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ActuAtor, a molecular tool for generating force in living cells

ActuAtor，一种在活细胞中产生力的分子工具

基本信息

批准号：
10473892
负责人：
Takanari Inoue
金额：
$ 32.75万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10473892
关键词：
Actins Address Affect Alzheimer&apos s Disease Animal Model Apoptosis Atomic Force Microscopy Bacteria Biochemical Reaction Biological Biological Assay Biology Cardiac Myocytes Cause of Death Cell Line Cell Nucleus Cell membrane Cell physiology Cell surface Cells Cellular biology Charcot-Marie-Tooth Disease Chemicals Chemotaxis Chimeric Proteins Collaborations Cultured Cells Cytokinesis Dependence Development Dimerization Disease Endoplasmic Reticulum Engineering Environment Event Eye Face Filament Fluorescence Microscopy Future Gene Expression Generations Golgi Apparatus Heart Heart failure Hypoglycemia Individual Inherited Interdisciplinary Study Investigation Lead Light Link Location Maintenance Malignant Neoplasms Measures Membrane Membrane Potentials Methodology Methods Microfilaments Microtubules Mitochondria Modeling Molecular Molecular Probes Morphology Movement Mus Nature Neurodegenerative Disorders Neurologic Neurons Nutrient Organelles Outer Mitochondrial Membrane Pathogenesis Patients Persons Pharmacological Treatment Phenotype Physiological Polymers Production Property Proteins Public Health Reactive Oxygen Species Regulation Reporting Research Research Personnel Role Series Shapes Skin Specificity Stimulus Stress Stress Fibers Subcellular structure Surface Techniques Testing Time Tissues Toxic effect Transportation Tubular formation base cell type constriction cytotoxicity design design and construction dimer experimental study feasibility testing fly genetic manipulation innovation insight laser tweezer mechanical force novel strategies operation optogenetics polymerization prototype response synergism technology development tool trafficking virtual

项目摘要

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.

项目概要机械力调节多种细胞事件，包括囊泡运输和基因表达。以前的关于机械反应的研究主要集中在细胞表面发生的事件，因为可用技术仅限于主要从细胞外部施加力。因此，我们建议开发和推进一种称为 ActuAtor 的方法，可以以受控方式在活细胞中产生力。执行器基于诱导工程化的、源自细菌的肌动蛋白成核剂在所需的亚细胞位置的积累，导致通过聚合肌动蛋白产生力。第一代ActuAtor探针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 ActuAtor 的生物学适用性，我们建议在细胞中实施该技术来解决细胞器的形式-功能相互作用。细胞内细胞器具有各种形状和大小。早已是怀疑这种变异性与它们的功能有关。然而，它们的形状之间存在因果关系。功能仍然很大程度上未知，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了它们的功能。在生理相关环境中使用 ActuAtor 也会带来有助于进一步改进其原始分子设计的信息。我们提案的关键创新是尖端技术的开发、推进和实施解决传统技术无法解决的基本生物学问题的技术单独的技术。尽管许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 ActuAtor 工具提供了极少数实现“物理”操纵的示例之一，即力在活细胞环境中生成，因此具有很高的意义。这种独特的跨学科研究由专门从事分子技术开发的 PI 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具有真正的实用性。由于 ActuAtor 的分子设计是模块化的，因此它们应用不限于线粒体或培养细胞。相反，它们很容易应用于其他细胞内内质网、细胞核等细胞器，以及果蝇、小鼠等模型动物，阐明了我们研究的令人兴奋的未来方向之一。