Molecular Determinants of Confined Migration

限制迁移的分子决定因素

• 批准号: 10204600
• 负责人: Cynthia A. Reinhart-King
• 金额: $ 1.93万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204600
• 关键词: 3-Dimensional; ATP Synthesis Pathway; Actins; Address; Adhesions; Adhesives; Affect; Anatomy; Architecture; Automobile Driving; Behavior; Biological Assay; Biosensor; Cell Adhesion; Cell Energetics; Cell-Matrix Junction; Cells; Cellular Metabolic Process; Chemicals; Collagen; Complex; Confined Spaces; Coupled; Cytoskeleton; Data; Decision Making; Development; Disease; Engineering; Environment; Equilibrium; Event; Extracellular Matrix; Fluorescence Resonance Energy Transfer; Focal Adhesions; Foundations; Gel; Heterogeneity; Image; Immune response; In Vitro; Individual; Intervention; Matrix Metalloproteinases; Measurement; Mechanics; Mediating; Metabolic; Metabolic Pathway; Metabolism; Metastatic to; Microfabrication; Modeling; Molds; Molecular; Monitor; Motion; Movement; Natural Products; Neoplasm Metastasis; Nutrient; Organ; Pattern; Pharmacology; Physiological; Play; Population; Process; Publishing; Research; Role; Shapes; Site; Structure; System; Talin; Techniques; Tissues; Vinculin; Work; base; cell motility; chemical property; cost; design and construction; in vitro Model; in vivo; interstitial; mechanical properties; migration; mutant; new therapeutic target; novel; optogenetics; rho GTP-Binding Proteins; therapeutic target; transmission process; tumor; 3-Dimensional; ATP Synthesis Pathway; Actins; Address; Adhesions; Adhesives; Affect; Anatomy; Architecture; Automobile Driving; Behavior; Biological Assay; Biosensor; Cell Adhesion; Cell Energetics; Cell-Matrix Junction; Cells; Cellular Metabolic Process; Chemicals; Collagen; Complex; Confined Spaces; Coupled; Cytoskeleton; Data; Decision Making; Development; Disease; Engineering; Environment; Equilibrium; Event; Extracellular Matrix; Fluorescence Resonance Energy Transfer; Focal Adhesions; Foundations; Gel; Heterogeneity; Image; Immune response; In Vitro; Individual; Intervention; Matrix Metalloproteinases; Measurement; Mechanics; Mediating; Metabolic; Metabolic Pathway; Metabolism; Metastatic to; Microfabrication; Modeling; Molds; Molecular; Monitor; Motion; Movement; Natural Products; Neoplasm Metastasis; Nutrient; Organ; Pattern; Pharmacology; Physiological; Play; Population; Process; Publishing; Research; Role; Shapes; Site; Structure; System; Talin; Techniques; Tissues; Vinculin; Work; base; cell motility; chemical property; cost; design and construction; in vitro Model; in vivo; interstitial; mechanical properties; migration; mutant; new therapeutic target; novel; optogenetics; rho GTP-Binding Proteins; therapeutic target; transmission process; tumor

Project Summary: In numerous processes including development and metastasis, cells can move in microtracks within the 3D microenvironment. These microtracks are formed by cells themselves through the use of matrix metalloproteinases that degrade matrix, or microtracks can exist as a product of the natural architecture of organs. While microtrack migration occurs in vivo, little is known about the specific mechanisms that cells employ to move in microtracks. We have developed a unique platform using microfabrication to recreate these microtracks in vitro by micromolding collagen. Microtracks can be made in various sizes, and they can be patterned into multiple different shapes including tapered channels and bifurcated channels. Our microfabricated microtracks are structurally indistinguishable from tracks found in vitro and in vivo. Moreover, they offer a distinct advantage over other PDMS-based platforms because the collagen is amenable to cell adhesion on all 4 walls of the track, the fibrous walls of the microtrack can be deformed by cells, and the tracks more closely mimic the mechanical and chemical properties found in vivo. Importantly, our work to-date has shown that the mechanisms driving movement in microtracks are not the same as those mediating cell migration on 2D substrates or in unmolded collagen. Here, we propose to build upon two of our major prior findings, which are that: 1. Vinculin is required for microtrack movement, 2. Cellular confinement alters migration and correlates with cell metabolism. Using this novel microtrack platform in concert with engineered probes to monitor adhesion and cellular energy, optogenetic probes to alter cell contractility and cellular protrusions, and novel force measurement techniques, we will investigate the molecular mechanisms driving cell migration and decision-making during migration in microtracks with a focus on adhesion dynamics and cellular energetics. In Aim 1, we investigate the role of focal adhesion dynamics and tension, focusing on vinculin-talin-actin interactions based on our preliminary showing vinculin mediates unidirectional motion. We will investigate the linkage between vinculin, talin and actin, and we will probe the force transmission occurring at the sites of cell-matrix adhesion. In Aim 2, we will investigate how cellular energetics and the availability of nutrients affects migration and migration decisions in confined spaces. Based on our prior work indicating that the extracellular matrix structure alters ATP utilization, we hypothesize that increased confinement will increase the energetic needs of the cell. In Aim 3, we will investigate the molecular and mechanical mechanisms governing cell migration decisions. Constructs designed to disrupt force transmission between the cell and the matrix and pharmacological interventions will be used to assess the effects of cell contractility and cell stiffness on cellular energy utilization, adhesion, and migration direction decisions in microtracks. Our understanding of metabolism is rapidly developing, and as such, therapeutics targeting metabolic pathways are emerging. Connecting migration behaviors to metabolism offers a potential new point of intervention in disease.

项目摘要：在包括开发和转移在内的许多过程中，细胞可以进入 3D微环境中的微裂片。这些微裂片是由细胞本身通过使用而形成的 可以作为天然建筑的产物存在降解基质或微裂片的基质金属蛋白酶的 器官。虽然微铁迁移发生在体内，但对细胞的特定机制知之甚少 雇用在微裂片中移动。我们已经开发了一个使用微加工来重新创建这些平台的独特平台 通过微胶原蛋白在体外的微裂片。可以用各种尺寸制成微钻，它们可以是 分为多种不同的形状，包括锥形通道和分叉通道。我们的微型制造 微裂片在结构上与在体外和体内发现的轨道无法区分。此外，它们提供了独特的 优于其他基于PDMS的平台，因为胶原蛋白均与所有4个墙壁上的细胞粘附相提并论 在轨道上，微裂片的纤维壁可能会被细胞变形，并且轨道更紧密地模仿 在体内发现的机械和化学特性。重要的是，我们迄今的工作表明了机制 微轨道中的驱动运动与介导细胞迁移的2D基板或中的细胞迁移不同 未镀胶蛋白。在这里，我们建议建立在我们的两个主要先前发现的基础上：1。Vinculin 2。细胞限制会改变迁移并与细胞代谢相关。 使用这个新颖的微钻平台与工程探针共同监测粘附和蜂窝能量， 光遗传学探针改变细胞收缩力和细胞突起，以及新型的力量测量技术， 我们将研究驱动细胞迁移和迁移过程中的决策的分子机制 微裂片专注于粘附动力学和细胞能量学。在AIM 1中，我们研究了焦点的作用 粘附动力和张力，重点介绍基于我们的初步显示 Vinculin介导单向运动。我们将研究Vinculin，Talin和actin之间的联系，我们 将探测在细胞矩阵粘附部位发生的力传播。在AIM 2中，我们将调查如何 细胞能量和养分的可用性会影响狭窄空间中的迁移和迁移决策。 基于我们先前的工作，表明细胞外基质结构改变了ATP的利用，我们假设 增加的限制将增加细胞的能量需求。在AIM 3中，我们将调查 控制细胞迁移决策的分子和机械机制。旨在破坏力的构造 细胞与基质和药理学干预措施之间的传播将用于评估影响 细胞收缩性和细胞刚度在细胞能量利用，粘附和迁移方向决策上的刚度 Microtracks。我们对新陈代谢的理解正在迅速发展，因此，治疗剂的目标 代谢途径正在出现。将迁移行为与新陈代谢联系起来提供了潜在的新点 干预疾病。