RESUMEN
Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 µm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion.
Asunto(s)
Movimiento Celular/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Seudópodos/fisiología , Cicatrización de Heridas/fisiología , Actomiosina/fisiología , Animales , Recuento de Células , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Perros , Uniones Intercelulares/fisiología , Riñón/citología , Quinasa de Cadena Ligera de Miosina/fisiología , Estrés Mecánico , Quinasas Asociadas a rho/fisiologíaRESUMEN
Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands. On the most rigid substrates, cells appear well-spread and present cadherin adhesions and cytoskeletal organization similar to those classically observed on cadherin-coated glass substrates. However, when cells are cultured on softer substrates, a change in morphology is observed: the cells are less spread, with a more disorganized actin network. A quantitative analysis of the cells adhering on the cadherin-coated surfaces shows that forces are correlated with the formation of cadherin adhesions. The stiffer the substrates, the larger are the average traction forces and the more developed are the cadherin adhesions. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechanosensitive regulation of cadherin-mediated intercellular junctions through the cellular contractile machinery.
Asunto(s)
Cadherinas/metabolismo , Actinas/metabolismo , Animales , Fenómenos Biomecánicos , Adhesión Celular , Línea Celular , Forma de la Célula , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Humanos , Uniones Intercelulares/metabolismo , Espacio Intracelular/metabolismo , Ratones , Miosina Tipo II/metabolismo , Propiedades de SuperficieRESUMEN
Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis. Microfabrication techniques have proven to be very useful for studies of collective cell migration in vitro. In this chapter, we briefly review the use of microfabricated substrates in providing new insights into collective cell behaviors. We first describe the development of micropatterned substrates to study the influence of geometrical constraints on cell migration and coordinated movements. Then, we present an alternative method based on microfabricated pillar substrates to create well-defined gaps within cell sheets and study gap closure. We also provide a discussion that presents possible pitfalls and sheds light onto the important parameters that allow the study of long-term cell culture on substrates of well-defined geometries.
Asunto(s)
Movimiento Celular , Microambiente Celular , Microtecnología/métodos , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Microambiente Celular/efectos de los fármacos , Dimetilpolisiloxanos/farmacología , Perros , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Fibronectinas/metabolismo , Células de Riñón Canino Madin DarbyRESUMEN
A fundamental feature of multicellular organisms is their ability to self-repair wounds through the movement of epithelial cells into the damaged area. This collective cellular movement is commonly attributed to a combination of cell crawling and "purse-string" contraction of a supracellular actomyosin ring. Here we show by direct experimental measurement that these two mechanisms are insufficient to explain force patterns observed during wound closure. At early stages of the process, leading actin protrusions generate traction forces that point away from the wound, showing that wound closure is initially driven by cell crawling. At later stages, we observed unanticipated patterns of traction forces pointing towards the wound. Such patterns have strong force components that are both radial and tangential to the wound. We show that these force components arise from tensions transmitted by a heterogeneous actomyosin ring to the underlying substrate through focal adhesions. The structural and mechanical organization reported here provides cells with a mechanism to close the wound by cooperatively compressing the underlying substrate.