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

摘要-体内细胞通过限制细胞外纤维之间的三维(3D)孔移动 基质(ECM)网络或通道状轨道，以ECM束、血管、肌纤维或神经为边界。这个 使细胞在不同微环境中移动的机制是自适应的，以响应物理和 生物化学指标，如约束、僵硬、粘弹性和ECM的成分。适应性 系统/模块包括细胞-细胞外基质相互作用、肌动蛋白细胞骨架和细胞体积调节。 最近，我们和其他人也发现了核在受限迁移中的关键作用。然而， 关于核能之间的串扰，许多基本问题和翻译问题仍然没有得到回答 机械传感、细胞骨架和细胞体积调节及其在健康受限迁移中的作用 和疾病。该项目的总体目标是使用最先进的生物工程、材料和 成像工具和活体模型，为在限制条件下高效迁移提供新的统一框架 通过破译核力学、细胞骨架和离子通道之间的相互作用。此R01应用程序 将检验这样一种假设，即核团通过微妙的调节来感知物理限制并对其做出反应 RhoA在密闭空间中沿纵向细胞轴的空间激活 限制诱导的核僵硬和苯丙素/ECT2核退出细胞质。这一假设是 由耐人寻味的初步数据支持，这些数据表明细胞进入限制µ通道会诱发核 硬化，激活RhoA，支持依赖离子通道的核泡化和破裂。核子 破裂导致芳香素和RhogeFect2从细胞核进入细胞质。阿尼林蓄积 特别是在细胞极点，在那里它局部连接Ect2，RhoA和肌动球蛋白，从而加剧RhoA- 肌球蛋白II的收缩性能。在目标1中，我们将破译苯青素进入细胞质的机制，并演示 它的关键作用是作为支架蛋白，连接ECT2、RhoA和细胞极点的肌动球蛋白，从而 调节RhoA的空间激活和禁闭中基于水泡的迁移。我们还将澄清 细胞体积调节与ANLYL/ECT2/RhoA在胞核起泡和破裂中的新串扰 禁闭。最后，我们将破译来自细胞后方的核推动与核拉动的贡献 从细胞前沿(即核活塞模型)到作为限制程度函数的迁移。在目标2中， 我们将把我们的发现扩展到3D凝胶和限制规定的生理微通道 相关力学性能的体外实验。我们还将可视化的不同的定位模式的苯青素，ect2和 在体内不同维度的自然组织中的关键离子通道，并测试这些扰动如何 分子影响体内局部和远程组织的侵袭。这项工作也将发展和建立小说 生物工程工具(如光遗传探针、微流体室)，以更好地了解健康中的细胞运动性 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.