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

摘要-体内细胞穿过纤维状细胞外之间的三维 (3D) 孔 基质（ECM）网络或以ECM束、血管、肌纤维或神经为边界的通道状轨道。的 使细胞在不同微环境中运动的机制能够适应物理和环境 生化线索，例如 ECM 的限制、刚度、粘弹性和成分。自适应 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，我们和其他人还确定了原子核在受限迁移中的关键作用。然而， 关于核之间的串扰，许多基本和转化问题仍未得到解答。 机械传感、细胞骨架和细胞体积调节及其对健康限制迁移的贡献 和疾病。该项目的总体目标是采用最先进的生物工程、材料和技术 成像工具以及体内模型，为限制中的有效迁移提供新颖的统一框架 通过破译核力学、细胞骨架和离子通道之间的相互作用。这个R01应用程序 将检验这样的假设：细胞核通过精细调节来感知和响应物理限制 通过协同作用，RhoA 在有限空间内沿细胞纵向轴进行空间激活 限制诱导的核硬化和 anillin/Ect2 核退出细胞质。这个假设是 有趣的初步数据表明，细胞进入限制性 µ 通道会诱导核 硬化可激活 RhoA 并支持离子通道依赖性核起泡和破裂。核能 破裂诱导 anillin 和 RhoGEF Ect2 从细胞核退出到细胞质。阿尼林积累 特别是在细胞两极，它局部桥接 Ect2、RhoA 和肌动球蛋白，从而加剧 RhoA- 肌球蛋白 II 的收缩性。在目标 1 中，我们将破译苯胺进入细胞质的机制，并证明 它作为支架蛋白的关键作用，在细胞两极桥接 Ect2、RhoA 和肌动球蛋白，从而 调节 RhoA 的空间激活和限制中基于气泡的迁移。我们还将阐明 细胞体积调节与 anillin/Ect2/RhoA 之间在核起泡和破裂中的新串扰 禁闭。最后，我们将解读细胞后部的核推力与核拉力的贡献 从细胞前沿（即核活塞模型）到迁移，作为限制程度的函数。在目标 2 中， 我们将把我们的研究结果的适用性扩展到 3D 凝胶并限制规定生理学的 µ 通道 相关的体外机械性能。我们还将可视化 anillin、Ect2 和 自然组织中的关键离子通道在体内跟踪不同维度，并测试这些扰动如何 分子影响体内局部和远处组织的侵袭。这项工作还将开发和建立新颖的 用于更好地了解健康细胞运动的生物工程工具（例如光遗传学探针、μ流体室） 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.