RESUMO
Endothelial monolayers physiologically adapt to flow and flow-induced wall shear stress, attaining ordered configurations in which elongation, orientation, and polarization are coherently organized over many cells. Here, with the flow direction unchanged, a peculiar bi-stable (along the flow direction or perpendicular to it) cell alignment is observed, emerging as a function of the flow intensity alone, while cell polarization is purely instructed by flow directionality. Driven by the experimental findings, the parallelism between endothelia is delineated under a flow field and the transition of dual-frequency nematic liquid crystals under an external oscillatory electric field. The resulting physical model reproduces the two stable configurations and the energy landscape of the corresponding system transitions. In addition, it reveals the existence of a disordered, metastable state emerging upon system perturbation. This intermediate state, experimentally demonstrated in endothelial monolayers, is shown to expose the cellular system to a weakening of cell-to-cell junctions to the detriment of the monolayer integrity. The flow-adaptation of monolayers composed of healthy and senescent endothelia is successfully predicted by the model with adjustable nematic parameters. These results may help to understand the maladaptive response of in vivo endothelial tissues to disturbed hemodynamics and the progressive functional decay of senescent endothelia.
Assuntos
Junções Intercelulares , Cristais Líquidos , Anisotropia , Endotélio , Cristais Líquidos/química , Estresse MecânicoRESUMO
Cell polarity refers to the intrinsic asymmetry of cells, including the orientation of the cytoskeleton. It affects cell shape and structure as well as the distribution of proteins and organelles. In migratory cells, front-rear polarity is essential and dictates movement direction. While the link between the cytoskeleton and nucleus is well-studied, we aim to investigate if front-rear polarity can be transmitted to the nucleus. We show that the knock-down of emerin, an integral protein of the nuclear envelope, abolishes preferential localization of several nuclear proteins. We propose that the frontally biased localization of the endoplasmic reticulum, through which emerin reaches the nuclear envelope, is sufficient to generate its observed bias. In primary emerin-deficient myoblasts, its expression partially rescues the polarity of the nucleus. Our results demonstrate that front-rear cell polarity is transmitted to the nucleus and that emerin is an important determinant of nuclear polarity.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Linhagem Celular , Núcleo Celular/ultraestrutura , Imunofluorescência , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mioblastos/metabolismo , Mioblastos/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Interferência de RNARESUMO
Droplet interactions with compliant materials are familiar, but surprisingly complex processes of importance to the manufacturing, chemical, and garment industries. Despite progress-previous research indicates that mesoscopic substrate deformations can enhance droplet drying or slow down spreading dynamics-our understanding of how the intertwined effects of transient wetting phenomena and substrate deformation affect drying remains incomplete. Here we show that above a critical receding contact line speed during drying, a previously not observed wetting transition occurs. We employ 4D confocal reference-free traction force microscopy (cTFM) to quantify the transient displacement and stress fields with the needed resolution, revealing high and asymmetric local substrate deformations leading to contact line pinning, illustrating a rate-dependent wettability on viscoelastic solids. Our study has significance for understanding the liquid removal mechanism on compliant substrates and for the associated surface design considerations. The developed methodology paves the way to study complex dynamic compliant substrate phenomena.
RESUMO
Traction force microscopy (TFM) derives maps of cell-generated forces, typically in the nanonewton range, transmitted to the extracellular environment upon actuation of complex biological processes. In traditional approaches, force rendering requires a terminal, time-consuming step of cell deadhesion to obtain a reference image. A conceptually opposite approach is provided by reference-free methods, opening to the on-the-fly generation of force maps from an ongoing experiment. This requires an image processing algorithm keeping the pace of the biological phenomena under investigation. Here, we introduce an integrated software pipeline rendering force maps from single reference-free TFM images seconds to minutes after their acquisition. The algorithm tackles image processing, reference image estimation, and finite element analysis as a single problem, yielding a robust and fully automatic solution. The method's capabilities are demonstrated in two applications. First, the mechanical annihilation of cancer cells is monitored as a function of rising environmental temperature, setting a population threshold at 45 °C. Second, the fast temporal correlation of forces produced across individual cells is used to map physically connected adhesion points, yielding typical lengths that vary as a function of the cell cycle phase.
RESUMO
Autologous epidermis grafts generated in vitro represent a promising option for the treatment of burn wounds. The procedure relies on of a sufficient number of cells harvested from healthy tissue, which are then sparsely seeded on a target surface. The time required to reconstitute a fully confluent and mature monolayer and the limited availability of cell seeds hinder the broad clinical application of this procedure. Here, a novel engineering approach to enhance the in vitro expansion of epithelial tissues is designed and experimentally validated. The method combines three independent elements supporting fast epithelialization. First, the tactical seeding of epithelial cells at high density in confined channels generated by means of magnetic silicon stencils. Second, the implementation of a curved interface along the channels, increasing the edge interface length. Third, a rationally developed and oriented anisotropic topography, in the form of gratings, aligned perpendicularly to the channels. Upon removal of the stencil, unconfined cell monolayers are free to expand and invade the open space, in a process of epithelialization that fully exploits the directional migration of epithelial collectives. As compared to sparse seeding, this approach attains an almost three times faster full epithelialization of a target surface with the same number of cells. Molecular signals triggered by cell-cell and cell-substrate contacts supported this enhanced response. In summary, we introduce a facile and scalable approach yielding fast in vitro epithelial tissue expansion with optimized yield.
RESUMO
The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was expressed in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by a recently developed reference-free confocal traction-force microscopy platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases than in the ensuing late S/G2, and locally colocalized with high levels of paxillin phosphorylation. Perturbation of paxillin phosphorylation at focal adhesions, obtained through the biochemical inhibition of focal adhesion kinase (FAK) or the transfection of nonphosphorylatable or phosphomimetic paxillin mutants, significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells, instrumental to their invasion of dense environments. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.
Assuntos
Ciclo Celular , Neoplasias/patologia , Fenômenos Biomecânicos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Invasividade Neoplásica , Paxilina/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.
Assuntos
Endocitose , Epitélio/metabolismo , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods.