ActuAtor, a molecular tool for generating force in living cells

项目总结 机械力调节多种细胞活动，包括囊泡运输和基因表达。上一首 对机械反应的研究主要集中在细胞表面发生的事件上，因为 技术受到限制，主要只能在细胞外施力。因此，我们建议发展和推进 一种称为致动器的方法，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核酸体积聚， 导致通过聚合的肌动蛋白产生力。第一代致动器探头成功变形 细胞内结构，包括线粒体和细胞核等细胞器。 为了评估致动器的生物学适用性，我们建议在细胞中实施这项技术，以解决 细胞器的形式-功能相互作用。细胞内细胞器的形状和大小各不相同。一直以来都是 怀疑这种差异与它们的功能有关。然而，它们的形状之间的因果关系 而功能在很大程度上仍然未知，主要是因为缺乏直接操纵细胞器的技术 形态学。通过使致动器适应一个模型细胞器，线粒体，我们将揭示线粒体是如何 形态决定了它们的功能。在与生理相关的环境中使用致动器也将带来 有助于进一步改进其原始分子设计的信息。 我们建议的关键创新是开发、推进和实施尖端技术 解决一个基本的生物学问题的技术，否则无法用常规方法解决 单单是技巧。尽管有很多现有的分子工具可以影响“生化”反应，但我们的基因-- 编码的致动器工具提供了为数不多的实现“物理”操纵的例子之一，即力 在活细胞环境中生成，因此具有很高的意义。这项独特的跨学科研究 由PI整合，其专长在于分子技术开发，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效应，这种工具具有 在广泛的细胞生物学实验中真正有用。由于执行器的分子设计是模块化的，他们的 应用并不局限于线粒体或培养细胞。相反，它们很容易适用于其他细胞内 细胞器，如内质网和细胞核，以及模式动物，如苍蝇和小鼠，它们 阐明了我们未来研究的一个令人兴奋的方向。

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.