CE13 - Biologie cellulaire, biologie du développement et de l’évolution

Shaping cells: The role of the external forces and constraints – ForcesOnCell

Submission summary

Epithelial cell shape depends on an internal balance between the actomyosin cytoskeleton, the Adherens Junctions and the Focal Adhesions. How these internal cell shape effectors are impacted by compressive forces generated by adjacent growing tissues is unknown. To tackle this question, we use the Drosophila ovarian follicle, in which cuboidal epithelial cells are apically compressed by germline cytoplasmic pressure, while simultaneously being basally constrained by a basement membrane. We previously demonstrated that these two surrounding tissues mechanically drives cuboidal cells to become either squamous or columnar. Here our aim is to determine the mechanisms by which compressive forces (germline cytoplasmic pressure and basement membrane stiffness) modify cell shape effectors (the cytoskeleton and adhesion proteins) to allow cell morphogenesis. Our hypothesis is that changes in any one of these molecules impact the others. This is supported by a few studies indicating that E-Cadherin and integrin are molecules that, when under tension, modify the actomyosin network, which in response may feed back on them.
To investigate this, we will perform experiments in both WT and mutant conditions where the mechanical properties of the germline or the BM are altered either positively or negatively. Importantly, we have already quantified and published the mechanical properties of these mutants. Specifically, we will first use 3D reconstructions and in-vivo live imaging to quantify cell shape changes and define the sequence of remodelling events at the apical, lateral and basal membranes. Second, we will characterise the differential subcellular localisation of E-Cad, integrin, actin and myosin and measure the dynamics of E-Cad and integrin through confocal and super-resolution microscopy. We will also decipher the interplay between E-Cad and integrin by monitoring each one in real time after forcing the clustering of the other through an optogenetic tool. Third, because the activities of E-Cadherin and Integrin depend on their low-level organisation into nano-clusters, we will perform homo-FRET experiments to obtain a detailed description of the nano-scale organisation of each subcellular accumulation (such as monomers or cis-oligomers). Fourth, we will determine the mechanical properties of cuboidal, squamous and columnar cells by performing micro-compression, laser ablation and force inference measurements. Finally, we will employ genetic and optogenetic tools to explore whether myosin activity differentially impacts adhesive complexes as a function of external compressive forces. Altogether, these experiments will 1) provide qualitative and quantitative descriptions of the impact of the surrounding mechanical properties in cell morphogenesis, 2) deliver a comprehensive 4D-representation of the subcellular organisation (levels, nanostructure, pooling and dynamics) of cell adhesion molecules as a function of mechanical impacts of the surrounding tissue and 3) establish how surrounding tissues tune internal mechanical properties and control the interplay between E-Cadherin, Integrin and actomyosin. To conclude, this will help elucidate the framework by which external forces modify intracellular mechanical properties to allow cell shape changes to occur. In addition, this work will give us a unique opportunity to establish the fundamental characteristics for the three central epithelial shapes at cell and nano-scales.
In summary, our project will generate fundamental insights into how cell shape develops as a function of the mechanical properties of the environment. We propose a multidisciplinary approach (including 4D reconstructions, homoFRET, biophysics and optogenetics) to achieve our objectives through a consortium that brings together complementary expertise in genetics, image analysis and mechanics (partner 1), in cell biology and optogenetics (partner 2) and in membrane dynamics and protein clustering (partner 3).

Project coordinator

Madame Muriel Grammont (Ecole Normale Supérieure de Lyon)

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.

Partner

LBMC Ecole Normale Supérieure de Lyon
MCD Unité de biologie moléculaire, cellulaire et du développement
NCBS

Help of the ANR 509,347 euros
Beginning and duration of the scientific project: March 2023 - 54 Months

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