21ENGBIO A versatile optogenetic toolbox to control cell mechanics for cell and tissue morphogenesis

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Our goal is to design a versatile toolbox to dynamically control cell and tissue shape. Inspired by soft robotics and the naturally occurring cell and tissue shape changes, we will design light-sensitive actuators that can control the activity of the proteins that control shape change. We will also develop software approaches to predict how cell mechanics and shape change based on the pattern of light actuation. These will be exploited to generate cells and tissues whose shape we can dynamically control in a predictable manner. One of the most striking properties of embryos is the complex shape changes that they undergo during development. These shape changes are actively driven by the collective behaviour of cells, which create internal stress within the tissue. Shape change requires spatiotemporally coordinated changes in the mechanics of cells within different regions of each tissue. This gives rise to gradients in tissue tension generated by motor protein activity. These gradients originate from spatial differences in gene expression that converge on a family of signalling proteins called small GTPases that control the cell skeleton and adhesion between cells. Whereas the signalling pathways controlling shape during cell and tissue shape change are the focus of much research, little is known about how signalling controls mechanics to change shape. Yet, it is the mechanical gradients at the cell surface that drive shape change.RhoGTPase signalling offers a high degree of control with 20 RhoGTPases with diverse function regulated by RhoGEFs (that activate RhoGTPases) and RhoGAPs (that inactivate them). RhoGEFs and RhoGAPs have diverse localizations and upstream regulators allowing fine control of cell mechanics and force generation. RhoGTPase activity modulates surface tension in the whole cell or subcellularly by acting on the activity of motor proteins, the organization of the cell skeleton, and adhesion between cells. However, our understanding of the link between signalling and cell surface tension remains poor and we lack a conceptual framework to predict the effect of signalling. As a result, we cannot predict the shape changes that would occur in response to a change in signalling.The goal of this proposal is to design a versatile toolbox to control mechanics and shape in cells and tissues. We will focus on the following objectives:(1) Design a modular toolbox to control signalling with light: We will design light-activated actuators based on the signalling naturally occurring during cell and tissue shape change. (2) Control single cell mechanics with actuators: We will characterize how individual actuators and combinations of actuators alter cell surface tension. Our toolbox relies on broadly applicable modular approaches that will allow the design of actuatable cellular building blocks to generate self-folding tissues of arbitrary shape for synthetic biology and allow control of the shape of single cells. Because of our expertise in cell and tissue mechanics, light-based actuators, and modelling, we are ideally placed to achieve those aims. Objective 1 will design a versatile toolbox to dynamically control cell mechanics with light based actuators at the cellular and subcellular scale. We will design actuators based on RhoGEFs and RhoGAPs to enable us to increase or decrease cell surface tensions with a high degree of spatial and temporal accuracy. Objective 2 will characterize how optogenetic actuators affect tension at the cell surface and at junctions between cells. We will also examine crosstalk between actuators to investigate non-linear effects. Finally, we will devise a conceptual framework to predict the mechanical changes expected from changes in signaling. This will allow to directly link shape changes to changes in signaling as well as predict the pattern of light actuation necessary to reach any chosen tissue or cell shape.