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.
Mechanical force underlies virtually all cellular processes involving movement or deformation, and altered mechanoresponses are linked to cancers (one of the most common causes of death), while abnormally deformed organelles are found in patients of Charcot-Marie-Tooth disease (the most common inherited neurologic condition worldwide, with no disease-modifying treatments) and Alzheimer's disease (a progressive neurodegenerative disease affecting 5.3 million persons in the US alone). Our molecular tools collectively termed ActuAtor that can exert ?physical force? specifically onto intended intracellular objects, to synthetically trigger mechanoresponses in a rapidly inducible manner, may become a powerful strategy for dissecting molecular mechanisms of mechanoresponses under physiologically relevant conditions, which cannot be readily achieved by existing methods alone. In the future, new findings obtained from the ActuAtor operation in patient and healthy cells may lead to insights into the pathogenesis of the above diseases.
