Institut Pasteur, Paris
Romain Levayer and his team work to figure out the mechanisms of the elimination of epithelial cells: the most common tissue in our body that forms coherent barriers.
More specifically, they explore how mechanical forces can influence cell survival and how this can be exploited by tumor cells to distort and eliminate their neighbours.
They use a combination of real-time microscopy and biophysical tools to study cell death in the Drosophila fly, and use the genetic power of this species to combine different genetic alterations and study the interactions between normal and tumour cells.
Living tissues have a remarkable plasticity illustrated by their capacity to regenerate and develop normal organs despite dramatic perturbations. This is based on the amazing capacity of every single cell to adapt its behavior to local information (e.g.: paracrine signal, contact dependant signaling, adhesive forces) and tissue scale information (e.g: tissue size, tissue density). Our laboratory is generally interested by the plasticity of cell behavior and more specifically by the adjustment and regulation of cell death in epithelia. Epithelia are two dimensional layers of adhesive cells that form barriers in the organism. Our group is currently focusing on two aspects of the regulation of cell death in epithelial context:
1. The influence of mechanical forces and cell shape on death induction in physiological and pathological contexts
2. The orchestration of epithelial cell clearance by effector caspases and the commitment to apoptosis
We use the fruitfly (Drosophila melanogaster) to tackle these questions through a combination of genetics, live imaging, quantitative image analysis, optogenetics and biophysics.
Despite our deep knowledge of the pathways regulating cell proliferation, growth and survival, we still do not understand how single cells can match their behavior to properties of the entire tissue (including its size and shape). Mechanical forces can convey tissue scale information (tissue size, density) to single cells. While the effects of mechanical forces on cell proliferation have been widely studied, their influence on cell survival have been poorly studied in vivo. Recently, it was shown that a local increase of cell density in an epithelium can induce cell extrusion and elimination. We recently showed that caspase activation is necessary for cell elimination in the pupal notum (a single layer epithelium). We are currently trying to identify new regulators of apoptosis induction which are sensitive to cell deformation and tissue crowding.
Left: Disitribution of cell death (orange dots) in the Drosophila pupal notum. Cell contours are shown with E-cad::GFP.
Right: Pseudocolour image of an ERK sensor (red: low ERK, blue: high ERK), a regulator of cell survival in the notum.
Timelaps in the Drosophila pupal notum showing the elimination of WT cells (purple) in the midline region (green)
We are also testing how spatial constrains could affect cell survival in various developmental contexts and test their contribution to morphogenesis and tissue size regulation. We use a combination of live imaging (particle image velocimetry, tissue segmentation and cell tracking) and clonal analysis to understand how this could help to fine tune the proportion of dying cells in growing tissues, during programmed tissue clearance and during tissue homeostasis.
Finally, we are trying to understand how crowding induced death can contribute to the competitive interactions between different cell types. Cell competition is a process inducing the elimination of slow proliferating cells by faster proliferative cells through apoptosis. Cell competition is a conserved mechanism required for the correction of developmental errors, to fine tune tissue size and could contribute to tumor expansion through the elimination and replacement of neighbouring healthy cells by pretumoral cells. We and others have recently shown that fast growing clones resistant for apoptosis can promote WT cell eliminations though their compaction and induction of apoptosis. We use a combination of genetics, live imaging, laser nanodissection and theory to understand how spatial constrains can contribute to cell competition and how confrontation of two cell populations can lead to cell compaction and elimination.
Left: WT cell elimination (green) near pretumoural cells (activation of Ras, purple). Orange arrows show tissue displacement analysed by PIV.
Right: Evolution of cell apical area (blue= compaction, red= expansion).
Timelaps in the Drosophila pupal notum showing the compaction and elimination of WT cells near a fast growing clone (UAS-RasV12 ,purple)
Despite the detailed characterisation of the molecular players of apoptosis, its orchestration and fine regulation in multicellular contexts is not well understood. While apoptosis is often considered as a simple binary process, there are nowadays many evidences showing that apoptosis is a complex decision making process, where cells can undergo transient caspase activation without proceeding to death. This is in agreement with the multiple non-apoptotic functions of caspases and the architecture of the apoptotic pathway which includes several negative feedback loops. This complexity increases further in epithelial cells, where cell clearance requires orchestration of successive remodelling events necessary to extrude the cell from the epithelial layer without impairing tissue barrier function.
We recently showed that cell extrusion in the Drosophila pupal notum always requires effector caspases activity and that caspase activation precedes extrusion. Moreover, we measured very various lagtimes between the onset of caspase activation and delamination (30 min to several hours) and also observed transient caspase activation which do not lead to cell delamination. These observations suggest that the commitment in extrusion and apoptosis is a complex decision making process. Moreover, our results suggest that yet uncharacterized substrate(s) of effector caspases are required for extrusion and that the same molecular player (the effector caspase) orchestrates all the remodelling events of apoptosis (such as extrusion, cytoplasmic compaction, DNA condensation, nucleus and cell fragmentation). We are currently studying the orchestration of cell death and extrusion in Drosophila epithelium in order to:
1) find new caspase substrates required for cell extrusion
2) dissect the mechanism that regulates the successive remodelling events
3) understand the cell decision making process leading to irreversible apoptosis
4) study the consequences of impaired cell extrusion for epithelial homeostasis.
Visualisation of caspase activity with a FRET marker (Scat3) in the Drosophila pupal notum. A diminution of FRET (right side, cold colours) indicates activation of caspases.
For this, we combine quantitative live imaging, proteomics, genetic, optogenetic, cell biology and theoretical approaches to build a predictive framework that will help to understand the orchestration of apoptosis in a living epithelium.
2011 : PhD, Development Biology Institute of Marseille, France, team of Thomas Lecuit
2012-2016 : Post-doctoral fellowship, Berne University, Swiss, team of Eduardo Moreno
2016 : Appointed group leader, Institut Pasteur, Paris, France
• 2018 : Laureate of the Jeune Chercheur price, Société Française de Biologie Cellulaire
• 2017 : Laureate of the Starting Grant program, European Research Council (ERC)
• 2012 : Laureate of the thesis price of the Fondation Réaumur