We first developed original experimental approaches from molecular to multicellular levels to quantify the remodeling of epithelial cell sheets and the dynamics of cell-cell junctions. The project is at the interface between biology, physics and nanotechnologies. Our collaborative program implies to further implement a panel of complementary micromechanical and visualization techniques with molecular biology tools to unravel the dynamics of cadherin associated proteins, the mechanical stability of multicellular cell assemblies such as epithelial cells in response to changes in their microenvironments as well as the molecular mechanisms responsible for force transduction.
One of the major technical issues is to develop methods to precisely map and/or apply local forces in multicellular system to study the mechanics of intercellular junctions. We have developed original microfabricated environments to map the contractile forces exerted by an epithelial monolayer and determine the force transmission mechanisms as well as original strategies to better understand collective dynamics due to tissue remodeling in terms of local forces, migration velocity and cell-cell rearrangements. Complementary experiments at the molecular level are performed to determine the possible mechanisms for force transduction at cell-cell contacts, such as the exposure of buried binding sites induced by stretching.
The development of these mechanical tools is coupled to the analysis of the biochemical mechanisms involved in the formation of cell-cell junctions and cell tension. Molecular tools are applied to determine the role of molecular components in the mechano-sensitivity of AJs in multicellular systems.
To further analyze quantitatively membrane associated and/or intracellular events at high spatial and temporal resolutions, standard optical methods can be used as well as more recent techniques which are based on correlation spectroscopy.
We have developed microfabricated substrates to probe the mechanics of cell-cell junctions. We have succeeded in developing a strategy to create magnetically actuated micropillar arrays.
Then we show that a large-scale mechanosensing mechanism leads to an adaptative response of cell migration to stiffness gradients. With S. Dufour (Institut Curie, Paris), we studied the cross-talk between cell-matrix and cell-cell achesions.
We used two different types of assays to probe the dynamics of epithelial cells under mechanical and geometrical constraints. We have shown 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.
We show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell–cell interactions using microfabrication techniques.
Molecular mechanics of AJ mechanotransduction
With Sylvie Dufour, we show that both a-catenin and vinculin are required for the force-dependent maturation of adherens junctions.
We produced also corresponding a-catenin mutants to be used or single molecule analysis.
We obtained the functional nanoparticle and set up also the experimental condition to visualize cadherin single molecule arrangement at the surface of cells by Electron microscopy imaging
Fluorescence fluctuation-based spectroscopy and adhesion proteins dynamics and interactions
We developed novel methods based on fluorescence lifetime correlation spectroscopy to quantify the interaction between two proteins within mobile complexes.
Cell sensitivity to mechanical tensions
By using optical tweezers combined to confocal fluorescence microscopy with dual objectives, mechanical cue is sufficient to bias cortical actomyosin instability and to impose cell polarity establishment in detached rounded cells (Bun & al.).
The contractile forces that allow cells to sense and respond to various different mechanical and structural contexts seem to be required for many steps in the maintenance and integrity of tissues. Because the characterization of forces in vivo is a complicated and daunting task, it is important to determine from in vitro mechanotransduction studies the important mechanisms that could help to explain complex pathological as well as developmental behaviours in vivo. Understanding how cells sense and respond to mechanical cues is important for diseases, such as cancer, in which the mechanical properties of the microenvironment are postulated to regulate tumorigenesis but also for our understanding of embryogenesis. From the viewpoint of cell and tissue engineering, the enhancement of our knowledge on epithelial cell growth and migration can help in the design and engineering of ECM and biomaterials with better control of cell migration, cell distribution, and morphogenesis, thus facilitating tissue repair and regeneration.
Clearly, addressing such outstanding issues falls outside the realm of traditional cell biology approaches and instead requires the cooperative effort of biologists, materials scientists, physicists and engineers. Indeed, this exciting force ‘frontier’ is fertile territory for scientific exploration of development and cancer biology that will undoubtedly yield new insights into cancer evolution and identify novel anticancer therapeutic targets.
1- B. Ladoux & A. Nicolas , Reports on Progress in Physics, in press (2012).
2- SRK. Vedula, MC. Leong, T. Lei Lai, P. Hersen, A. J. Kabla,
CT. Lim & B. Ladoux Proc Natl Acad Sci U S A. 109, 12974-79 (2012).
3- A. Jasaitis, M. Estevez, J
How living cells are able to sense their environment and adequately respond to maintain cell, tissue and organ homeostasis remains one of the more puzzling issues in cell biology. Cellular responses to mechanical factors are critical in normal development but alterations in critical proteins will alter mechanical functions resulting for example in abnormally highly migratory cells. The analysis of the adhesion and mechanical response and adaptation of normal and altered cells can provide important insights into the roles of those proteins in the motile processes and their roles in the overall cell functions in disease states. Nevertheless, transduction of mechanical stress is an amazingly underestimated mode of cell signalling. This mechano-transduction intimately associated to cell adhesion processes targets primarily cell-matrix and cell-cell contacts. The traction forces developed and transmitted via integrins by cells toward the extracellular matrix and substratum have been proposed a long time ago and only recently characterized in details. The transmitted mechanical stresses through their effects on cell tension have high incidence on cell shape, migration and differentiation. In contrast, very little is known on mechano-transduction at cell-cell contacts. The objective of the present program is to understand how cadherins, the major actors of intercellular adhesions (adherens junctions) will sense, adapt and transmit mechanical stress from cell to cell. In the context of epithelial cell tissues, we plan to study the mechanics of mechanotransduction and mechanosentivity at cell-cell junctions from molecular up to multicellular levels thanks to the combination of cell biology approaches with existing and forthcoming microfabrication techniques, biophysics developments allowing to map, quantify and even apply forces in the range of the ones sensed and developed by cells. The precise spatial and temporal coordination of intracellular signalling events in relation to mechanical cues, will be tackled through the spatial maps of molecular interactions and dynamics of key proteins by using spatial and temporal fluorescence high resolution imaging techniques. To our knowledge the transmission of mechanical strength from cell to cell by cadherins is a fully new research area, and only a few groups in the world including partners 1 and 2 in collaboration have initiated such programs. To address this question absolutely needs highly complementary scientific backgrounds and skills gathered by the 3 partners to conduct appropriate multidisciplinary approaches. Indeed, beside the central classical biochemistry, and cell biology approaches mastered by Partner 2, this program envision the implementation and development of a panel of microfabricated microforce captors and AFM-based single molecules approaches (Partner 1) as well as advanced live cell imaging brought by Partner 3. This interdisciplinary association is based on already solid collaborations and gathers most of the equipments and skills necessary to carry out these novel studies. All 3 partners have a previous experience of such interdisciplinary work and thus most of the planned experiments are directly involving at least to complementary skills either cell biology and micromechanics, cell biology and advanced microscopies… Although our project deals with fundamental research, the output of the current project will raise the level of fundamental knowledge of how biological systems actually work as biomechanical structures in response to the micro-environment, which will have direct relevance to biomedical and translational science as well as the biotechnological industry. It has major implications for the normal development of multicellular organisms as well as the understanding of disease-associated deregulations such misslocalisation of cell progenitors leading to congenital malformations or loss of contact and migratory phenotypes acquired by cancer cells.
Monsieur Benoît Ladoux (CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – Benoit.Ladoux@univ-paris-diderot.fr
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IFM INSERM- DELEGATION PARIS VI
IJM - CNRS CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
MSC - CNRS CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
Help of the ANR 380,053 euros
Beginning and duration of the scientific project: - 36 Months