Campas+Project

= Force patterns in cell monolayers and living embryonic tissues = = = Friedhelm Serwain, Madhav Mani and Otger Campas

= Biological problem =

Cellular forces and, more specifically, the spatiotemporal patterns of cellular forces during embryonic development are known to be critical for the shaping of tissues and organs into their functional morphologies. Despite the relevance of cellular forces in embryo morphogenesis, very little is know about how they guide the shaping of embryonic tissues, mostly because of a lack in experimental techniques to directly quantify physical forces //in vivo//. Several methods exist to measure traction forces //in vitro//, with cultured cells, but they cannot be used to measure forces in living embryonic tissues. Force inference methods and laser ablation have allowed estimations of cellular tensions in epithelial tissues during development. However, it remains unclear if these estimates correspond to the real tensions in the system. Using a new technique developed by Prof. Campàs and coworkers, which allows a direct readout of local cellular stresses, we will measure the local cellular stresses in cultured epithelial cells and, time allowing, in living embryonic tissue (Zebrafish and Drosophila). The technique consists of using oil droplets, similar in size to individual cells, with defined mechanical properties and displaying ligands for cell surface adhesion receptors, as force transducers. When introduced between cells, local cellular forces deform the droplets; local cellular forces are then obtained directly from droplet deformations. Once direct measures of cellular stresses are obtained in cultured epithelial cells and living embryonic tissues, we will test whether force inference methods and laser ablation provide faithful estimates of cellular tensions. These measurements should allow us to quantify the mechanical state of embryonic epithelial tissues in space and time, providing key information to understand embryonic morphogenesis.

= Microscopy and microsurgery =

- LSM Zeiss 780 and LSM Zeiss 700 with environmental control for live cell and embryo imaging. - 40x, NA= 1.1, LD water immersion objective. - Record time-lapse z-stacks of cell monolayers and, in some cases, embryos containing florescent droplets, with high spatial resolution (dx,dy < .6 µm, dz < 1 µm). - Laser ablation of cell junctions surrounding oil droplets.

= Data Analysis =

- Segment cell boundaries to perform estimates of the mechanical state using force inference methods. - Reconstruct the shape of different droplets in 3D to measure local stresses performed by cells. - Use force inference methods to estimate the mechanical state of the tissue from the cell shapes, and compare the predictions of the estimations to the local stresses developed by cells (measured using the droplet’s technique).

= Physical Model =

There currently exist several models of force inference from cell shape in epithelial monolayers (refs.). However, no direct measure of cell-cell forces have been done so far and it is unclear if force inference methods actually capture the mechanical state of the system. We will use direct measurement of local cellular stresses to test the proposed inference methods.

= Skills that students acquire =

Cell culture, laser ablation, embryo microinjection, embryo staging and mounting, confocal microscopy, live cell and embryo imaging, image segmentation and force inference methods.