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

21ENGBIO 多功能光遗传学工具箱，用于控制细胞和组织形态发生的细胞力学

• 批准号: BB/W011123/1
• 负责人: Guillaume Charras
• 金额: $ 12.85万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW011123%2F1
• 关键词: 21ENGBIO; versatile; optogenetic; toolbox; control

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.

我们的目标是设计一个多功能工具箱，以动态控制细胞和组织形状。受软机器人技术的启发以及天然存在的细胞和组织形状变化，我们将设计光敏的执行器，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，以根据光致动模式来预测细胞力学和形状变化。这些将被利用以产生我们可以以可预测方式动态控制的细胞和组织。胚胎最引人注目的特性之一是它们在发育过程中发生的复杂形状变化。这些形状的变化是由细胞的集体行为积极驱动的，这些行为会在组织内部产生内部应力。形状变化需要在每个组织不同区域内细胞力学的时空协调变化。这引起了由运动蛋白活性产生的组织张力梯度。这些梯度源于基因表达的空间差异，这些空间差异在一种称为小GTPases的信号蛋白上收敛，该蛋白质控制细胞骨架和细胞之间的粘附。尽管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重点，但对信号如何控制力学改变形状的焦点知之甚少。然而，驱动形状变化的是细胞表面的机械梯度。RhogTPase信号传导具有20个由Rhogefs（激活Rhogtpase）和Rhogaps（激活它们灭活的）调节功能的Rhogtpase的高度控制。 Rhogefs和Rhogaps具有不同的局部定位和上游调节剂，可以很好地控制细胞力学和力的产生。 Rhogtpase活性通过作用于运动蛋白的活性，细胞骨骼的组织以及细胞之间的粘附来调节整个细胞中的表面张力或下细胞。但是，我们对信号传导与细胞表面张力之间联系的理解仍然很差，我们缺乏预测信号效果的概念框架。结果，我们无法预测因信号变化而发生的形状变化。该提案的目的是设计一种多功能工具箱来控制细胞和组织中的力学和形状。我们将重点关注以下目标：（1）设计一个模块化工具箱，以光线控制信号：我们将根据细胞和组织形状变化过程中自然发生的信号来设计光激活的执行器。 （2）用执行器来控制单细胞力学：我们将表征执行器的单个执行器和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力的细胞构建块的设计生成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，基于光的执行器和建模方面的专业知识，因此我们可以选择实现这些目标。目标1将设计一种多功能工具箱，以在细胞和亚细胞尺度上使用基于光的执行器动态控制细胞力学。我们将根据Rhogefs和Rhogaps设计执行器，以使我们能够以高度的空间和时间准确性来增加或减少细胞表面张力。目标2将表征光遗传学的致动器如何影响细胞表面和细胞之间的连接处的张力。我们还将检查执行器之间的串扰，以研究非线性效应。最后，我们将设计一个概念框架，以预测信号变化预期的机械变化。这将允许将形状变化与信号的变化联系起来，并预测达到任何选择的组织或细胞形状所需的光驱动模式。