Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.973
Filtrar
1.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638607

RESUMO

Asymmetric cell division (ACD) of neural stem cells and progenitors not only renews the stem cell population but also ensures the normal development of the nervous system, producing various types of neurons with different shapes and functions in the brain. One major mechanism to achieve ACD is the asymmetric localization and uneven segregation of intracellular proteins and organelles into sibling cells. Recent studies have demonstrated that liquid-liquid phase separation (LLPS) provides a potential mechanism for the formation of membrane-less biomolecular condensates that are asymmetrically distributed on limited membrane regions. Moreover, mechanical forces have emerged as pivotal regulators of asymmetric neural stem cell division by generating sibling cell size asymmetry. In this review, we will summarize recent discoveries of ACD mechanisms driven by LLPS and mechanical forces.


Assuntos
Divisão Celular Assimétrica/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Animais , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Tamanho Celular , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Modelos Neurológicos , Miosinas/fisiologia , Neurogênese/fisiologia , Organelas/fisiologia
2.
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
4.
Arch Biochem Biophys ; 706: 108923, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34029559

RESUMO

A highly organized and densely packed lattice of molecular machinery within the sarcomeres of muscle cells powers contraction. Although many of the proteins that drive contraction have been studied extensively, the mechanical impact of fluid shearing within the lattice of molecular machinery has received minimal attention. It was recently proposed that fluid flow augments substrate transport in the sarcomere, however, this analysis used analytical models of fluid flow in the molecular machinery that could not capture its full complexity. By building a finite element model of the sarcomere, we estimate the explicit flow field, and contrast it with analytical models. Our results demonstrate that viscous drag forces on sliding filaments are surprisingly small in contrast to the forces generated by single myosin molecular motors. This model also indicates that the energetic cost of fluid flow through viscous shearing with lattice proteins is likely minimal. The model also highlights a steep velocity gradient between sliding filaments and demonstrates that the maximal radial fluid velocity occurs near the tips of the filaments. To our knowledge, this is the first computational analysis of fluid flow within the highly structured sarcomere.


Assuntos
Análise de Elementos Finitos , Modelos Biológicos , Miosinas/fisiologia , Sarcômeros/fisiologia , Animais , Fenômenos Biomecânicos , Simulação por Computador , Humanos , Contração Muscular/fisiologia , Miosinas/ultraestrutura , Reologia , Sarcômeros/ultraestrutura , Termodinâmica , Viscosidade
5.
Annu Rev Biophys ; 50: 373-400, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33637009

RESUMO

Two groundbreaking papers published in 1954 laid out the theory of the mechanism of muscle contraction based on force-generating interactions between myofilaments in the sarcomere that cause filaments to slide past one another during muscle contraction. The succeeding decades of research in muscle physiology have revealed a unifying interest: to understand the multiscale processes-from atom to organ-that govern muscle function. Such an understanding would have profound consequences for a vast array of applications, from developing new biomimetic technologies to treating heart disease. However, connecting structural and functional properties that are relevant at one spatiotemporal scale to those that are relevant at other scales remains a great challenge. Through a lens of multiscale dynamics, we review in this article current and historical research in muscle physiology sparked by the sliding filament theory.


Assuntos
Contração Muscular/fisiologia , Citoesqueleto de Actina , Animais , Humanos , Miofibrilas/fisiologia , Miosinas/fisiologia , Sarcômeros/fisiologia
6.
Plant J ; 104(6): 1685-1697, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33067901

RESUMO

F-actin and myosin XI play important roles in plant organelle movement. A few myosin XI genes in the genome of Arabidopsis are mainly expressed in mature pollen, which suggests that they may play a crucial role in pollen germination and pollen tube tip growth. In this study, a genetic complementation assay was conducted in a myosin xi-c (myo11c1) myosin xi-e (myo11c2) double mutant, and fluorescence labeling combined with microscopic observation was applied. We found that myosin XI-E (Myo11C2)-green fluorescent protein (GFP) restored the slow pollen tube growth and seed deficiency phenotypes of the myo11c1 myo11c2 double mutant and Myo11C2-GFP partially colocalized with mitochondria, peroxisomes and Golgi stacks. Furthermore, decreased mitochondrial movement and subapical accumulation were detected in myo11c1 myo11c2 double mutant pollen tubes. Fluorescence recovery after photobleaching experiments showed that the fluorescence recoveries of GFP-RabA4d and AtPRK1-GFP at the pollen tube tip of the myo11c1 myo11c2 double mutant were lower than those of the wild type were after photobleaching. These results suggest that Myo11C2 may be associated with mitochondria, peroxisomes and Golgi stacks, and play a crucial role in organelle movement and apical accumulation of secretory vesicles in pollen tubes of Arabidopsis thaliana.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Miosinas/fisiologia , Organelas/fisiologia , Tubo Polínico/fisiologia , Vesículas Secretórias/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Complexo de Golgi/metabolismo , Mitocôndrias/metabolismo , Miosinas/metabolismo , Organelas/metabolismo , Peroxissomos/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Vesículas Secretórias/metabolismo
7.
J Neurosci ; 40(48): 9169-9185, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33097641

RESUMO

Myosin X (Myo X) transports cargos to the tips of filopodia for cell adhesion, migration, and neuronal axon guidance. Deleted in Colorectal Cancer (DCC) is one of the Myo X cargos that is essential for Netrin-1-regulated axon pathfinding. The function of Myo X in axon development in vivo and the underlying mechanisms remain elusive. Here, we provide evidence for the role of Myo X in Netrin-1-DCC-regulated axon development in developing mouse neocortex. The knockout (KO) or knockdown (KD) of Myo X in cortical neurons of embryonic mouse brain impairs axon initiation and contralateral branching/targeting. Similar axon deficits are detected in Netrin-1-KO or DCC-KD cortical neurons. Further proteomic analysis of Myo X binding proteins identifies KIF13B (a kinesin family motor protein). The Myo X interaction with KIF13B is induced by Netrin-1. Netrin-1 promotes anterograde transportation of Myo X into axons in a KIF13B-dependent manner. KIF13B-KD cortical neurons exhibit similar axon deficits. Together, these results reveal Myo X-KIF13B as a critical pathway for Netrin-1-promoted axon initiation and branching/targeting.SIGNIFICANCE STATEMENT Netrin-1 increases Myosin X (Myo X) interaction with KIF13B, and thus promotes axonal delivery of Myo X and axon initiation and contralateral branching in developing cerebral neurons, revealing unrecognized functions and mechanisms underlying Netrin-1 regulation of axon development.


Assuntos
Axônios/fisiologia , Proteínas de Membrana/fisiologia , Miosinas/fisiologia , Netrina-1/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Receptor DCC/genética , Receptor DCC/fisiologia , Feminino , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosinas/genética , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Netrina-1/genética , Gravidez
8.
Cell Death Dis ; 11(8): 654, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811811

RESUMO

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Contração Muscular/fisiologia , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Actinas/fisiologia , Animais , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Músculo Liso/fisiologia , Miosinas/fisiologia
9.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
10.
Cancer Rep (Hoboken) ; 3(1): e1157, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32671955

RESUMO

BACKGROUND: Mammalian cells must constantly reprogram the distribution of mitochondria in order to meet the local demands for energy, calcium, redox balance, and other mitochondrial functions. Mitochondrial localization inside the cell is a result of a combination of movement along the microtubule tracks plus anchoring to actin filaments. RECENT FINDINGS: Recent advances show that subcellular distribution of mitochondria can regulate tumor cell growth, proliferation/motility plasticity, metastatic competence, and therapy responses in tumors. In this review, we discuss our current understanding of the mechanisms by which mitochondrial subcellular distribution is regulated in tumor cells. CONCLUSIONS: Mitochondrial trafficking is dysregulated in tumors. Accumulation of mitochondria at the leading edge of the cell supports energy expensive processes of focal adhesion dynamics, cell membrane dynamics, migration, and invasion.


Assuntos
Mitocôndrias/fisiologia , Metástase Neoplásica , Trifosfato de Adenosina/metabolismo , Animais , Movimento Celular , Adesões Focais , Humanos , Proteínas de Membrana/fisiologia , Microtúbulos/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Invasividade Neoplásica , Proteínas do Tecido Nervoso/fisiologia , Transdução de Sinais
11.
PLoS Comput Biol ; 16(6): e1007693, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520928

RESUMO

Understanding cellular remodeling in response to mechanical stimuli is a critical step in elucidating mechanical activation of biochemical signaling pathways. Experimental evidence indicates that external stress-induced subcellular adaptation is accomplished through dynamic cytoskeletal reorganization. To study the interactions between subcellular structures involved in transducing mechanical signals, we combined experimental data and computational simulations to evaluate real-time mechanical adaptation of the actin cytoskeletal network. Actin cytoskeleton was imaged at the same time as an external tensile force was applied to live vascular smooth muscle cells using a fibronectin-functionalized atomic force microscope probe. Moreover, we performed computational simulations of active cytoskeletal networks under an external tensile force. The experimental data and simulation results suggest that mechanical structural adaptation occurs before chemical adaptation during filament bundle formation: actin filaments first align in the direction of the external force by initializing anisotropic filament orientations, then the chemical evolution of the network follows the anisotropic structures to further develop the bundle-like geometry. Our findings present an alternative two-step explanation for the formation of actin bundles due to mechanical stimulation and provide new insights into the mechanism of mechanotransduction.


Assuntos
Citoesqueleto de Actina/fisiologia , Resistência à Tração , Actinas/fisiologia , Animais , Anisotropia , Fenômenos Biomecânicos , Células Cultivadas , Simulação por Computador , Fibronectinas/fisiologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Mecanotransdução Celular , Microscopia de Força Atômica , Miócitos de Músculo Liso/metabolismo , Miosinas/fisiologia , Ratos , Estresse Mecânico
12.
Soft Matter ; 16(22): 5177-5194, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32459252

RESUMO

In cell migration, polarization is the process by which a stationary cell breaks symmetry and initiates motion. Although a lot is known about the mechanisms involved in cell polarization, the role played by myosin contraction remains unclear. In addition, cell polarization by mechanical impact has received little attention. Here, we study the influence of myosin activity on cell polarization and the initiation of motion induced by mechanical cues using a computational model for keratocytes. The model accounts for cell deformation, the dynamics of myosin and the signaling protein RhoA (a member of the Rho GTPases family), as well as the forces acting on the actomyosin network. Our results show that the attainment of a steady polarized state depends on the strength of myosin down- or up-regulation and that myosin upregulation favors cell polarization. Our results also confirm the existence of a threshold level for cell polarization, which is determined by the level of polarization of the Rho GTPases at the time the external stimuli vanish. In all, this paper shows that capturing the interactions between the signaling proteins (Rho GTPases for keratocytes) and the compounds of the motile machinery in a moving cell is crucial to study cell polarization.


Assuntos
Polaridade Celular , Queratinócitos/fisiologia , Modelos Biológicos , Miosinas/fisiologia , Movimento Celular , Fenômenos Físicos , Estimulação Física , Proteínas rho de Ligação ao GTP/fisiologia
13.
Mol Biol Cell ; 31(13): 1355-1369, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32320320

RESUMO

During organogenesis, different cell types need to work together to generate functional multicellular structures. To study this process, we made use of the genetically tractable fly retina, with a focus on the mechanisms that coordinate morphogenesis between the different epithelial cell types that make up the optical lens. Our work shows that these epithelial cells present contractile apical-medial MyosinII meshworks, which control the apical area and junctional geometry of these cells during lens development. Our study also suggests that these MyosinII meshworks drive cell shape changes in response to external forces, and thus they mediate part of the biomechanical coupling that takes place between these cells. Importantly, our work, including mathematical modeling of forces and material stiffness during lens development, raises the possibility that increased cell stiffness acts as a mechanism for limiting this mechanical coupling. We propose this might be required in complex tissues, where different cell types undergo concurrent morphogenesis and where averaging out of forces across cells could compromise individual cell apical geometry and thereby organ function.


Assuntos
Drosophila/crescimento & desenvolvimento , Miosinas/metabolismo , Organogênese , Retina/crescimento & desenvolvimento , Animais , Drosophila/metabolismo , Modelos Biológicos , Miosinas/fisiologia , Retina/metabolismo
14.
Biomech Model Mechanobiol ; 19(6): 2683-2692, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32346808

RESUMO

Finite element (FE) modeling is becoming increasingly prevalent in the world of cardiac mechanics; however, many existing FE models are phenomenological and thus do not capture cellular-level mechanics. This work implements a cellular-level contraction scheme into an existing nonlinear FE code to model ventricular contraction. Specifically, this contraction model incorporates three myosin states: OFF-, ON-, and an attached force-generating state. It has been speculated that force-dependent transitions from the OFF- to ON-state may contribute to length-dependent activation at the cellular level. The current work investigates the contribution of force-dependent recruitment out of the OFF-state to ventricular-level function, specifically the Frank-Starling relationship, as seen through the end-systolic pressure-volume relationship (ESPVR). Five FE models were constructed using geometries of rat left ventricles obtained via cardiac magnetic resonance imaging. FE simulations were conducted to optimize parameters for the cellular contraction model such that the differences between FE predicted ventricular pressures for the models and experimentally measured pressures were minimized. The models were further validated by comparing FE predicted end-systolic strain to experimentally measured strain. Simulations mimicking vena cava occlusion generated descending pressure volume loops from which ESPVRs were calculated. In simulations with the inclusion of the OFF-state, using a force-dependent transition to the ON-state, the ESPVR calculated was steeper than in simulations excluding the OFF-state. Furthermore, the ESPVR was also steeper when compared to models that included the OFF-state without a force-dependent transition. This suggests that the force-dependent recruitment of thick filament heads from the OFF-state at the cellular level contributes to the Frank-Starling relationship observed at the organ level.


Assuntos
Ventrículos do Coração/patologia , Estresse Mecânico , Sístole , Função Ventricular Esquerda , Animais , Pressão Sanguínea , Simulação por Computador , Feminino , Análise de Elementos Finitos , Coração/fisiologia , Imageamento Tridimensional , Fenômenos Mecânicos , Modelos Cardiovasculares , Contração Miocárdica/fisiologia , Miocárdio , Miosinas/fisiologia , Ratos , Ratos Sprague-Dawley , Volume Sistólico/fisiologia
15.
Proc Natl Acad Sci U S A ; 117(14): 8177-8186, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32220962

RESUMO

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the "C-zone." Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.


Assuntos
Proteínas de Transporte/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miosinas/fisiologia , Sarcômeros/metabolismo , Animais , Proteínas de Transporte/ultraestrutura , Masculino , Miosinas/ultraestrutura , Domínios Proteicos/fisiologia , Ratos , Sarcômeros/ultraestrutura , Síncrotrons , Difração de Raios X/instrumentação
16.
J Assoc Res Otolaryngol ; 21(2): 121-135, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152769

RESUMO

Mammalian hair cells develop their mechanosensory bundles through consecutive phases of stereocilia elongation, thickening, and retraction of supernumerary stereocilia. Many molecules involved in stereocilia elongation have been identified, including myosin-XVa. Significantly less is known about molecular mechanisms of stereocilia thickening and retraction. Here, we used scanning electron microscopy (SEM) to quantify postnatal changes in number and diameters of the auditory hair cell stereocilia in shaker-2 mice (Myo15sh2) that lack both "long" and "short" isoforms of myosin-XVa, and in mice lacking only the "long" myosin-XVa isoform (Myo15∆N). Previously, we observed large mechanotransduction current in young postnatal inner (IHC) and outer (OHC) hair cells of both these strains. Stereocilia counts showed nearly identical developmental retraction of supernumerary stereocilia in control heterozygous, Myo15sh2/sh2, and Myo15∆N/∆N mice, suggesting that this retraction is largely unaffected by myosin-XVa deficiency. However, myosin-XVa deficiency does affect stereocilia diameters. In control, the first (tallest) and second row stereocilia grow in diameter simultaneously. However, the third row stereocilia in IHCs grow only until postnatal day 1-2 and then become thinner. In OHCs, they also grow slower than taller stereocilia, forming a stereocilia diameter gradation within a hair bundle. The sh2 mutation disrupts this gradation and makes all stereocilia nearly identical in thickness in both IHCs and OHCs, with only subtle residual diameter differences. All Myo15sh2/sh2 stereocilia grow postnatally including the third row, which is not a part of normal development. Serial sections with focused ion beam (FIB)-SEM confirmed that diameter changes of Myo15sh2/sh2 IHC and OHC stereocilia resulted from corresponding changes of their actin cores. In contrast to Myo15sh2/sh2, Myo15∆N/∆N hair cells develop prominent stereocilia diameter gradation. Thus, besides building the staircase, the short isoform of myosin-XVa is essential for controlling the diameter of the third row stereocilia and formation of the stereocilia diameter gradation in a hair bundle.


Assuntos
Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/ultraestrutura , Miosinas/fisiologia , Estereocílios/fisiologia , Actinas/metabolismo , Animais , Camundongos , Camundongos Knockout , Isoformas de Proteínas , Estereocílios/ultraestrutura
17.
Curr Biol ; 30(9): 1762-1769.e5, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32220324

RESUMO

Traction forces are generated by cellular actin-myosin system and transmitted to the environment through adhesions. They are believed to drive cell motion, shape changes, and extracellular matrix remodeling [1-3]. However, most of the traction force analysis has been performed on stationary cells, investigating forces at the level of individual focal adhesions or linking them to static cell parameters, such as area and edge curvature [4-10]. It is not well understood how traction forces are related to shape changes and motion, e.g., forces were reported to either increase or drop prior to cell retraction [11-15]. Here, we analyze the dynamics of traction forces during the protrusion-retraction cycle of polarizing fish epidermal keratocytes and find that forces fluctuate together with the cycle, increasing during protrusion and reaching maximum at the beginning of retraction. We relate force dynamics to the recently discovered phenomenological rule [16] that governs cell-edge behavior during keratocyte polarization: both traction forces and probability of switch from protrusion to retraction increase with the distance from the cell center. Diminishing forces with cell contractility inhibitor leads to decreased edge fluctuations and abnormal polarization, although externally applied force can induce protrusion-retraction switch. These results suggest that forces mediate distance sensitivity of the edge dynamics and organize cell-edge behavior, leading to spontaneous polarization. Actin flow rate did not exhibit the same distance dependence as traction stress, arguing against its role in organizing edge dynamics. Finally, using a simple model of actin-myosin network, we show that force-distance relationship might be an emergent feature of such networks.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Fibroblastos/fisiologia , Actinas/fisiologia , Animais , Adesão Celular , Fenômenos Fisiológicos Celulares , Células Cultivadas , Characidae , Feminino , Masculino , Miosinas/fisiologia
18.
Am J Physiol Cell Physiol ; 318(1): C103-C110, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618078

RESUMO

In the present study we evaluated the load dependence of force produced by isolated muscle myosin filaments interacting with fluorescently labeled actin filaments, using for the first time whole native myosin filaments. We used a newly developed approach that allowed the use of physiological levels of ATP. Single filaments composed of either skeletal or smooth muscle myosin and single filaments of actin were attached between pairs of nano-fabricated cantilevers of known stiffness. The filaments were brought into contact to produce force, which caused sliding of the actin filaments over the myosin filaments. We applied load to the system by either pushing or pulling the filaments during interactions and observed that increasing the load increased the force produced by myosin and decreasing the load decreased the force. We also performed additional experiments in which we clamped the filaments at predetermined levels of force, which caused the filaments to slide to adjust the different loads, allowing us to measure the velocity of length changes to construct a force-velocity relation. Force values were in the range observed previously with myosin filaments and molecules. The force-velocity curves for skeletal and smooth muscle myosins resembled the relations observed for muscle fibers. The technique can be used to investigate many issues of interest and debate in the field of muscle biophysics.


Assuntos
Citoesqueleto de Actina/fisiologia , Contração Muscular , Força Muscular , Músculo Liso/fisiologia , Miofibrilas/fisiologia , Miosinas/fisiologia , Músculos Psoas/fisiologia , Citoesqueleto de Actina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Músculo Liso/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Mytilus edulis , Músculos Psoas/metabolismo , Coelhos , Fatores de Tempo
19.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801239

RESUMO

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Contração Isométrica/fisiologia , Músculo Estriado/fisiologia , Miosinas/fisiologia , Sarcômeros/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Simulação por Computador , Conectina/fisiologia , Conectina/ultraestrutura , Modelos Biológicos , Método de Monte Carlo , Músculo Estriado/diagnóstico por imagem , Miosinas/ultraestrutura , Rana catesbeiana/fisiologia , Sarcômeros/ultraestrutura , Espalhamento a Baixo Ângulo , Técnicas de Cultura de Tecidos , Difração de Raios X
20.
J Gen Physiol ; 151(11): 1265-1271, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31570503

RESUMO

In a recent JGP article, Heeley et al. (2019. J. Gen. Physiol https://doi.org/10.1085/jgp.201812198) reopened the debate about two- versus three-state models of thin filament regulation. The authors review their work, which measures the rate constant of Pi release from myosin.ADP.Pi activated by actin or thin filaments under a variety of conditions. They conclude that their data can be described by a two-state model and raise doubts about the generally accepted three-state model as originally formulated by McKillop and Geeves (1993. Biophys. J. https://doi.org/10.1016/S0006-3495(93)81110-X). However, in the following article, we follow Plato's dictum that "twice and thrice over, as they say, good it is to repeat and review what is good." We have therefore reviewed the evidence for the three- and two-state models and present our view that the evidence is overwhelmingly in favor of three structural states of the thin filament, which regulate access of myosin to its binding sites on actin and, hence, muscle contractility.


Assuntos
Citoesqueleto , Modelos Biológicos , Citoesqueleto de Actina , Actinas/química , Animais , Sítios de Ligação , Cinética , Contração Muscular , Miosinas/química , Miosinas/fisiologia , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...