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1.
EMBO J ; 42(9): e112717, 2023 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-36912152

RESUMEN

Intracellular organization is largely mediated by actin turnover. Cellular actin networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called "dynamic steady state," allows cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled by an actin comet in a microwell containing a limited amount of components. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly, and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated protein (CAP) is the key step in allowing the reuse of monomers for multiple assembly cycles. ATP supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.


Asunto(s)
Citoesqueleto de Actina , Actinas , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo
2.
Development ; 151(17)2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-39136544

RESUMEN

Hematopoietic stem and progenitor cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes, including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that specific heterotypic interactions with osteoblast and endothelial cells promote asymmetric divisions of human HSPCs. Upon interaction, HSPCs polarize in interphase with the centrosome, the Golgi apparatus, and lysosomes positioned close to the site of contact. Subsequently, during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division mode gives rise to siblings with unequal amounts of lysosomes and of the differentiation marker CD34. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to contribute to the plasticity of the early steps of hematopoiesis.


Asunto(s)
Células Madre Hematopoyéticas , Humanos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis/fisiología , Diferenciación Celular , Mitosis , Osteoblastos/citología , Osteoblastos/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , División Celular Asimétrica , Lisosomas/metabolismo , Centrosoma/metabolismo , Antígenos CD34/metabolismo , Aparato de Golgi/metabolismo , División Celular
3.
EMBO J ; 41(20): e111631, 2022 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-35916262

RESUMEN

The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.


Asunto(s)
Actinas , Centrosoma , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Centrosoma/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo
4.
Proc Natl Acad Sci U S A ; 120(39): e2300416120, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37725653

RESUMEN

The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we used glass or lipids surface micropatterning to control the initial shape of the network. We adapted the concentration of Nucleating Promoting Factor on each surface to induce the assembly of actin networks of similar densities and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction, both the active and resistive forces are essential to direct the actin network deformation.


Asunto(s)
Actinas , Actomiosina , Fricción , Contracción Muscular , Membrana Dobles de Lípidos
5.
J Cell Sci ; 136(22)2023 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-37870087

RESUMEN

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.


Asunto(s)
Actinas , Microtúbulos , Actinas/fisiología , Microtúbulos/fisiología , Citoesqueleto de Actina/química , Polaridad Celular , Seudópodos
6.
EMBO J ; 39(23): e103957, 2020 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-33089509

RESUMEN

Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage.


Asunto(s)
Diferenciación Celular , Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Microtúbulos , Línea Celular , Linaje de la Célula , Núcleo Celular/genética , Núcleo Celular/fisiología , Citocinas , Células Madre Hematopoyéticas/citología , Histonas , Humanos , Transcriptoma
7.
Nat Mater ; 21(3): 366-377, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34663953

RESUMEN

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.


Asunto(s)
Mecanotransducción Celular , Microtúbulos , Citoesqueleto de Actina/metabolismo , Adhesión Celular , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo
8.
Exp Dermatol ; 32(7): 1096-1107, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37148203

RESUMEN

Keloid scars are hypertrophic and proliferating pathological scars extending beyond the initial lesion and without tendency to regression. Usually, keloids are considered and treated as a single entity but clinical observations suggest heterogeneity in keloid morphologies with distinction of superficial/extensive and nodular entities. Within a keloid, heterogeneity could also be detected between superficial and deep dermis or centre and periphery. Focusing on fibroblasts as main actors of keloid formation, we aimed at evaluating intra- and inter-keloid fibroblast heterogeneity by analysing their gene expression and functional capacities (proliferation, migration, traction forces), in order to improve our understanding of keloid pathogenesis. Fibroblasts were obtained from centre, periphery, papillary and reticular dermis from extensive or nodular keloids and were compared to control fibroblasts from healthy skin. Transcriptional profiling of fibroblasts identified a total of 834 differentially expressed genes between nodular and extensive keloids. Quantification of ECM-associated gene expression by RT-qPCR brought evidence that central reticular fibroblasts of nodular keloids are the population which synthesize higher levels of mature collagens, TGFß, HIF1α and αSMA as compared to control skin, suggesting that this central deep region is the nucleus of ECM production with a centrifuge extension in keloids. Although no significant variations were found for basal proliferation, migration of peripheral fibroblasts from extensive keloids was higher than that of central ones and from nodular cells. Moreover, these peripheral fibroblasts from extensive keloids exhibited higher traction forces than central cells, control fibroblasts and nodular ones. Altogether, studying fibroblast features demonstrate keloid heterogeneity, leading to a better understanding of keloid pathophysiology and treatment adaptation.


Asunto(s)
Queloide , Humanos , Queloide/metabolismo , Piel/metabolismo , Dermis/metabolismo , Fibroblastos/metabolismo , Colágeno/metabolismo , Células Cultivadas
9.
Biol Cell ; 110(4): 91-96, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29388708

RESUMEN

BACKGROUND INFORMATION: Tissue morphogenesis results from the interplay between cell growth and mechanical forces. While the impact of geometrical confinement and mechanical forces on cell proliferation has been fairly well characterised, the inverse relationship is much less understood. Here, we investigated how traction forces vary during cell cycle progression. RESULTS: Cell shape was constrained on micropatterned substrates in order to distinguish variations in cell contractility from cell size increase. We performed traction force measurements of asynchronously dividing cells expressing a cell-cycle reporter, to obtain measurements of contractile forces generated during cell division. We found that forces tend to increase as cells progress through G1, before reaching a plateau in S phase, and then decline during G2. CONCLUSIONS: While cell size increases regularly during cell cycle progression, traction forces follow a biphasic behaviour based on specific and opposite regulation of cell contractility during early and late growth phases. SIGNIFICANCE: These results highlight the key role of cellular signalling in the regulation of cell contractility, independently of cell size and shape. Non-monotonous variations of cell contractility during cell cycle progression are likely to impact the mechanical regulation of tissue homoeostasis in a complex and non-linear manner.


Asunto(s)
Ciclo Celular , Proteínas Luminiscentes/metabolismo , Mecanotransducción Celular , Modelos Biológicos , Epitelio Pigmentado de la Retina/citología , Fenómenos Fisiológicos Celulares , Células Cultivadas , Simulación por Computador , Humanos , Epitelio Pigmentado de la Retina/fisiología
10.
Biophys J ; 108(10): 2437-2447, 2015 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-25992722

RESUMEN

The mechanical interaction between adherent cells and their substrate relies on the formation of adhesion sites and on the stabilization of contractile acto-myosin bundles, or stress fibers. The shape of the cell and the orientation of these fibers can be controlled by adhesive patterning. On nonadhesive gaps, fibroblasts develop thick peripheral stress fibers, with a concave curvature. The radius of curvature of these arcs results from the balance of the line tension in the arc and of the surface tension in the cell bulk. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, is not clear. To get insight into the force balance, we inhibit myosin activity and simultaneously monitor the dynamics of peripheral arc radii and traction forces. We use these measurements to estimate line and surface tension. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that both line tension and surface tension dropped, but the line tension decreased to a lesser extent than surface tension. These results suggest that myosin-independent force contributes to tension in the peripheral arcs. We propose a simple physical model in which the peripheral arc line tension is due to the combination of myosin II contractility and a passive elastic component, while surface tension is largely due to active contractility. Numerical solutions of this model reproduce well the experimental data and allow estimation of the contributions of elasticity and contractility to the arc line tension.


Asunto(s)
Forma de la Célula , Elasticidad , Fibras de Estrés/metabolismo , Animales , Fenómenos Biomecánicos , Adhesión Celular , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Modelos Biológicos , Miosinas/química , Miosinas/metabolismo , Ratas , Fibras de Estrés/química
11.
Curr Biol ; 33(1): 122-133.e4, 2023 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-36565699

RESUMEN

Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 µm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.


Asunto(s)
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Citoplasma/metabolismo , Polímeros/metabolismo , Citoesqueleto de Actina/metabolismo , Guanosina Trifosfato/metabolismo
12.
Methods Mol Biol ; 2308: 263-278, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34057729

RESUMEN

The bone marrow (BM) is a complex microenvironment in which hematopoietic stem and progenitor cells (HSPCs) interact with multiple cell types that regulate their quiescence, growth, and differentiation. These cells constitute local niches where HSPCs are confined and subjected to specific set of physical and biochemical cues. Endothelial cells forming the walls of blood capillaries have been shown to establish a vascular niche, whereas osteoblasts lying along the bone matrix organize the endosteal niche with distinct and specific impact on HSPC fate. The observation of the interaction of HSPCs with niche cells, and the investigation of its impact on HSPCs behavior in vivo is hindered by the opacity of the bone matrix. Therefore, various experimental strategies have been devised to reconstitute in vitro the interaction of HSPCs with distinct sets of BM-derived cells. In this chapter, we present a method to manufacture a pseudo BM-on-a-chip with separated compartments mimicking the vascular and the endosteal niches. Such a configuration with connected but distant compartments allowed the investigation of the specific contribution of each niche to the regulation of HSPC behavior. We describe the microfabrication of the chip with a maskless photolithography method that allows the iterative improvement of the geometric design of the chip in order to optimize the adaptation of the multicellular architecture to the specific aim of the study. We also describe the loading and culture of the various cell types in each compartment.


Asunto(s)
Células de la Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Nicho de Células Madre , Ingeniería de Tejidos/instrumentación , Diferenciación Celular , Línea Celular , Técnicas de Cocultivo , Células Endoteliales/fisiología , Diseño de Equipo , Humanos , Hidrogeles , Osteoblastos/fisiología , Fenotipo
13.
Curr Biol ; 31(6): 1206-1220.e5, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33609453

RESUMEN

The centrosome is the main organizer of microtubules and as such, its position is a key determinant of polarized cell functions. As the name says, the default position of the centrosome is considered to be the cell geometrical center. However, the mechanism regulating centrosome positioning is still unclear and often confused with the mechanism regulating the position of the nucleus to which it is linked. Here, we used enucleated cells plated on adhesive micropatterns to impose regular and precise geometrical conditions to centrosome-microtubule networks. Although frequently observed there, the equilibrium position of the centrosome is not systematically at the cell geometrical center and can be close to cell edge. Centrosome positioning appears to respond accurately to the architecture and anisotropy of the actin network, which constitutes, rather than cell shape, the actual spatial boundary conditions the microtubule network is sensitive to. We found that the contraction of the actin network defines a peripheral margin in which microtubules appear bent by compressive forces. The progressive disassembly of the actin network at distance from the cell edges defines an inner zone where actin bundles were absent, where microtubules were more radially organized and where dynein concentration was higher. We further showed that the production of dynein-based forces on microtubules places the centrosome at the center of this zone. In conclusion, the spatial distribution of cell adhesion and the production of contractile forces define the architecture of the actin network with respect to which the centrosome-microtubule network is centered.


Asunto(s)
Actinas , Centrosoma , Dineínas , Miosinas , Actinas/metabolismo , Centrosoma/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo
14.
J Cell Biol ; 220(11)2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34570198

RESUMEN

The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.


Asunto(s)
Médula Ósea/metabolismo , Médula Ósea/fisiología , Quimiocina CXCL12/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos
15.
Phys Rev Lett ; 105(12): 128101, 2010 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-20867675

RESUMEN

When spreading onto a protein microlattice living cells spontaneously acquire simple shapes determined by the lattice geometry. This suggests that, on a lattice, living cells' shapes are in thermodynamic metastable states. Using a model at thermodynamic equilibrium we are able to reproduce the observed shapes. We build a phase diagram based on two adimensional parameters characterizing essential cellular properties involved in spreading: the cell's compressibility and fluctuations.


Asunto(s)
Citoesqueleto/ultraestructura , Proteínas de la Matriz Extracelular/metabolismo , Actinas/metabolismo , Fenómenos Biomecánicos , Adhesión Celular/fisiología , Forma de la Célula/fisiología , Fuerza Compresiva , Citoesqueleto/metabolismo , Elasticidad , Modelos Biológicos , Método de Montecarlo , Termodinámica
16.
J Cell Biol ; 217(9): 3031-3044, 2018 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-29980627

RESUMEN

Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell-cell interactions.


Asunto(s)
Astrocitos/fisiología , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Filamentos Intermedios/metabolismo , Animales , Astrocitos/metabolismo , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/metabolismo , Nestina/metabolismo , Ratas , Vimentina/metabolismo , Cicatrización de Heridas/fisiología
17.
Mol Biol Cell ; 28(14): 1825-1832, 2017 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-28684608

RESUMEN

Mechanical forces are key regulators of cell and tissue physiology. The basic molecular mechanism of fiber contraction by the sliding of actin filament upon myosin leading to conformational change has been known for decades. The regulation of force generation at the level of the cell, however, is still far from elucidated. Indeed, the magnitude of cell traction forces on the underlying extracellular matrix in culture is almost impossible to predict or experimentally control. The considerable variability in measurements of cell-traction forces indicates that they may not be the optimal readout to properly characterize cell contractile state and that a significant part of the contractile energy is not transferred to cell anchorage but instead is involved in actin network dynamics. Here we discuss the experimental, numerical, and biological parameters that may be responsible for the variability in traction force production. We argue that limiting these sources of variability and investigating the dissipation of mechanical work that occurs with structural rearrangements and the disengagement of force transmission is key for further understanding of cell mechanics.


Asunto(s)
Actinas/fisiología , Contracción Muscular/fisiología , Citoesqueleto de Actina/fisiología , Animales , Fenómenos Biomecánicos , Movimiento Celular/fisiología , Matriz Extracelular/fisiología , Humanos , Miosinas/fisiología
18.
Sci Rep ; 6: 23722, 2016 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-27025817

RESUMEN

Actin-myosin filament bundles (stress fibers) are critical for tension generation and cell shape, but their mechanical properties are difficult to access. Here we propose a novel approach to probe individual peripheral stress fibers in living cells through a microsurgically generated opening in the cytoplasm. By applying large deformations with a soft cantilever we were able to fully characterize the mechanical response of the fibers and evaluate their tension, extensibility, elastic and viscous properties.


Asunto(s)
Fibras de Estrés/fisiología , Animales , Línea Celular , Técnicas Citológicas , Elasticidad , Microcirugia , Ratas , Viscosidad
19.
Lab Chip ; 14(14): 2539-47, 2014 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-24867825

RESUMEN

Actin-myosin microfilament bundles or stress-fibers are the principal tension-generating structures in the cell. Their mechanical properties are critical for cell shape, motion, and interaction with other cells and extracellular matrix, but were so far difficult to access in a living cell. Here we propose a micro-fabricated two-component setup for direct tension measurement on a peripheral bundle within an intact cell. We used 3-D substrates made of silicon elastomer to elevate the cell making the filament bundle at its border accessible from the side, and employed an ultra-soft (spring constant 0.78 nN µm(-1)) epoxy-based cantilever for mechanical probing. With this setup we were able for the first time to measure the tension in peripheral actin bundles in living primary fibroblasts spread on a rigid substrate.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Técnicas de Cultivo de Célula , Matriz Extracelular/química , Fibroblastos , Miosinas/metabolismo , Elastómeros de Silicona/química , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Ratas
20.
Cell Calcium ; 54(6): 436-45, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24183802

RESUMEN

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca2+ concentration ([Ca2+]i) which propagates as Ca2+ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca2+ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca2+ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca2+]i dynamics and membrane potential events along the network of cells. The rate of [Ca2+]i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca2+ channels (VOCCs) inhibition suppressed the ultrafast Ca2+ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca2+ entry from the external medium through VOCCs, modeled as an ultrafast Ca2+ wave. This wave may trigger and drive slower Ca2+ waves observed ex vivo and in vivo.


Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Canales de Calcio/metabolismo , Células Cultivadas , Uniones Comunicantes/metabolismo , Iones/química , Iones/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Arterias Mesentéricas/citología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Estrés Mecánico , Propiedades de Superficie
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