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1.
Parasitol Int ; 86: 102444, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34464754

RESUMO

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.


Assuntos
Endocitose , Miosinas/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/fisiologia , Miosinas/metabolismo , Proteínas de Protozoários/metabolismo
2.
Elife ; 102021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34698017

RESUMO

A key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, we identify, for the first time, that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in: increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascin's actin bundling activity is required to limit Myosin activation. Surprisingly, we find that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to uncover that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This understudied means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration.


Assuntos
Proteínas de Transporte/genética , Movimento Celular/fisiologia , Drosophila melanogaster/fisiologia , Proteínas dos Microfilamentos/genética , Miosinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Drosophila melanogaster/genética , Proteínas dos Microfilamentos/metabolismo
3.
Nat Commun ; 12(1): 5272, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489440

RESUMO

The normal function of heart muscle depends on its ability to contract more strongly at longer length. Increased venous filling stretches relaxed heart muscle cells, triggering a stronger contraction in the next beat- the Frank-Starling relation. Conversely, heart muscle cells are inactivated when they shorten during ejection, accelerating relaxation to facilitate refilling before the next beat. Although both effects are essential for the efficient function of the heart, the underlying mechanisms were unknown. Using bifunctional fluorescent probes on the regulatory light chain of the myosin motor we show that its N-terminal domain may be captured in the folded OFF state of the myosin dimer at the end of the working-stroke of the actin-attached motor, whilst its C-terminal domain joins the OFF state only after motor detachment from actin. We propose that sequential folding of myosin motors onto the filament backbone may be responsible for shortening-induced de-activation in the heart.


Assuntos
Miocárdio/metabolismo , Miosinas/metabolismo , Animais , Cálcio/metabolismo , Masculino , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Ratos Wistar , Sarcômeros/metabolismo
4.
PLoS One ; 16(9): e0244768, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495964

RESUMO

To gain insight on the impact of preventive exercise during pulmonary arterial hypertension (PAH), we evaluated the gene expression of myosins and gene-encoding proteins associated with the extracellular matrix remodeling of right hypertrophied ventricles. We used 32 male Wistar rats, separated in four groups: Sedentary Control (S, n = 8); Control with Training (T, n = 8); Sedentary with Pulmonary Arterial Hypertension (SPAH, n = 8); and Pulmonary Arterial Hypertension with Training (TPAH, n = 8). All rats underwent a two-week adaptation period; T and TPAH group rats then proceeded to an eight-week training period on a treadmill. At the beginning of the 11th week, S and T groups received an intraperitoneal injection of saline, and SPAH and TPAH groups received an injection of monocrotaline (60 mg/kg). Rats in the T and TPAH groups then continued with the training protocol until the 13th week. We assessed exercise capacity, echocardiography analysis, Fulton's index, cross-sectional areas of cardiomyocytes, collagen content and types, and fractal dimension (FD). Transcript abundance of myosins and extracellular matrix genes were estimated through reverse transcription-quantitative PCR (RT-qPCR). When compared to the SPAH group, the TPAH group showed increases in functional capacity and pulmonary artery acceleration time/pulmonary ejection time ratio and decreases in Fulton's index and cross-sectional areas of myocyte cells. However, preventive exercise did not induce alterations in col1a1 and myh7 gene expression. Our findings demonstrate that preventive exercise improved functional capacity, reduced cardiac hypertrophy, and attenuated PH development without interfering in mRNA-encoding myosin and collagen expression during PAH.


Assuntos
Hipertensão Arterial Pulmonar , Animais , Colágeno/metabolismo , Hipertensão Pulmonar , Masculino , Miosinas/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Remodelação Ventricular
5.
Elife ; 102021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34569933

RESUMO

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.


Assuntos
Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Imagem Individual de Molécula/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Músculo Estriado/metabolismo , Ligação Proteica , Processos Estocásticos , Troponina/metabolismo
6.
J Biol Chem ; 297(4): 101157, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34481842

RESUMO

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.


Assuntos
Benzilaminas/química , Transferência Ressonante de Energia de Fluorescência , Miosinas/química , Uracila/análogos & derivados , Motivos de Aminoácidos , Animais , Benzilaminas/uso terapêutico , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/metabolismo , Bovinos , Cinética , Miosinas/metabolismo , Uracila/química , Uracila/uso terapêutico
7.
Elife ; 102021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34519272

RESUMO

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Animais , Diferenciação Celular , Fusão Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos Esqueléticos/patologia , Miosinas/genética , Pseudópodes/genética , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Fatores de Tempo
8.
J Biol Chem ; 297(3): 101066, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34384781

RESUMO

The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input "open" state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured "closed" state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/fisiologia , Dineínas/metabolismo , Proteínas de Choque Térmico/fisiologia , Hidrólise , Cinesina/metabolismo , Cinética , Modelos Moleculares , Miosinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Relação Estrutura-Atividade
9.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204776

RESUMO

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin's ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin-tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin-tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.


Assuntos
Contração Muscular/genética , Mutação/genética , Oximas/farmacologia , Sulfonamidas/farmacologia , Tropomiosina/genética , Actinas/metabolismo , Animais , Cálcio/metabolismo , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miosinas/metabolismo , Coelhos , Troponina/metabolismo
10.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299379

RESUMO

Myofibroblasts are contractile cells found in multiple tissues. They are physiological cells as in the human placenta and can be obtained from bone marrow mesenchymal stem cells after differentiation by transforming growth factor-ß (TGF-ß). They are also found in the stroma of cancerous tissues and can be located in non-muscle contractile tissues. When stimulated by an electric current or after exposure to KCl, these tissues contract. They relax either by lowering the intracellular Ca2+ concentration (by means of isosorbide dinitrate or sildenafil) or by inhibiting actin-myosin interactions (by means of 2,3-butanedione monoxime or blebbistatin). Their shortening velocity and their developed tension are dramatically low compared to those of muscles. Like sarcomeric and smooth muscles, they obey Frank-Starling's law and exhibit the Hill hyperbolic tension-velocity relationship. The molecular motor of the myofibroblast is the non-muscle myosin type IIA (NMIIA). Its essential characteristic is the extreme slowness of its molecular kinetics. In contrast, NMIIA develops a unitary force similar to that of muscle myosins. From a thermodynamic point of view, non-muscle contractile tissues containing NMIIA operate extremely close to equilibrium in a linear stationary mode.


Assuntos
Contração Muscular/fisiologia , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Miofibroblastos/metabolismo , Miofibroblastos/fisiologia , Miosinas/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Humanos , Cinética , Termodinâmica
11.
Nat Commun ; 12(1): 4595, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321459

RESUMO

Constriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that anillin, a non-motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. Our results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring.


Assuntos
Actinas/metabolismo , Proteínas Contráteis/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Divisão Celular , Proteínas Contráteis/genética , Citocinese , Drosophila melanogaster/metabolismo , Humanos , Proteínas dos Microfilamentos
12.
J Vis Exp ; (173)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34279513

RESUMO

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force. By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.


Assuntos
Actinas , Miosinas , Actinas/metabolismo , Dineínas , Miosinas/metabolismo , Pinças Ópticas , Análise Espectral
13.
Elife ; 102021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34212861

RESUMO

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1-PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1, and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Membrana Celular/fisiologia , Citoesqueleto/fisiologia , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Miosinas/genética , Miosinas/metabolismo , Fosfoproteínas Fosfatases/genética , Proteína Quinase C/genética , Quinases Ativadas por p21/genética
14.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064895

RESUMO

Skeletal muscles, being one of the most abundant tissues in the body, are involved in many vital processes, such as locomotion, posture maintenance, respiration, glucose homeostasis, etc. Hence, the maintenance of skeletal muscle mass is crucial for overall health, prevention of various diseases, and contributes to an individual's quality of life. Prolonged muscle inactivity/disuse (due to limb immobilization, mechanical ventilation, bedrest, spaceflight) represents one of the typical causes, leading to the loss of muscle mass and function. This disuse-induced muscle loss primarily results from repressed protein synthesis and increased proteolysis. Further, prolonged disuse results in slow-to-fast fiber-type transition, mitochondrial dysfunction and reduced oxidative capacity. Glycogen synthase kinase 3ß (GSK-3ß) is a key enzyme standing at the crossroads of various signaling pathways regulating a wide range of cellular processes. This review discusses various important roles of GSK-3ß in the regulation of protein turnover, myosin phenotype, and oxidative capacity in skeletal muscles under disuse/unloading conditions and subsequent recovery. According to its vital functions, GSK-3ß may represent a perspective therapeutic target in the treatment of muscle wasting induced by chronic disuse, aging, and a number of diseases.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Elevação dos Membros Posteriores , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Miosinas/metabolismo , Estresse Oxidativo , Proteólise , Animais , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Fenótipo
15.
J Gen Physiol ; 153(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34115104

RESUMO

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.


Assuntos
Proteínas de Ligação a Calmodulina , Bexiga Urinária , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Camundongos , Contração Muscular , Músculo Liso/metabolismo , Miosinas/metabolismo , Fosforilação
16.
PLoS One ; 16(6): e0248256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34106923

RESUMO

Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on in vitro substrate stiffness, which is an analog for in vivo tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also investigated two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both substrate stiffness and contractile phenotype, with intermediate contractility and high substrate stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of substrate stiffness and cell contractility on FN fibril assembly.


Assuntos
Actinas/química , Fibronectinas/ultraestrutura , Miofibrilas/ultraestrutura , Simulação por Computador , Elasticidade , Fibronectinas/química , Fibronectinas/fisiologia , Contração Muscular , Miofibrilas/química , Miofibrilas/fisiologia , Miosinas/metabolismo
17.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34088833

RESUMO

Changes in the molecular properties of cardiac myosin strongly affect the interactions of myosin with actin that result in cardiac contraction and relaxation. However, it remains unclear how myosin molecules work together in cardiac myofilaments and which properties of the individual myosin molecules impact force production to drive cardiac contractility. Here, we measured the force production of cardiac myofilaments using optical tweezers. The measurements revealed that stepwise force generation was associated with a higher frequency of backward steps at lower loads and higher stall forces than those of fast skeletal myofilaments. To understand these unique collective behaviors of cardiac myosin, the dynamic responses of single cardiac and fast skeletal myosin molecules, interacting with actin filaments, were evaluated under load. The cardiac myosin molecules switched among three distinct conformational positions, ranging from pre- to post-power stroke positions, in 1 mM ADP and 0 to 10 mM phosphate solution. In contrast to cardiac myosin, fast skeletal myosin stayed primarily in the post-power stroke position, suggesting that cardiac myosin executes the reverse stroke more frequently than fast skeletal myosin. To elucidate how the reverse stroke affects the force production of myofilaments and possibly heart function, a simulation model was developed that combines the results from the single-molecule and myofilament experiments. The results of this model suggest that the reversal of the cardiac myosin power stroke may be key to characterizing the force output of cardiac myosin ensembles and possibly to facilitating heart contractions.


Assuntos
Contração Miocárdica , Miocárdio/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Animais , Suínos
18.
Elife ; 102021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34121660

RESUMO

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.


Assuntos
Contração Muscular/fisiologia , Músculo Esquelético , Miosinas , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/química , Músculo Esquelético/fisiologia , Miosinas/química , Miosinas/metabolismo , Miosinas/fisiologia , Sarcômeros/química , Sarcômeros/fisiologia
19.
Proc Biol Sci ; 288(1950): 20202895, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33975478

RESUMO

Muscle contraction results from force-generating cross-bridge interactions between myosin and actin. Cross-bridge cycling kinetics underlie fundamental contractile properties, such as active force production and energy utilization. Factors that influence cross-bridge kinetics at the molecular level propagate through the sarcomeres, cells and tissue to modulate whole-muscle function. Conversely, movement and changes in the muscle length can influence cross-bridge kinetics on the molecular level. Reduced, single-molecule and single-fibre experiments have shown that increasing the strain on cross-bridges may slow their cycling rate and prolong their attachment duration. However, whether these strain-dependent cycling mechanisms persist in the intact muscle tissue, which encompasses more complex organization and passive elements, remains unclear. To investigate this multi-scale relationship, we adapted traditional step-stretch protocols for use with mouse soleus muscle during isometric tetanic contractions, enabling novel estimates of length-dependent cross-bridge kinetics in the intact skeletal muscle. Compared to rates at the optimal muscle length (Lo), we found that cross-bridge detachment rates increased by approximately 20% at 90% of Lo (shorter) and decreased by approximately 20% at 110% of Lo (longer). These data indicate that cross-bridge kinetics vary with whole-muscle length during intact, isometric contraction, which could intrinsically modulate force generation and energetics, and suggests a multi-scale feedback pathway between whole-muscle function and cross-bridge activity.


Assuntos
Contração Isométrica , Miosinas , Animais , Cinética , Camundongos , Contração Muscular , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Sarcômeros
20.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34013964

RESUMO

Myosin XIX (Myo19) is an actin-based motor that competes with adaptors of microtubule-based motors for binding to the outer mitochondrial transmembrane proteins Miro1 and Miro2 (collectively Miro, also known as RhoT1 and RhoT2, respectively). Here, we investigate which mitochondrial and cellular processes depend on the coordination of Myo19 and microtubule-based motor activities. To this end, we created Myo19-deficient HEK293T cells. Mitochondria in these cells were not properly fragmented at mitosis and were partitioned asymmetrically to daughter cells. Respiratory functions of mitochondria were impaired and ROS generation was enhanced. On a cellular level, cell proliferation, cytokinesis and cell-matrix adhesion were negatively affected. On a molecular level, Myo19 regulates focal adhesions in interphase, and mitochondrial fusion and mitochondrially associated levels of fission protein Drp1 and adaptor proteins dynactin and TRAK1 at prometaphase. These alterations were due to a disturbed coordination of Myo19 and microtubule-based motor activities by Miro.


Assuntos
Actinas , Miosinas , Actinas/genética , Actinas/metabolismo , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miosinas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
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