Synergistic action of actin binding proteins regulate actin network organization and cell shape

Animal cell shape is largely determined by the organization of the actin cytoskeleton. Spread shapes result from a balance between protrusive actin networks and contractile stress fibers, while rounded shapes are supported by a contractile actomyosin cortex. The assembly and regulation of distinct types of actin networks have been extensively studied, yet, what determines which networks dominate in a given cell remains unclear. In this Brief Report, we explore the molecular regulation of overall actin organization and resulting cell shape. We use our recently published comparison of the F-actin interactome in spread interphase and rounded mitotic cells to establish a list of candidate regulators of actin networks in spread cells. Utilizing micropatterning and automated image analysis we quantitatively analyze how these candidates affect actin organization. Out of our initial 16 candidates, we identify subsets of proteins promoting stress fibers or regulating their arrangement. Interestingly, no single regulator depletion caused significant cell shape change. However, perturbing two hits simultaneously, supervillin and myosin II, led to stress fiber disassembly and cell rounding. Overall, our systematic investigation shows that actin networks are robust to perturbations, and identifies regulatory modules controlling overall actin organization and resulting cell shape.