RESUMEN

Morphogenesis requires spatiotemporal regulation of proliferation, both by biochemical and mechanical cues. In epithelia, this regulation is called contact inhibition of proliferation, but disentangling biochemical from mechanical cues remains challenging. Here, we show that epithelia growing under confinement accumulate pressure that inhibits proliferation above a threshold value. During growth, epithelia spontaneously buckle, and cell proliferation is transiently reactivated within the fold. Reactivation of proliferation within folds correlated with the local reactivation of the mechano-sensing YAP/TAZ pathway. At late time points, when the pressure is highest, ß-catenin activity increases. The threshold pressure increases when ß-catenin is overactivated and decreases when ß-catenin is inhibited. Altogether, our results suggest that different mechanical cues resulting from pressure inhibition of proliferation are at play through different mechano-sensing pathways: the ß-catenin pathway sustains cell division under high pressure, and the YAP pathway senses local curvature.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , beta Catenina , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ciclo Celular , División Celular , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP , beta Catenina/metabolismo

RESUMEN

Many organs are formed through folding of an epithelium. This change in shape is usually attributed to tissue heterogeneities, for example, local apical contraction. In contrast, compressive stresses have been proposed to fold a homogeneous epithelium by buckling. While buckling is an appealing mechanism, demonstrating that it underlies folding requires measurement of the stress field and the material properties of the tissue, which are currently inaccessible in vivo. Here, we show that monolayers of identical cells proliferating on the inner surface of elastic spherical shells can spontaneously fold. By measuring the elastic deformation of the shell, we infer the forces acting within the monolayer and its elastic modulus. Using analytical and numerical theories linking forces to shape, we find that buckling quantitatively accounts for the shape changes of our monolayers. Our study shows that forces arising from epithelial growth in three-dimensional confinement are sufficient to drive folding by buckling.

Asunto(s)

Fenómenos Biomecánicos/fisiología , Módulo de Elasticidad/fisiología , Epitelio/crecimiento & desarrollo , Adhesión Celular/fisiología , Proliferación Celular/fisiología , Simulación por Computador , Humanos , Modelos Biológicos

RESUMEN

We present here a microfluidic device that generates sub-millimetric hollow hydrogel spheres, encapsulating cells and coated internally with a layer of reconstituted extracellular matrix (ECM) of a few microns thick. The spherical capsules, composed of alginate hydrogel, originate from the spontaneous instability of a multi-layered jet formed by co-extrusion using a coaxial flow device. We provide a simple design to manufacture this device using a DLP (digital light processing) 3D printer. Then, we demonstrate how the inner wall of the capsules can be decorated with a continuous ECM layer that is anchored to the alginate gel and mimics the basal membrane of a cellular niche. Finally, we used this approach to encapsulate human Neural Stem Cells (hNSC) derived from human Induced Pluripotent Stem Cells (hIPSC), which were further differentiated into neurons within the capsules with negligible loss of viability. Altogether, we show that these capsules may serve as cell micro-containers compatible with complex cell culture conditions and applications. These developments widen the field of research and biomedical applications of the cell encapsulation technology.

Asunto(s)

Técnicas de Cultivo de Célula/instrumentación , Células Inmovilizadas/citología , Microambiente Celular , Dispositivos Laboratorio en un Chip , Células-Madre Neurales/citología , Neurogénesis , Neuronas/citología , Alginatos/química , Automatización de Laboratorios , Línea Celular , Supervivencia Celular , Células Inmovilizadas/metabolismo , Colágeno/química , Combinación de Medicamentos , Matriz Extracelular/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Hidrogeles , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Laminina/química , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Impresión Tridimensional , Prueba de Estudio Conceptual , Proteoglicanos/química , Nicho de Células Madre , Propiedades de Superficie

RESUMEN

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.

Asunto(s)

Microesferas , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Resistencia a la Tracción/fisiología , Proteínas de Xenopus/metabolismo , Animales , Cinética , Microtúbulos/química , Microtúbulos/metabolismo , Polimerizacion , Unión Proteica , Porcinos , Tubulina (Proteína)/metabolismo , Xenopus laevis

RESUMEN

In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.

Asunto(s)

Procesamiento de Imagen Asistido por Computador/métodos , Microtúbulos/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Color , Colorantes Fluorescentes/farmacología , Humanos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Modelos Biológicos , Coloración y Etiquetado/métodos , Tubulina (Proteína)/metabolismo