be instrumental in the regulation of basal vascular tone and blood pressure. Here, we take advantage of a MLCK1 null mouse (mylk1 -/-) to further test our hypothesis that RSK2 can function as an MLCK, playing a significant physiological role in SM contractility. Methods: Using fetal (E14.5-18.5) SM tissues, as embryos die at birth, we investigated the necessity of MLCK for contractility and fetal development and determined the ability of RSK2 kinase to compensate for the lack of MLCK and characterized its signaling pathway in SM. Results and Discussion: Agonists induced contraction and RLC20 phosphorylation in mylk1 -/- SM was attenuated by RSK2 inhibition. The pCa-tension relationships in permeabilized strips of bladder showed no difference in Ca2+ sensitivity in WT vs mylk1 -/- muscles, although the magnitude of force responses was considerably smaller in the absence of MLCK. The magnitude of contractile responses was similar upon addition of GTPγS to activate the RhoA/ROCK pathway or calyculinA to inhibit the myosin phosphatase. The Ca2+-dependent tyrosine kinase, Pyk2, contributed to RSK2-mediated contractility and RLC20 phosphorylation. Proximity-ligation and immunoprecipitation assays demonstrated an association of RSK2, PDK1 and ERK1/2 with MLCK and actin. RSK2, PDK1, ERK1/2 and MLCK formed a signaling complex on the actin filament, positioning them for interaction with adjacent myosin heads. The Ca2+-dependent component reflected the agonist mediated increases in Ca2+, which activated the Pyk2/PDK1/RSK2 signaling cascade. The Ca2+-independent component was through activation of Erk1/2/PDK1/RSK2 leading to direct phosphorylation of RLC20, to increase contraction. Overall, RSK2 signaling constitutes a new third signaling pathway, in addition to the established Ca2+/CaM/MLCK and RhoA/ROCK pathways to regulate SM contractility.

Isolation and Cultivation of Vascular Smooth Muscle Cells from the Mouse Circle of Willis

Introduction: Culturing cerebrovascular smooth muscle cells (CVSMCs) in vitro can provide a model for studying many cerebrovascular diseases. This study describes a convenient and efficient method to obtain mouse CVSMCs by enzyme digestion. Methods: Mouse circle of Willis was isolated, digested, and cultured with platelet-derived growth factor-BB (PDGF-BB) to promote CVSMC growth, and CVSMCs were identified by morphology, immunofluorescence analysis, and flow cytometry. The effect of PDGF-BB on vascular smooth muscle cell (VSMC) proliferation was evaluated by cell counting kit (CCK)-8 assay, morphological observations, Western blotting, and flow cytometry. Results: CVSMCs cultured in a PDGF-BB-free culture medium had a typical peak-to-valley growth pattern after approximately 14 days. Immunofluorescence staining and flow cytometry detected strong positive expression of the cell type-specific markers alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain 11 (SMMHC), smooth muscle protein 22 (SM22), calponin, and desmin. In the CCK-8 assay and Western blotting, cells incubated with PDGF-BB had significantly enhanced proliferation compared to those without PDGF-BB. Conclusion: We obtained highly purified VSMCs from the mouse circle of Willis using simple methods, providing experimental materials for studying the pathogenesis and treatment of neurovascular diseases in vitro. Moreover, the experimental efficiency improved with PDGF-BB, shortening the cell cultivation period.

Sex differences in skeletal muscle size, function, and myosin heavy chain isoform expression during post-injury regeneration in mice.

Skeletal muscle regeneration is an essential process to restore muscle function after injury and is influenced by various factors. Despite the known importance of sex hormones in muscle regeneration, whether and what sex difference exists in this process is still unclear. In this study, we provide evidence for a clear sex difference in muscle regeneration in mice. At 7 and 14 days after barium chloride-induced muscle injury, female mice showed a faster recovery of muscle fiber size than males. Consistently, muscle force in female mice was restored faster than in males after injury, and this functional difference was maintained at 14 months of age when regenerative capacity declined. Myosin heavy chain isoform profiling and fatigability test revealed dynamic remodeling of myosin heavy chain isoform expression including a type IIB to IIA/X MHC transition and reduced fatigability in regenerated muscles compared to uninjured muscles. A significant sex difference was detected in myosin heavy chain IIX content, although this did not lead to different fatigability. Together, our results suggest that sex is an important determinant of the recovery of regenerating skeletal muscle and is partially involved in the remodeling of myosin heavy chain isoforms during muscle regeneration.

First report of primary testicular leiomyosarcoma in two dogs

BackgroundTesticular tumours are common in dogs and, among them, interstitial cell tumours, seminomas and sustentacular cell tumours are the most reported. Mesenchymal testicular tumours are rarely reported in humans as in veterinary medicine where only three cases of sarcomas (leiomyomas and leomyosarcomas) have been described in two stallions and in a ram.Case presentationThe present cases regarded a 12-year-old mixed-breed dog and a 10-year-old American Staffordshire Terrier that underwent bilateral orchiectomy. Formalin fixed testes were referred for histopathological diagnosis. At gross examination, in one of the testes of both dogs, a white, firm and variably cystic testicular mass, effacing and replacing the testicular parenchyma was detected. Samples were collected from both neoplastic and contralateral testes, routinely processed for histology and serial sections were also examined immunohistochemically with primary antibodies against cytokeratins, vimentin, Von Willebrand factor, inhibin-α, α-smooth muscle actin, smooth muscle myosin and desmin. Histopathological features as well as the immunohistochemical results, positive for vimentin, actin, myosin and desmin, confirmed the mesenchymal origin and the myoid phenotype of both testicular tumours supporting the diagnoses of leiomyosarcoma.ConclusionsTo the authors knowledge these are the first cases of primary testicular sarcoma reported in the canine species. However, even rare, these tumours deserve to be considered in routine diagnosis when a testicular spindle cell tumour is observed. The immunohistochemical panel applied was useful to distinguish the present tumours from undifferentiated Sertoli cell tumours confirming the diagnosis of leiomyosarcoma.

Reversible suspension culture of human vascular smooth muscle cells using the functional biopolymer FP003.

Human vascular smooth muscle cells (SMCs) are adherent cells, and they cannot survive without scaffolds in suspension culture. Here, we aimed to establish a suspension culture of SMCs using the functional biopolymer FP003 and to investigate the proliferation status of the cells. When SMCs were suspension cultured with FP003, their proliferation was inhibited with a viability of 75% until day 15. When SMCs were re-plated on plastic plates after suspension culture with FP003 for 48h, the SMCs proliferated as in a normal plate culture. The SMCs cultured in suspension with FP003 showed a relatively low phosphorylation of retinoblastoma protein, low expression of cyclin D1, high proportion of G0/G1 phase cells, low proportion of S phase cells, and no obvious signs of apoptosis, indicating that this culture system inhibited progression from the G1 to S phase. This growth arrest was a reversible property that showed no significant changes in the expressions of the marker proteins α-smooth muscle actin and smooth muscle myosin heavy chain. These results suggest that human SMCs can be stably cultured in suspension with FP003 without losing their characteristics when they are cultured on plastic plates again.

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

Theoretical efficiency limits and speed-efficiency trade-off in myosin motors.

Muscle myosin is a non-processive molecular motor generates mechanical work when cooperating in large ensembles. During its cyle, each individual motor keeps attaching and detaching from the actin filament. The random nature of attachment and detachment inevitably leads to losses and imposes theoretical limits on the energetic efficiency. Here, we numerically determine the theoretical efficiency limit of a classical myosin model with a given number of mechano-chemical states. All parameters that are not bounded by physical limits (like rate limiting steps) are determined by numerical efficiency optimization. We show that the efficiency is limited by the number of states, the stiffness and the rate-limiting kinetic steps. There is a trade-off between speed and efficiency. Slow motors are optimal when most of the available free energy is allocated to the working stroke and the stiffness of their elastic element is high. Fast motors, on the other hand, work better with a lower and asymmetric stiffness and allocate a larger fraction of free energy to the release of ADP. Overall, many features found in myosins coincide with the findings from the model optimization: there are at least 3 bound states, the largest part of the working stroke takes place during the first transition, the ADP affinity is adapted differently in slow and fast myosins and there is an asymmetry in elastic elements.