Cell mechanobiology in confinement using an integration of bioengineering, materials systems and in vivo models

总结-细胞在体内穿过纤维状细胞外基质之间的限制性三维（3D）孔 基质（ECM）网络或由ECM束、血管、肌纤维或神经界定的通道样轨迹。的 使细胞在不同微环境中运动的机制是适应性的， 生物化学线索，如限制，刚度，粘弹性和ECM的组成。自适应 这些系统/模块包括细胞-ECM相互作用、肌动球蛋白细胞骨架和细胞体积调节。 最近，我们和其他人也确定了核在有限迁移中的关键作用。然而，在这方面， 关于核武器与核武器之间的串扰，许多基本的和翻译的问题仍然没有答案。 机械感应，细胞骨架和细胞体积调节，以及它们对健康受限迁移的贡献 和疾病该项目的总体目标是采用最先进的生物工程，材料和 成像工具以及体内模型，以提供用于限制中的有效迁移的新颖的统一框架 通过破译核力学，细胞骨架和离子通道之间的相互作用。R 01应用程序 将检验这样一个假设，即细胞核通过精细地调节 RhoA沿着细胞纵轴在密闭空间中的空间激活，通过以下协同作用： 限制诱导的核硬化和anillin/Ect 2核退出细胞质。这种假设是 有趣的初步数据表明，细胞进入限制性的μ通道， 硬化，其激活RhoA并支持离子通道依赖性核起泡和破裂。核 破裂诱导苯胺醛和RhoGEF Ect 2从细胞核离开到细胞质。苯胺醛积累 特别是在细胞两极，在那里它局部桥接Ect 2，RhoA和肌动球蛋白，从而加剧RhoA。 肌球蛋白II收缩性。在目标1中，我们将破译苯胺醛退出细胞质的机制，并证明 它作为支架蛋白的关键作用，在细胞两极桥接Ect 2，RhoA和肌动球蛋白，从而 调节RhoA的空间激活和限制中基于水泡的迁移。我们亦会阐释 细胞体积调节与核泡形成和破裂中的苯胺醛/Ect 2/RhoA之间的新串扰 禁闭。最后，我们将破译的贡献，核推动从细胞后部与核拉 从电池正面（即，核活塞模型）迁移作为约束程度的函数。在目标2中， 我们将把我们的研究结果的适用性扩展到3D凝胶和限制规定的生理微通道， 体外相关机械性能。我们还将观察到苯胺醛、Ect 2和 在体内不同尺寸的天然组织中的关键离子通道，并测试这些通道的扰动如何影响 分子影响体内的局部和远端组织侵入。这项工作也将发展和建立新的 生物工程工具（例如，光遗传学探针，μ-流体室），以更好地了解健康中的细胞运动 和疾病

Summary- Cells in vivo travel through confining three-dimensional (3D) pores between fibrillar extracellular matrix (ECM) networks or channel-like tracks bordered by ECM bundles, vessels, myofibers or nerves. The mechanisms enabling cell locomotion in diverse microenvironments are adaptive in response to the physical and biochemical cues, such as confinement, stiffness, viscoelastic properties and composition of ECM. Adaptive systems/modules include cell-ECM interactions, the actomyosin cytoskeleton and cell volume regulation. Recently, we and others have also identified the key role of the nucleus in confined migration. However, numerous fundamental and translational questions remain unanswered on the crosstalk between nuclear mechanosensing, cytoskeleton and cell volume regulation, and their contributions to confined migration in health and disease. The overarching goal of this project is to employ state-of-the-art bioengineering, materials and imaging tools as well as in vivo models to provide a novel unified framework for efficient migration in confinement by deciphering the interplay between nuclear mechanics, cytoskeleton and ion channels. This R01 application will test the hypothesis that the nucleus senses and responds to physical confinement by exquisitely regulating the spatial activation of RhoA along the longitudinal cell axis in confined spaces via the synergistic roles of confinement-induced nuclear stiffening and anillin/Ect2 nuclear exit to the cytoplasm. This hypothesis is supported by intriguing preliminary data showing that cell entry into confining µ-channels induces nuclear stiffening which activates RhoA and supports ion channel-dependent nuclear blebbing and rupture. Nuclear rupture induces the exit of anillin and the RhoGEF Ect2 from the nucleus to the cytoplasm. Anillin accumulates specifically at the cell poles, where it locally bridges Ect2, RhoA and actomyosin, thereby exacerbating RhoA- myosin II contractility. In Aim 1, we will decipher the mechanisms of anillin exit to the cytoplasm, and demonstrate its critical role as a scaffolding protein, which bridges Ect2, RhoA and actomyosin at the cell poles, thereby regulating the spatial activation of RhoA and bleb-based migration in confinement. We will also elucidate the novel crosstalk between cell volume regulation and anillin/Ect2/RhoA in nuclear blebbing and rupture in confinement. Lastly, we will decipher the contributions of nuclear pushing from the cell rear versus nuclear pulling from the cell front (i.e., nuclear piston model) to migration as a function of the degree of confinement. In Aim 2, we will extend the applicability of our findings to 3D gels and confining µ-channels of prescribed physiologically relevant mechanical properties in vitro. We will also visualize the distinct localization patterns of anillin, Ect2 and key ion channels in natural tissue tracks of different dimensions in vivo, and test how perturbations of these molecules impact local and distant tissue invasion in vivo. This work will also develop and establish novel bioengineering tools (e.g., optogenetic probes, µ-fluidic chamber) for better understanding cell motility in health and disease.