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1.
PLoS Comput Biol ; 15(7): e1007156, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287817

RESUMO

Bundled actin structures play a key role in maintaining cellular shape, in aiding force transmission to and from extracellular substrates, and in affecting cellular motility. Recent studies have also brought to light new details on stress generation, force transmission and contractility of actin bundles. In this work, we are primarily interested in the question of what determines the stability of actin bundles and what network geometries do unstable bundles eventually transition to. To address this problem, we used the MEDYAN mechano-chemical force field, modeling several micron-long actin bundles in 3D, while accounting for a comprehensive set of chemical, mechanical and transport processes. We developed a hierarchical clustering algorithm for classification of the different long time scale morphologies in our study. Our main finding is that initially unipolar bundles are significantly more stable compared with an apolar initial configuration. Filaments within the latter bundles slide easily with respect to each other due to myosin activity, producing a loose network that can be subsequently severely distorted. At high myosin concentrations, a morphological transition to aster-like geometries was observed. We also investigated how actin treadmilling rates influence bundle dynamics, and found that enhanced treadmilling leads to network fragmentation and disintegration, while this process is opposed by myosin and crosslinking activities. Interestingly, treadmilling bundles with an initial apolar geometry eventually evolve to a whole gamut of network morphologies based on relative positions of filament ends, such as sarcomere-like organization. We found that apolar bundles show a remarkable sensitivity to environmental conditions, which may be important in enabling rapid cytoskeletal structural reorganization and adaptation in response to intracellular and extracellular cues.


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Animais , Fenômenos Biomecânicos , Biologia Computacional , Simulação por Computador , Reagentes para Ligações Cruzadas , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Miosinas/química , Miosinas/fisiologia , Estabilidade Proteica
2.
Anim Sci J ; 90(7): 801-807, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31134719

RESUMO

Skeletal muscle consists of bundles of myofibers containing millions of myofibrils, each of which is formed of longitudinally aligned sarcomere structures. Sarcomeres are the minimum contractile unit, which mainly consists of four components: Z-bands, thin filaments, thick filaments, and connectin/titin. The size and shape of the sarcomere component is strictly controlled. Surprisingly, skeletal muscle cells not only synthesize a series of myofibrillar proteins but also regulate the assembly of those proteins into the sarcomere structures. However, authentic sarcomere structures cannot be reconstituted by combining purified myofibrillar proteins in vitro, therefore there must be an elaborate mechanism ensuring the correct formation of myofibril structure in skeletal muscle cells. This review discusses the role of myosin, a main component of the thick filament, in thick filament formation and the dynamics of myosin in skeletal muscle cells. Changes in the number of myofibrils in myofibers can cause muscle hypertrophy or atrophy. Therefore, it is important to understand the fundamental mechanisms by which myofibers control myofibril formation at the molecular level to develop approaches that effectively enhance muscle growth in animals.


Assuntos
Citoesqueleto/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miosinas/fisiologia , Animais , Atrofia , Hipertrofia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Miofibrilas/metabolismo , Miofibrilas/patologia , Miosinas/metabolismo , Sarcômeros/metabolismo
3.
Invest Ophthalmol Vis Sci ; 60(2): 843-851, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30807639

RESUMO

Purpose: The actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM. Methods: Short hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed. Results: CK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake. Conclusions: Inhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.


Assuntos
Humor Aquoso/fisiologia , Inativação Gênica , Microvasos/fisiologia , Miosinas/fisiologia , Malha Trabecular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Proteínas da Matriz Extracelular/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Indóis/farmacologia , Lentivirus/genética , Masculino , Metaloproteinases da Matriz/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Nanotubos , Fagocitose , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Interferência de RNA/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
4.
Plant Physiol ; 179(4): 1537-1555, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705068

RESUMO

In plants, cellulose is synthesized at the cell surface by plasma membrane (PM)-localized cellulose synthase (CESA) complexes (CSCs). The molecular and cellular mechanisms that underpin delivery of CSCs to the PM, however, are poorly understood. Cortical microtubules have been shown to interact with CESA-containing compartments and mark the site for CSC delivery, but are not required for the delivery itself. Here, we demonstrate that myosin XI and the actin cytoskeleton mediate CSC delivery to the PM by coordinating the exocytosis of CESA-containing compartments. Measurement of cellulose content indicated that cellulose biosynthesis was significantly reduced in a myosin xik xi1 xi2 triple-knockout mutant. By combining genetic and pharmacological disruption of myosin activity with quantitative live-cell imaging, we observed decreased abundance of PM-localized CSCs and reduced delivery rate of CSCs in myosin-deficient cells. These phenotypes correlated with a significant increase in failed vesicle secretion events at the PM as well as an abnormal accumulation of CESA-containing compartments at the cell cortex. Through high-resolution spatiotemporal assays of cortical vesicle behavior, we identified defects in CSC vesicle tethering and fusion at the PM. Furthermore, disruption of myosin activity reduced the delivery of several other secretory markers to the PM and reduced constitutive and receptor-mediated endocytosis. These findings reveal a previously undescribed role for myosin in vesicle secretion and cellulose production at the cytoskeleton-PM-cell wall nexus.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Exocitose , Glucosiltransferases/metabolismo , Miosinas/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular , Celulose/metabolismo , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Modelos Moleculares , Miosinas/genética , Miosinas/metabolismo
6.
Nat Commun ; 10(1): 52, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604763

RESUMO

The presence of aberrant number of centrioles is a recognized cause of aneuploidy and hallmark of cancer. Hence, centriole duplication needs to be tightly regulated. It has been proposed that centriole separation limits centrosome duplication. The mechanism driving centriole separation is poorly understood and little is known on how this is linked to centriole duplication. Here, we propose that actin-generated forces regulate centriole separation. By imposing geometric constraints via micropatterns, we were able to prove that precise acto-myosin force arrangements control direction, distance and time of centriole separation. Accordingly, inhibition of acto-myosin contractility impairs centriole separation. Alongside, we observed that organization of acto-myosin force modulates specifically the length of S-G2 phases of the cell cycle, PLK4 recruitment at the centrosome and centriole fidelity. These discoveries led us to suggest that acto-myosin forces might act in fundamental mechanisms of aneuploidy prevention.


Assuntos
Actinas/metabolismo , Ciclo Celular/fisiologia , Centríolos/metabolismo , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Actinas/fisiologia , Aneuploidia , Ciclo Celular/efeitos dos fármacos , Centríolos/fisiologia , Células HeLa , Humanos , Microscopia Intravital/métodos , Microscopia Confocal , Miosinas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Timidina/farmacologia , Imagem com Lapso de Tempo/métodos
7.
Dev Biol ; 445(1): 90-102, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30414844

RESUMO

Skeletal muscle is generated by the successive incorporation of primary (embryonic), secondary (fetal), and tertiary (adult) fibers into muscle. Conditional excision of Pitx2 function by an MCKCre driver resulted in animals with histological and ultrastructural defects in P30 muscles and fibers, respectively. Mutant muscle showed severe reduction in mitochondria and FoxO3-mediated mitophagy. Both oxidative and glycolytic energy metabolism were reduced. Conditional excision was limited to fetal muscle fibers after the G1-G0 transition and resulted in altered MHC, Rac1, MEF2a, and alpha-tubulin expression within these fibers. The onset of excision, monitored by a nuclear reporter gene, was observed as early as E16. Muscle at this stage was already severely malformed, but appeared to recover by P30 by the expansion of adjoining larger fibers. Our studies demonstrate that the homeodomain transcription factor Pitx2 has a postmitotic role in maintaining skeletal muscle integrity and energy homeostasis in fetal muscle fibers.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Feminino , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/embriologia , Miosinas/fisiologia
8.
J Cell Sci ; 132(4)2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30404824

RESUMO

Cytoskeletal networks of actin filaments and myosin motors drive many dynamic cell processes. A key characteristic of these networks is their contractility. Despite intense experimental and theoretical efforts, it is not clear what mechanism favors network contraction over expansion. Recent work points to a dominant role for the nonlinear mechanical response of actin filaments, which can withstand stretching but buckle upon compression. Here, we present an alternative mechanism. We study how interactions between actin and myosin-2 at the single-filament level translate into contraction at the network scale by performing time-lapse imaging on reconstituted quasi-2D networks mimicking the cell cortex. We observe myosin end-dwelling after it runs processively along actin filaments. This leads to transport and clustering of actin filament ends and the formation of transiently stable bipolar structures. Further, we show that myosin-driven polarity sorting produces polar actin asters, which act as contractile nodes that drive contraction in crosslinked networks. Computer simulations comparing the roles of the end-dwelling mechanism and a buckling-dependent mechanism show that the relative contribution of end-dwelling contraction increases as the network mesh-size decreases.


Assuntos
Actinas/fisiologia , Simulação por Computador , Citoesqueleto/fisiologia , Miosinas/fisiologia , Citoesqueleto de Actina/química , Actomiosina/fisiologia , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Modelos Biológicos , Contração Muscular/fisiologia
9.
Nat Commun ; 9(1): 3381, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30139971

RESUMO

Establishing left-right asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia-driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event. Here, we report that myosin 1d (myo1d) is essential for establishing left-right asymmetry in zebrafish. Using super-resolution microscopy, we show that the zebrafish LRO, Kupffer's vesicle (KV), fails to form a spherical lumen and establish proper unidirectional flow in the absence of myo1d. This process requires directed vacuolar trafficking in KV epithelial cells. Interestingly, the vacuole transporting function of zebrafish Myo1d can be substituted by myosin1C derived from an ancient eukaryote, Acanthamoeba castellanii, where it regulates the transport of contractile vacuoles. Our findings reveal an evolutionary conserved role for an unconventional myosin in vacuole trafficking, lumen formation, and determining laterality.


Assuntos
Morfogênese/fisiologia , Miosinas/fisiologia , Vacúolos/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Morfolinos/metabolismo , Miosinas/genética , Proteínas de Protozoários/metabolismo , Imagem com Lapso de Tempo , Proteínas de Peixe-Zebra/genética
10.
Bull Math Biol ; 80(11): 2789-2827, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30159856

RESUMO

We study in this paper the filament-based lamellipodium model (FBLM) and the corresponding finite element method (FEM) used to solve it. We investigate fundamental numerical properties of the FEM and justify its further use with the FBLM. We show that the FEM satisfies a time step stability condition that is consistent with the nature of the problem and propose a particular strategy to automatically adapt the time step of the method. We show that the FEM converges with respect to the (two-dimensional) space discretization in a series of characteristic and representative chemotaxis and haptotaxis experiments. We embed and couple the FBLM with a complex and adaptive extracellular environment comprised of chemical and adhesion components that are described by their macroscopic density and study their combined time evolution. With this combination, we study the sensitivity of the FBLM on several of its controlling parameters and discuss their influence in the dynamics of the model and its future evolution. We finally perform a number of numerical experiments that reproduce biological cases and compare the results with the ones reported in the literature.


Assuntos
Citoesqueleto de Actina/fisiologia , Modelos Biológicos , Pseudópodes/fisiologia , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Simulação por Computador , Espaço Extracelular/fisiologia , Análise de Elementos Finitos , Humanos , Conceitos Matemáticos , Miosinas/fisiologia , Polimerização
11.
Integr Comp Biol ; 58(2): 163-173, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137314

RESUMO

Biological movement is an inherently dynamic process, characterized by large spatiotemporal variations in force and mechanical energy. Molecular level interactions between the contractile proteins actin and myosin do work, generating forces and transmitting them to the environment via the muscle's and supporting tissues' complex structures. Most existing theories of muscle contraction are derived from observations of muscle performance under simple, tightly controlled, in vitro or in situ conditions. These theories provide predictive power that falls off as we examine the more complicated action and movement regimes seen in biological movement. Our early and heavy focus on actin and myosin interactions have lead us to overlook other interactions and sources of force regulation. It increasingly appears that the structural heterogeneity, and micro-to-macro spatial scales of the force transmission pathways that exist between actin and myosin and the environment, determine muscle performance in ways that manifest most clearly under the dynamic conditions occurring during biological movement. Considering these interactions, along with the dynamics of force transmission tissues, actuators, and environmental physics have enriched our understanding of biological motion and force generation. This symposium brings together diverse investigators to consolidate our understanding of the role of spatial scale and structural heterogeneity role in muscle performance, with the hope of updating frameworks for understanding muscle contraction and predicting muscle performance in biological movement.


Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Humanos , Miosinas/fisiologia
12.
Nat Commun ; 9(1): 2844, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30030431

RESUMO

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.


Assuntos
Cálcio/química , Cadeias Pesadas de Miosina/química , Miosina Tipo V/química , Miosinas/química , Animais , Biotinilação , DNA/química , Homeostase , Cinesina/química , Cinética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Neurônios/metabolismo , Pontos Quânticos , Ratos , Estresse Mecânico , Potenciais Sinápticos
13.
Sci Rep ; 8(1): 10449, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29993000

RESUMO

Myosin X (Myo10), an actin-associated molecular motor, has a clear role in filopodia induction and cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of Myo10 in melanocyte lineage and melanoma induction. We found that Myo10 knockout (Myo10KO) mice exhibit a white spot on their belly caused by reduced melanoblast migration. Myo10KO mice crossed with available mice that conditionally express in melanocytes the BRAFV600E mutation combined with Pten silencing exhibited reduced melanoma development and metastasis, which extended medial survival time. Knockdown of Myo10 (Myo10kd) in B16F1 mouse melanoma cell lines decreased lung colonization after tail-vein injection. Myo10kd also inhibited long protrusion (LP) formation by reducing the transportation of its cargo molecule vasodilator-stimulated phosphoprotein (VASP) to the leading edge of migrating cells. These findings provide the first genetic evidence for the involvement of Myo10 not only in melanoblast migration, but also in melanoma development and metastasis.


Assuntos
Carcinogênese/patologia , Melanoma/patologia , Miosinas/fisiologia , Metástase Neoplásica/patologia , Animais , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Inativação Gênica , Melanócitos/patologia , Melanoma/etiologia , Melanoma Experimental , Camundongos , Proteínas dos Microfilamentos/metabolismo , Mutação , PTEN Fosfo-Hidrolase/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética
14.
J Comp Physiol B ; 188(6): 919-927, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30056509

RESUMO

The soluble Ca2+-binding protein (SCBP) from the earthworm Lumbricus terrestris was analyzed with regard to its role as a soluble muscle relaxation factor. The actomyosin ATPase activity was inhibited by the addition of decalcified SCBP as it binds Ca2+ stronger than the regulatory proteins associated with the actomyosin. Competitive 45Ca2+-binding assays with decalcified actomyosin and SCBP showed that 45Ca2+ is first bound to actomyosin and is subsequently taken over by SCBP with increasing incubation time. Ca2+ competition experiments carried out with 45Ca2+ loaded SCBP and fragmented sarcoplasmic reticulum vesicles revealed that 45Ca2+ bound to SCBP can be deprived by the ATP-dependent Ca2+ uptake of the sarcoplasmic reticulum. Furthermore, experiments in a diffusion chamber showed that the addition of SCBP significantly enhances the 45Ca2+ flux in a concentration dependent manner. The amount of the Ca2+ flux increase tends to reach a maximum value of about 70%. With all protein components isolated from the obliquely striated muscle, our in vitro experiments consistently show that SCBP may accelerate muscle relaxation similar as assumed for vertebrate parvalbumin.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/fisiologia , Relaxamento Muscular/fisiologia , Músculos/fisiologia , Oligoquetos/fisiologia , Actomiosina/fisiologia , Animais , Miosinas/antagonistas & inibidores , Miosinas/fisiologia
15.
J Biomech ; 76: 263-268, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-29954596

RESUMO

OBJECTIVE: To perform comparative analysis of the role of scavenger receptor CD36 on endothelial vs. sub-endothelial elastic modulus (stiffness) in the aortas of young and aged mice. APPROACHES AND RESULTS: Elastic moduli of endothelial and sub-endothelial layers of freshly isolated mouse aortas were quantified using atomic force microscopy. In young mice (4-6 months old), we found that while endothelial stiffness is markedly reduced in aortas of CD36-/-mice, as compared to WT controls, no difference between CD36-/- and WT aortas is observed in the stiffness of the sub-endothelial layer in denuded arteries. Additionally, inhibition of myosin phosphorylation also decreases the elastic modulus in the EC, but not the sub-EC layer in WT mice. Moreover, inhibiting CD36 mediated uptake of oxLDL in intact WT aortas abrogated oxLDL-induced endothelial stiffening. Further analysis of aged mice (22-25 months) revealed that aging resulted not only in significant stiffening of the denuded arteries, as was previously known, but also a comparable increase in the elastic modulus of the endothelial layer. Most significantly, this stiffening in the EC layer is dependent on CD36, whereas the denuded layer is not affected. CONCLUSIONS: Our results show that the role CD36 in stiffening of cellular components of intact aortas is endothelial-specific and that genetic deficiency of CD36 protects against endothelial stiffening in aged mice. Moreover, these data suggest that endothelial stiffness in intact mouse aortas depends more on the expression of CD36 than on the stiffness of the sub-endothelial layer.


Assuntos
Envelhecimento/fisiologia , Artérias/fisiologia , Antígenos CD36/fisiologia , Células Endoteliais/fisiologia , Animais , Transporte Biológico , Módulo de Elasticidade , Lipoproteínas LDL/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Força Atômica , Miosinas/fisiologia
16.
Int J Mol Sci ; 19(5)2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29734671

RESUMO

The most straightforward way to get information on the performance of individual myosin heads producing muscle contraction may be to record their movement, coupled with ATP hydrolysis, electron-microscopically using the gas environmental chamber (EC). The EC enables us to visualize and record ATP-induced myosin head movement in hydrated skeletal muscle myosin filaments. When actin filaments are absent, myosin heads fluctuate around a definite neutral position, so that their time-averaged mean position remains unchanged. On application of ATP, myosin heads are found to move away from, but not towards, the bare region, indicating that myosin heads perform a recovery stroke (average amplitude, 6 nm). After exhaustion of ATP, myosin heads return to their neutral position. In the actin⁻myosin filament mixture, myosin heads form rigor actin myosin linkages, and on application of ATP, they perform a power stroke by stretching adjacent elastic structures because of a limited amount of applied ATP ≤ 10 µM. The average amplitude of the power stroke is 3.3 nm and 2.5 nm at the distal and the proximal regions of the myosin head catalytic domain (CAD), respectively. The power stroke amplitude increases appreciably at low ionic strength, which is known to enhance Ca2+-activated force in muscle. In both the power and recovery strokes, myosin heads return to their neutral position after exhaustion of ATP.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Contração Muscular/fisiologia , Músculo Esquelético/ultraestrutura , Miosinas/ultraestrutura , Citoesqueleto de Actina/fisiologia , Trifosfato de Adenosina/metabolismo , Humanos , Hidrólise , Fenômenos Mecânicos , Microscopia Eletrônica , Músculo Esquelético/fisiologia , Miosinas/fisiologia
17.
Artigo em Inglês | MEDLINE | ID: mdl-29716949

RESUMO

Myosin motors power movements on actin filaments, whereas dynein and kinesin motors power movements on microtubules. The mechanisms of these motor proteins differ, but, in all cases, ATP hydrolysis and subsequent release of the hydrolysis products drives a cycle of interactions with the track (either an actin filament or a microtubule), resulting in force generation and directed movement.


Assuntos
Dineínas/fisiologia , Cinesina/fisiologia , Miosinas/fisiologia , Citoesqueleto de Actina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico/fisiologia , Dineínas/ultraestrutura , Cinesina/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Miosinas/ultraestrutura
18.
Ann Biomed Eng ; 46(9): 1348-1361, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29789970

RESUMO

Dendritic cell (DC) migration is required for efficient presentation of antigen to T cells and the initiation of an adaptive immune response. In spite of its importance, many aspects of DC migration have not been characterized. DCs encounter a variety of environments with different stiffness and geometry, but the effect of these parameters on DC migration has not yet been determined. We addressed this question by comparing DC motility on standard migration surfaces (polydimethylsiloxane (PDMS)-coated coverslips) and micropost array detectors (mPADs). These two surfaces differ in both stiffness and geometry. We found that DC migration was affected by substrate type, with significant increases in speed and significant decreases in persistence time on mPADs made of PDMS as compared to spin-coated PDMS coverslips. To determine whether the geometry or compliance of the post arrays was responsible for these changes in DC migration, we quantified DC motility on mPADs of identical geometry but different stiffness. Migration was indistinguishable on these mPADs, suggesting that DCs are responsive to geometry of ligand presentation and not stiffness. Further, by micropatterning ligands on flat PDMS surfaces in similar geometries to the mPAD arrays, we determined that DCs respond to the geometry of printed ligand. Finally, we used a variety of small molecule inhibitors to identify pathways involved in geometry sensing. We saw a significant role for myosin contractility and α5ß1 integrin engagement. We also noted significant reorganization of the actin cytoskeleton into dynamic actin rings when DCs were motile on posts. From these experiments, we conclude that DCs are insensitive to substrate compliance in the range tested but respond to changes in geometry via a mechanism that involves integrin function, myosin contractility, and remodeling of the actin cytoskeleton. As a possible explanation, we postulate a consistent role for filopodial extension and contraction as the driver of DC motility.


Assuntos
Movimento Celular , Células Dendríticas/fisiologia , Citoesqueleto de Actina , Animais , Fenômenos Biomecânicos , Adesão Celular , Dimetilpolisiloxanos , Integrinas/fisiologia , Camundongos Endogâmicos C57BL , Miosinas/fisiologia
19.
J Physiol ; 596(13): 2581-2596, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29714038

RESUMO

KEY POINTS: Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. ABSTRACT: The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+ ]o from 1 to 2.5 mm at sarcomere length ∼2.2 µm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.


Assuntos
Cálcio/metabolismo , Espaço Extracelular/metabolismo , Contração Muscular , Fibras Musculares Esqueléticas/fisiologia , Miosinas/fisiologia , Animais , Masculino , Ratos , Ratos Wistar
20.
Compr Physiol ; 8(2): 631-709, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29687901

RESUMO

Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018.


Assuntos
Proteínas Musculares/fisiologia , Miofibrilas/metabolismo , Sarcômeros/fisiologia , Animais , Humanos , Contração Muscular/fisiologia , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Doenças Musculares/metabolismo , Mutação , Miofibrilas/fisiologia , Miosinas/genética , Miosinas/fisiologia
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