Cell Shape and Actin Cytoskeleton
Cell shape is the outcome of a complex interaction between cell cytoskeleton and external conditions. Cells in tissue have reproducible shape and cytoskeleton organization in response to cell-cell or cell ECM contacts. The reproducibility of cell cytoskeleton architecture is lost when cells are plated on culture dishes. Under such conditions, the permanent assembly and disassembly of focal adhesions and actin fibers during cell migration prevent the cytoskeleton network dynamics achieving a steady state structure and activity. Cells adopt random morphologies and random walk, a highly artefactual situation compared to the tissue environment.
Fully adhesive micropatterns (such as full disks, squares and triangles) prevent migration and impose cell shape, but distribution of focal adhesions remain variable. By restricting cell adhesion and controlling locations of adhesive and non adhesive areas under a cell of a given shape, focal adhesions are more restricted to certain areas and cells are closer to the conditions that they encounter in tissues where cell attachment to each other or to the ECM are also restricted. On a concave micropattern, cells spread out and develop a highly reproducible actin organization with large and contractile stress fibers upon non-adhesive regions to adopt the convex shape of the underlying micropattern.
By modifying the geometry of the adhesive microenvironment, CYTOO’s micropatterns control cell adhesion and cell shape independently.
Identical cell shapes, distinct cell adhesion patterns. Dissecting the impact of cell adhesion on cell shape and cytoskeleton architecture
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Adhesive pattern
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Cell distribution of stress fibres in response to the geometry of the adhesive environment
The figure above shows how it is possible to control the size and location of stress fibers and focal adhesion points within cells by adapting the micropattern geometry and while maintaining a similar cell shape.
By screening a panel of adhesive geometries, parameters influencing the cell architecture can be identified, allowing to uncouple the role of cell shape versus cell adhesion.
A few examples:
Stress fiber strength* versus local adhesiveness Using micropatterns, it was found that fiber strength is inversely related to local adhesiveness. Stronger fibers are built over non adhesive edges and support the free edge. If adhesion is allowed along a cell’s edge, stress fibers are considerably weaker. |
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Stress fiber strength* versus length As the length between the two anchoring points decreases the stress fibers becomes weaker. |
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Stress fiber strength* versus number of stress fibers per cell In addition, the group of images on the left shows that when stress fiber bundles are more numerous they are also weaker. The 3 stress fibers on the Y are individually each weaker than the 2 on the T which are individually each weaker than the single one on the V. Actin bundles are thinner and focal adhesions are smaller. The total amount of F actin per cell on all three geometries is constant. |
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Polymerization versus contraction in the actin network |
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*Strength is inferred from the area and intensity of both phalloidin and vinculin labeling |
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Easy analysis of cell contractility in response to drugs
In the standard assay, 10µM Y27632 is the minimum concentration of the ROCK inhibitor showing effect on RPE1 cells |
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On adhesive micropatterns, the 10µM effect of Y27632 on RPE1 cells is immediately visible and quantifiable. An automated methodology can be easily programmed on existing image analysis platforms by using simple tools of thresholding and measuring the curvature of the free membrane
Cell distribution of stress fibres in response to the geometry of the adhesive environment.
Thery M, Pepin A, Dressaire E, Chen Y, Bornens M.
Cell Motil Cytoskeleton 63(6):341-55.2006.