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

我们的目标是设计一个多功能的工具箱来动态控制细胞和组织的形状。受软机器人和自然发生的细胞和组织形状变化的启发，我们将设计光敏致动器，可以控制控制形状变化的蛋白质的活性。我们还将开发软件方法来预测细胞力学和形状如何根据光致动模式变化。这些将被用来产生细胞和组织，我们可以以可预测的方式动态控制它们的形状。胚胎最引人注目的特性之一是它们在发育过程中经历的复杂形状变化。这些形状变化是由细胞的集体行为主动驱动的，这在组织内产生内应力。形状变化需要每个组织的不同区域内的细胞力学的时空协调变化。这引起由运动蛋白活性产生的组织张力的梯度。这些梯度源于基因表达的空间差异，这些差异聚集在一个称为小GTP酶的信号蛋白家族上，该家族控制细胞骨架和细胞之间的粘附。虽然在细胞和组织形状变化过程中控制形状的信号通路是许多研究的焦点，但对信号如何控制力学改变形状知之甚少。然而，正是细胞表面的机械梯度驱动形状变化。RhoGTPases信号传导提供了高度的控制，其中20种RhoGTPases具有不同的功能，由RhoGEFs（激活RhoGTPases）和RhoGAP（激活它们）调节。RhoGEF和RhoGAP具有不同的定位和上游调节器，允许精细控制细胞力学和力的产生。RhoGT3活性通过作用于马达蛋白的活性、细胞骨架的组织和细胞之间的粘附来调节整个细胞或亚细胞中的表面张力。然而，我们对信号和细胞表面张力之间的联系的理解仍然很差，我们缺乏一个概念框架来预测信号的影响。因此，我们无法预测响应信号变化而发生的形状变化。该提案的目标是设计一个多功能工具箱来控制细胞和组织中的力学和形状。我们将专注于以下目标：（1）设计一个模块化工具箱来控制光信号：我们将设计基于细胞和组织形状变化过程中自然发生的信号的光激活致动器。(2)用执行器控制单细胞力学：我们将描述单个执行器和执行器组合如何改变细胞表面张力。我们的工具箱依赖于广泛适用的模块化方法，这些方法将允许设计可致动的细胞构建块，以生成用于合成生物学的任意形状的自折叠组织，并允许控制单细胞的形状。由于我们在细胞和组织力学，光基执行器和建模方面的专业知识，我们非常适合实现这些目标。目标1将设计一个多功能的工具箱，在细胞和亚细胞尺度上用基于光的致动器动态控制细胞力学。我们将设计基于RhoGEFs和RhoGAP的致动器，使我们能够以高度的空间和时间精度增加或减少细胞表面张力。目标2将描述光遗传致动器如何影响细胞表面和细胞间连接处的张力。我们还将研究致动器之间的串扰，以研究非线性效应。最后，我们将设计一个概念框架来预测信号变化所带来的机械变化。这将允许直接将形状变化与信号变化联系起来，并预测到达任何选定组织或细胞形状所需的光致动模式。

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.