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1.
Proc Natl Acad Sci U S A ; 121(2): e2300174121, 2024 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-38175870

RESUMEN

Microtubules and molecular motors are essential components of the cellular cytoskeleton, driving fundamental processes in vivo, including chromosome segregation and cargo transport. When reconstituted in vitro, these cytoskeletal proteins serve as energy-consuming building blocks to study the self-organization of active matter. Cytoskeletal active gels display rich emergent dynamics, including extensile flows, locally contractile asters, and bulk contraction. However, it is unclear how the protein-protein interaction kinetics set their contractile or extensile nature. Here, we explore the origin of the transition from extensile bundles to contractile asters in a minimal reconstituted system composed of stabilized microtubules, depletant, adenosine 5'-triphosphate (ATP), and clusters of kinesin-1 motors. We show that the microtubule-binding and unbinding kinetics of highly processive motor clusters set their ability to end-accumulate, which can drive polarity sorting of the microtubules and aster formation. We further demonstrate that the microscopic time scale of end-accumulation sets the emergent time scale of aster formation. Finally, we show that biochemical regulation is insufficient to fully explain the transition as generic aligning interactions through depletion, cross-linking, or excluded volume interactions can drive bundle formation despite end-accumulating motors. The extensile-to-contractile transition is well captured by a simple self-assembly model where nematic and polar aligning interactions compete to form either bundles or asters. Starting from a five-dimensional organization phase space, we identify a single control parameter given by the ratio of the different component concentrations that dictates the material-scale organization. Overall, this work shows that the interplay of biochemical and mechanical tuning at the microscopic level controls the robust self-organization of active cytoskeletal materials.


Asunto(s)
Citoesqueleto , Microtúbulos , Microtúbulos/metabolismo , Citoesqueleto/metabolismo , Cinesinas/metabolismo , Movimiento Celular , Segregación Cromosómica
2.
Phys Rev Lett ; 125(25): 257801, 2020 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-33416339

RESUMEN

Spontaneous growth of long-wavelength deformations is a defining feature of active liquid crystals. We investigate the effect of confinement on the instability of 3D active liquid crystals in the isotropic phase composed of extensile microtubule bundles and kinesin molecular motors. When shear aligned, such fluids exhibit finite-wavelength self-amplifying bend deformations. By systematically changing the channel size we elucidate how the instability wavelength and its growth rate depend on the channel dimensions. Experimental findings are qualitatively consistent with a minimal hydrodynamic model, where the fastest growing deformation is set by a balance of active driving and elastic relaxation. Our results demonstrate that confinement determines the structure and dynamics of active fluids on all experimentally accessible length scales.

3.
Proc Natl Acad Sci U S A ; 112(19): 5944-9, 2015 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-25922533

RESUMEN

In a wide range of epithelial tissues such as kidney tubules or breast acini, cells organize into bidimensional monolayers experiencing an out-of-plane curvature. Cancer cells can also migrate collectively from epithelial tumors by wrapping around vessels or muscle fibers. However, in vitro experiments dealing with epithelia are mostly performed on flat substrates, neglecting this out-of-plane component. In this paper, we study the development and migration of epithelial tissues on glass wires of well-defined radii varying from less than 1 µm up to 85 µm. To uncouple the effect of out-of-plane curvature from the lateral confinement experienced by the cells in these geometries, we compare our results to experiments performed on narrow adhesive tracks. Because of lateral confinement, the velocity of collective migration increases for radii smaller than typically 20 µm. The monolayer dynamics is then controlled by front-edge protrusions. Conversely, high curvature is identified as the inducer of frequent cell detachments at the front edge, a phenotype reminiscent of the Epithelial-Mesenchymal Transition. High curvature also induces a circumferential alignment of the actin cytoskeleton, stabilized by multiple focal adhesions. This organization of the cytoskeleton is reminiscent of in vivo situations such as the development of the trachea of the Drosophila embryo. Finally, submicron radii halt the monolayer, which then reconfigures into hollow cysts.


Asunto(s)
Citoesqueleto de Actina/fisiología , Epitelio/fisiología , Animales , Adhesión Celular , Movimiento Celular , Citoesqueleto/metabolismo , Perros , Drosophila/embriología , Transición Epitelial-Mesenquimal , Adhesiones Focales , Vidrio/química , Rayos Láser , Células de Riñón Canino Madin Darby , Ratones , Microscopía Fluorescente , Músculos/fisiología , Células 3T3 NIH , Fenotipo , Tráquea/embriología
4.
Proc Natl Acad Sci U S A ; 112(31): 9546-51, 2015 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-26199417

RESUMEN

Tissue fusion eliminates physical voids in a tissue to form a continuous structure and is central to many processes in development and repair. Fusion events in vivo, particularly in embryonic development, often involve the purse-string contraction of a pluricellular actomyosin cable at the free edge. However, in vitro, adhesion of the cells to their substrate favors a closure mechanism mediated by lamellipodial protrusions, which has prevented a systematic study of the purse-string mechanism. Here, we show that monolayers can cover well-controlled mesoscopic nonadherent areas much larger than a cell size by purse-string closure and that active epithelial fluctuations are required for this process. We have formulated a simple stochastic model that includes purse-string contractility, tissue fluctuations, and effective friction to qualitatively and quantitatively account for the dynamics of closure. Our data suggest that, in vivo, tissue fusion adapts to the local environment by coordinating lamellipodial protrusions and purse-string contractions.


Asunto(s)
Organogénesis , Actomiosina/metabolismo , Animales , Adhesión Celular , Perros , Células Epiteliales/citología , Epitelio/fisiología , Terapia por Láser , Células de Riñón Canino Madin Darby , Modelos Biológicos , Procesos Estocásticos , Propiedades de Superficie , Cicatrización de Heridas
5.
Soft Matter ; 13(45): 8474-8482, 2017 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-29091088

RESUMEN

We study spreading on soft substrates of cellular aggregates using CT26 cells that produce an extracellular matrix (ECM). Compared to our previous work on the spreading of S180 cellular aggregates, which did not secrete ECMs, we found that the spreading velocity of the precursor film is also maximal for intermediate rigidities, but new striking features show up. First, we observed a cascade of liquid-gas-liquid (L/G/L) transitions of the precursor film as the substrate rigidity is decreased. We attribute the L/G transition to a decrease of cell/cell adhesion resulting from the weakening of the cell/substrate adhesion. We attribute the reentrant liquid phase (G/L) observed on soft substrates to the slow spreading of the aggregates on ultra-soft substrates, which gives time to the cells to secrete more ECM proteins and stick together. Second, a nematic order appears in the cohesive (liquid) states of the precursor film, attributed to the gradient of cell's velocities.


Asunto(s)
Agregación Celular , Humectabilidad , Resinas Acrílicas/química , Fenómenos Biomecánicos , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Vidrio/química , Humanos , Transición de Fase
6.
Proc Natl Acad Sci U S A ; 110(5): 1686-91, 2013 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-23319630

RESUMEN

Metastasis, the truly lethal aspect of cancer, occurs when metastatic cancer cells in a tumor break through the basement membrane and penetrate the extracellular matrix. We show that MDA-MB-231 metastatic breast cancer cells cooperatively invade a 3D collagen matrix while following a glucose gradient. The invasion front of the cells is a dynamic one, with different cells assuming the lead on a time scale of 70 h. The front cell leadership is dynamic presumably because of metabolic costs associated with a long-range strain field that precedes the invading cell front, which we have imaged using confocal imaging and marker beads imbedded in the collagen matrix. We suggest this could be a quantitative assay for an invasive phenotype tracking a glucose gradient and show that the invading cells act in a cooperative manner by exchanging leaders in the invading front.


Asunto(s)
Movimiento Celular , Colágeno/metabolismo , Glucosa/metabolismo , Termodinámica , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Quimiotaxis , Matriz Extracelular/metabolismo , Femenino , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Células MCF-7 , Microscopía Confocal , Microscopía Fluorescente , Invasividad Neoplásica , Metástasis de la Neoplasia , Factores de Tiempo , Microambiente Tumoral
7.
Phys Rev Lett ; 110(15): 158103, 2013 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-25167315

RESUMEN

The collective chemosensing of nonexcitable mammalian cells involves a biochemical network that features gap junction communications and heterogeneous single cell activities. To understand the integrated multicellular chemosensing, we study the calcium dynamics of micropatterned fibroblast cell colonies in response to adenosine triphosphate (ATP) stimulation. We find that the cross-correlation function between the responses of individual cells decays with topological distance as a power law for large colonies and much faster for smaller colonies. Furthermore, the strongly correlated cell pairs tend to form clusters and are more likely to exceed the percolation threshold. At a given topological distance, the cross-correlations exhibit characteristics of Poisson distributions, which allows us to estimate the unitary conductance of a single gap junction which is in good agreement with direct experimental measurements.


Asunto(s)
Adenosina Trifosfato/farmacología , Comunicación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Modelos Biológicos , Animales , Calcio/metabolismo , Comunicación Celular/fisiología , Fibroblastos/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Ratones , Células 3T3 NIH , Distribución de Poisson
8.
Nat Commun ; 13(1): 6465, 2022 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-36309493

RESUMEN

How active stresses generated by molecular motors set the large-scale mechanics of the cell cytoskeleton remains poorly understood. Here, we combine experiments and theory to demonstrate how the emergent properties of a biomimetic active crosslinked gel depend on the properties of its microscopic constituents. We show that an extensile nematic elastomer exhibits two distinct activity-driven instabilities, spontaneously bending in-plane or buckling out-of-plane depending on its composition. Molecular motors play a dual antagonistic role, fluidizing or stiffening the gel depending on the ATP concentration. We demonstrate how active and elastic stresses are set by each component, providing estimates for the active gel theory parameters. Finally, activity and elasticity were manipulated in situ with light-activable motor proteins, controlling the direction of the instability optically. These results highlight how cytoskeletal stresses regulate the self-organization of living matter and set the foundations for the rational design and optogenetic control of active materials.


Asunto(s)
Citoesqueleto , Microtúbulos , Citoesqueleto/fisiología , Elasticidad , Geles , Elastómeros
9.
Science ; 367(6482): 1120-1124, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32139540

RESUMEN

Topological structures are effective descriptors of the nonequilibrium dynamics of diverse many-body systems. For example, motile, point-like topological defects capture the salient features of two-dimensional active liquid crystals composed of energy-consuming anisotropic units. We dispersed force-generating microtubule bundles in a passive colloidal liquid crystal to form a three-dimensional active nematic. Light-sheet microscopy revealed the temporal evolution of the millimeter-scale structure of these active nematics with single-bundle resolution. The primary topological excitations are extended, charge-neutral disclination loops that undergo complex dynamics and recombination events. Our work suggests a framework for analyzing the nonequilibrium dynamics of bulk anisotropic systems as diverse as driven complex fluids, active metamaterials, biological tissues, and collections of robots or organisms.

10.
J Phys Condens Matter ; 32(19): 193001, 2020 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-32058979

RESUMEN

Activity and autonomous motion are fundamental in living and engineering systems. This has stimulated the new field of 'active matter' in recent years, which focuses on the physical aspects of propulsion mechanisms, and on motility-induced emergent collective behavior of a larger number of identical agents. The scale of agents ranges from nanomotors and microswimmers, to cells, fish, birds, and people. Inspired by biological microswimmers, various designs of autonomous synthetic nano- and micromachines have been proposed. Such machines provide the basis for multifunctional, highly responsive, intelligent (artificial) active materials, which exhibit emergent behavior and the ability to perform tasks in response to external stimuli. A major challenge for understanding and designing active matter is their inherent nonequilibrium nature due to persistent energy consumption, which invalidates equilibrium concepts such as free energy, detailed balance, and time-reversal symmetry. Unraveling, predicting, and controlling the behavior of active matter is a truly interdisciplinary endeavor at the interface of biology, chemistry, ecology, engineering, mathematics, and physics. The vast complexity of phenomena and mechanisms involved in the self-organization and dynamics of motile active matter comprises a major challenge. Hence, to advance, and eventually reach a comprehensive understanding, this important research area requires a concerted, synergetic approach of the various disciplines. The 2020 motile active matter roadmap of Journal of Physics: Condensed Matter addresses the current state of the art of the field and provides guidance for both students as well as established scientists in their efforts to advance this fascinating area.

11.
Methods Mol Biol ; 1749: 387-399, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29526012

RESUMEN

Confinement and substrate topology strongly affect the behavior of cell populations and, in particular, their collective migration. In vitro experiments dealing with these aspects require strategies of surface patterning that remain effective over long times (typically several days) and ways to control the surface topology in three dimensions. Here, we describe protocols addressing these two aspects. High-resolution patterning of a robust cell-repellent coating is achieved by etching the coating through a photoresist mask patterned directly on the coated surface. Out-of-plane curvature can be controlled using glass wires or corrugated "wavy" surfaces.


Asunto(s)
Movimiento Celular/fisiología , Animales , Línea Celular , Humanos , Polietilenglicoles/química
12.
Nat Commun ; 7: 11373, 2016 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-27109213

RESUMEN

Cell competition is a quality control mechanism that eliminates unfit cells. How cells compete is poorly understood, but it is generally accepted that molecular exchange between cells signals elimination of unfit cells. Here we report an orthogonal mechanism of cell competition, whereby cells compete through mechanical insults. We show that MDCK cells silenced for the polarity gene scribble (scrib(KD)) are hypersensitive to compaction, that interaction with wild-type cells causes their compaction and that crowding is sufficient for scrib(KD) cell elimination. Importantly, we show that elevation of the tumour suppressor p53 is necessary and sufficient for crowding hypersensitivity. Compaction, via activation of Rho-associated kinase (ROCK) and the stress kinase p38, leads to further p53 elevation, causing cell death. Thus, in addition to molecules, cells use mechanical means to compete. Given the involvement of p53, compaction hypersensitivity may be widespread among damaged cells and offers an additional route to eliminate unfit cells.


Asunto(s)
Comunicación Celular , Células de Riñón Canino Madin Darby/química , Células de Riñón Canino Madin Darby/citología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Fenómenos Biomecánicos , Perros , Drosophila/citología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células de Riñón Canino Madin Darby/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteína p53 Supresora de Tumor/genética , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo
13.
Sci Rep ; 5: 11759, 2015 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-26152517

RESUMEN

RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis. Herein we investigated the mechanistic contribution of Ral proteins to invasion of lung cancer A549 cells after induction of epithelial-mesenchymal transition (EMT) with TGFß. We show that TGFß-induced EMT promotes dissemination of A549 cells in a 2/3D assay, independently of proteolysis, by activating the Rho/ROCK pathway which generates actomyosin-dependent contractility forces that actively remodel the extracellular matrix, as assessed by Traction Force microscopy. RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector. Indeed, uncoupling of the Exocyst subunit Sec5 from GEF-H1 impairs RhoA activation, generation of traction forces and cell dissemination. These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.


Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Proteínas de Unión al GTP ral/metabolismo , Amidas/farmacología , Línea Celular Tumoral , Humanos , Microscopía de Fuerza Atómica , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/genética , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo
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