ActuAtor, a molecular tool for generating force in living cells

项目摘要 机械力调节不同的细胞事件，包括囊泡运输和基因表达。先前 对机械反应的研究集中在细胞表面发生的事件，因为可用的 技术限于主要从细胞外部施加力。因此，我们建议发展和推进 一种被称为致动器的方法，可以以受控的方式在活细胞中产生力。执行器基于 在所需的亚细胞位置诱导工程化的、细菌衍生的肌动蛋白成核剂的积累， 导致通过聚合肌动蛋白产生力。第一代执行器探针成功变形 细胞内结构包括细胞器如线粒体和细胞核。 为了评估ActuAtor的生物适用性，我们建议在细胞中实施该技术以解决 细胞器的形式-功能相互作用。细胞内的细胞器有各种形状和大小。人们早就 我怀疑这种变化与它们的功能有关。然而，它们的形状之间的因果关系 和功能仍然在很大程度上是未知的，主要是由于缺乏直接操纵细胞器的技术 形态学通过使ActuAtor适应模型细胞器，线粒体，我们将揭示线粒体如何 形态决定其功能。在生理相关设置中使用致动器还将带来 有助于从其原始分子设计进一步改进的信息。 我们的建议的关键创新是发展，推进和实施一个尖端的 技术来解决一个基本的生物学问题，否则无法解决传统的 技术独自。尽管有许多现有的分子工具可以影响“生化”反应，我们的遗传- 编码的执行器工具是极少数能够实现“物理”操纵的例子之一，即力 在活细胞环境中产生，因此构成高意义。这项独特的跨学科研究 由PI整合，PI的专业知识在于分子技术开发，作为与 线粒体生物学家Hiromi Sesaki博士预计将在开发分子工具方面产生协同作用， 在广泛的细胞生物学实验中真正实用。由于致动器的分子设计是模块化的， 应用不限于线粒体或培养的细胞。相反，它们容易适用于其他细胞内 细胞器如内质网和细胞核，以及模型动物如苍蝇和小鼠， 阐明了我们研究的一个令人兴奋的未来方向。

PROJECT SUMMARY Mechanical force regulates diverse cellular events including vesicular trafficking and gene expression. Previous studies on mechanoresponses have focused on events taking place at the cell surface, because available techniques are limited to exert force mostly from outside of cells. We therefore propose to develop and advance a methodology termed ActuAtor that can generate force in living cells in a controlled manner. ActuAtor is based on induced accumulation of an engineered, bacteria-derived actin nucleator at a desired subcellular location, leading to force generation through polymerized actin. A first generation ActuAtor probe successfully deformed intracellular structures including organelles such as mitochondria and nucleus. To assess the biological applicability of ActuAtor, we propose to implement the technique in cells to address the form-function interplay of organelles. Intracellular organelles take various shapes and sizes. It has long been suspected that this variability relates to their functions. However, the causal relationship between their shape and function remains largely unknown, primarily due to a lack of techniques to directly manipulate the organelle morphology. By adapting ActuAtor to a model organelle, mitochondria, we will reveal how mitochondrial morphology determines their functions. Use of ActuAtor in a physiologically relevant setting will also bring about information helpful for further improvement from its original molecular design. The key innovation of our proposal is development, advancement and implementation of a cutting-edge technique to tackle a fundamental biological question that cannot otherwise be addressed with conventional techniques alone. Despite many existing molecular tools that can affect “biochemical” reactions, our genetically- encoded ActuAtor tools present one of the very few examples of enabling “physical” manipulation, namely force generation, in a live-cell environment, thus constituting high significance. This unique interdisciplinary study integrated by the PI whose expertise lies in molecular technology development as a collaboration with a mitochondria biologist, Dr. Hiromi Sesaki, is expected to generate synergy in developing molecular tools that has bona fide utility in broad cell biology experiments. As the molecular design of ActuAtor is modular, their application is not limited to mitochondria or cultured cells. Rather, they are readily applicable to other intracellular organelles such as endoplasmic reticulum and nucleus, as well as model animals such as flies and mice, which illuminates one of the exciting future directions of our research.