Direction of cellular protrusions by curvature
Martin, Kimberly Cordwint
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Developmental processes involving symmetry-breaking of homogeneous cell populations into leaders and followers are found in many important contexts. Cells constrained by culture on two-dimensional scaffolds, as well as in three-dimensional shapes, appear to respond to convex curves with an increasing propensity to protrude, while concave curves in contrast appear to inhibit protrusion. This has interesting implications in terms of a potential positive feedback loop. This feedback may act in symmetry-breaking, through amplification of initial stochastic differences in cell shape, and also in collective migration, through reinforcing and directing the coherent movement of collectives. In this study, epithelial cells were cultured on two-dimensional micropatterns with variable curvatures to examine the effect of edge geometry and other variables on the likelihood of protrusions forming. This platform allowed the quantification of F-actin-based protrusions at the periphery of multicellular epithelial clusters, in segments defined by cluster edge curvature. The initial observations confirmed reports in the literature of preferential localisation of protrusions at more convex regions, and relative inhibition at more concave regions. A previously-published work has postulated a role for secreted modulators of motility, with the shape of a group of cells determining the concentration of diffusing morphogen each individual cell is exposed to. To test this hypothesis, a low-shear flow culture chamber was used to disrupt the putative gradients. Despite theoretical and empirical support for the sufficiency of the flow condition to disrupt autocrine signalling, micropatterned cells cultured under flow showed no significant differences from the control condition. These findings form the basis of a manuscript which has been accepted for publication by the Journal of Anatomy. The results of an Atomic Force Microscopy (AFM) study carried out by collaborators were suggestive of a role for cellular mechanotransduction in sensing and responding to micropattern curvature. Differential calcium channel mechanoactivation was hypothesised as being one potential mechanism underlying the response to curvature, given the known involvement of mechanosensitive ion channels in cellular responses to force and substrate stiffness, and the multiple roles of calcium in cellular motility. Artificially increasing cytosolic calcium levels with Ionomycin reduced protrusion rates at convex curves. However, treatment with BAPTA-AM to sequester intracellular calcium had no effect on protrusion rates. ROCK inhibitor, in contrast, increased protrusion rates at concave curves, and Blebbistatin increased protrusion rates globally. These results together are suggestive of differential control of myosin depending on local curvature: cyclic and driven by calcium-activation of MLCK in the convex regions (with lamellipodia undergoing protrusion-retraction cycles), versus sustained and controlled by ROCK in the concave regions (where lamellipodia are inhibited). The unexpected finding that protrusions at convex regions were resistant to the actin cytoskeleton-disrupting drug Cytochalasin D may point to a role for a tropomyosin isoform in defining the differing mechanical characteristics of the actin cytoskeleton in response to local curvature. In addition, the previously-noted lack of effect of BAPTA-AM treatment (which has been shown to interfere with dynamic microtubules) is suggestive of a role for stabilised microtubules in protrusions at convex regions. These indications of unique characteristics to the protrusions promoted by convex curvature give added support to the curvature-protrusion feedback model, and its relevance to tissue morphogenesis. In summary, this work provides evidence against a previously-published suggested mechanism for the curvature-protrusion feedback loop that is proposed to act during epithelial morphogenesis, and evidence in support of a role for a calcium-based mechanism in driving the initiation and maintenance of leader cells in migrating epithelial sheets. Further work is called for in characterising the protrusions promoted by convex curvature, and the mechanisms controlling them. This area is of significance in gaining greater understanding of tissue morphogenesis in pathogenesis and development, and of potential value in tissue engineering applications.