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

结合生物工程、材料系统和体内模型的限制细胞力学生物学

• 批准号: 10374917
• 负责人: Konstantinos Konstantopoulos
• 金额: $ 38.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10374917
• 关键词: 3-Dimensional; Actomyosin; Alginates; Anatomy; Anions; Automobile Driving; Biochemical; Biological Process; Biomedical Engineering; Bulla; Cancer Biology; Cell Nucleus; Cell Volumes; Cells; Cellular biology; Collagen; Confined Spaces; Cues; Cytokinesis; Cytoplasm; Cytoskeleton; Data; Developmental Biology; Dimensions; Disease; Disease Progression; Distant; Embryonic Development; Event; Extracellular Matrix; Fiber; Gel; Goals; Health; Imaging Device; In Vitro; Ion Channel; Lamin Type A; Light; Liquid substance; Mechanical Stress; Mechanics; Mediating; Modeling; Molecular; Myosin Type II; Nerve; Nuclear; Nuclear Translocation; Organism; Pathway interactions; Pattern; Phenotype; Physiological; Process; Property; Regulation; Role; Rupture; Scaffolding Protein; Speed; Supporting Cell; Surface; System; Technology; Testing; Tissues; Travel; Work; Yeasts; anillin; base; cell motility; in vivo; in vivo Model; in vivo evaluation; insight; knock-down; mechanical properties; mechanotransduction; migration; novel; optogenetics; polyacrylamide; pressure; response; tool; viscoelasticity

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.

体内的摘要 - 细胞通过纤维细胞外的三维（3D）孔的限制 矩阵（ECM）网络或类似通道的轨道，其轨道与ECM束，容器，肌纤维或神经接壤。这 潜水微环境中可以使细胞运动的机制适应于物理和 生化提示，例如限制，刚度，粘弹性和ECM的组成。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细胞核在受到约束迁移中的关键作用。然而， 在核之间的串扰上，许多基本和翻译问题仍未得到答复 机械感应，细胞骨架和细胞体积调节及其对健康迁移的贡献 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，为有效迁移提供新颖的统一框架 通过解读核力学，细胞骨架和离子通道之间的相互作用。此R01应用程序 将检验以下假设，即细胞核感觉并通过精确调节物理限制做出反应 RhoA沿着纵向细胞轴的空间激活通过狭窄空间中的协同作用 限制引起的核僵硬和Anillin/ECT2核出口到细胞质。这个假设是 通过有趣的初步数据支持，表明细胞进入限制µ通道会诱导核 僵化，激活RhoA并支持离子通道依赖性的核溢和破裂。核 破裂诱导了阿尼林的出口和Rhogef ECT2从细胞核到细胞质的出口。阿尼林积聚 特别是在局部桥梁ECT2，RhoA和肌动蛋白的细胞两极，从而加剧了Rhoa- 肌球蛋白II收缩力。在AIM 1中，我们将解读Anillin退出的机制，并证明 它作为脚手架蛋白的关键作用，在细胞杆上桥接ECT2，RhoA和肌动蛋白的蛋白质 调节在禁闭中基于RHOA和基于BLEB的迁移的空间激活。我们还将阐明 细胞体积调节与核爆发和破裂中的anillin/ect2/rhoA之间的新型串扰 监禁。最后，我们将破译从细胞后与核拉的核推动的贡献 从细胞正面（即核活塞模型）到迁移，作为限制程度的函数。在AIM 2中， 我们将把发现的适用性扩展到3D凝胶，并在生理上限制µ通道 体外相关的机械性能。我们还将可视化anillin，ect2和 体内不同维度的自然组织轨迹中的关键离子通道，并测试这些轨道的扰动 分子会影响体内局部和遥远的组织侵袭。这项工作还将发展并建立小说 生物工程工具（例如，光遗传学问题，µ-富集室），以更好地了解健康的细胞运动性 和疾病。