Molecular Determinants of Confined Migration

限制迁移的分子决定因素

• 批准号: 10386588
• 负责人: Cynthia A. Reinhart-King
• 金额: $ 18.01万
• 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/10386588
• 关键词: 3-Dimensional; Actins; Adhesions; Affect; Anatomy; Architecture; Automobile Driving; Behavior; Cell Adhesion; Cell-Matrix Junction; Cells; Cellular Metabolic Process; Chemicals; Collagen; Confined Spaces; Decision Making; Development; Disease; Engineering; Extracellular Matrix; Focal Adhesions; Immune response; In Vitro; Intervention; Matrix Metalloproteinases; Measurement; Mechanics; Mediating; Metabolic Pathway; Metabolism; Microfabrication; Molds; Molecular; Monitor; Motion; Movement; Natural Products; Neoplasm Metastasis; Nutrient; Organ; Pattern; Pharmacology; Process; Role; Shapes; Site; Structure; System; Talin; Techniques; Tissues; Vinculin; Work; base; cell motility; chemical property; design and construction; in vivo; mechanical properties; migration; novel; optogenetics; therapeutic target; transmission process

PROJECT SUMMARY/ABSTRACT 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.

项目概要/摘要 在包括发育和转移在内的许多过程中，细胞可以在 3D 内的微轨迹中移动 微环境。这些微轨道是细胞本身通过使用基质形成的 降解基质或微径的金属蛋白酶可以作为自然结构的产物存在 器官。虽然微轨迁移发生在体内，但人们对细胞采用的具体机制知之甚少 以微轨道移动。我们开发了一个独特的平台，使用微加工来重新创建这些 通过微模塑胶原蛋白在体外形成微轨迹。微轨可以制成各种尺寸，并且可以 图案化为多种不同的形状，包括锥形通道和分叉通道。我们的微加工 微轨迹在结构上与体外和体内发现的轨迹没有区别。此外，它们还提供独特的 优于其他基于 PDMS 的平台，因为胶原蛋白易于细胞粘附在所有 4 个壁上 在轨道上，微轨道的纤维壁可以被细胞变形，并且轨道更接近地模仿 体内发现的机械和化学特性。重要的是，我们迄今为止的工作表明，该机制 微轨道中的驱动运动与在 2D 基质或二维基底上介导细胞迁移的驱动运动不同。 未成型的胶原蛋白。在这里，我们建议以我们先前的两个主要发现为基础，即： 1. 纽蛋白是 微轨迹运动所需的，2。细胞限制改变迁移并与细胞代谢相关。 使用这种新颖的微轨道平台与工程探针配合监测粘附和细胞能量， 改变细胞收缩性和细胞突起的光遗传学探针，以及新颖的力测量技术， 我们将研究驱动细胞迁移的分子机制以及迁移过程中的决策 微轨迹，重点关注粘附动力学和细胞能量学。在目标 1 中，我们研究了焦点的作用 粘附动力学和张力，根据我们的初步显示，重点关注纽蛋白-达林-肌动蛋白相互作用 纽蛋白介导单向运动。我们将研究纽蛋白、踝蛋白和肌动蛋白之间的联系，并且我们 将探测细胞-基质粘附部位发生的力传递。在目标 2 中，我们将研究如何 细胞能量学和营养物质的可用性影响有限空间内的迁移和迁移决策。 根据我们之前的工作表明细胞外基质结构改变 ATP 利用率，我们假设 增加限制将增加细胞的能量需求。在目标 3 中，我们将调查 控制细胞迁移决策的分子和机械机制。旨在破坏力量的结构 细胞和基质之间的传输和药物干预将用于评估效果 细胞收缩性和细胞刚度对细胞能量利用、粘附和迁移方向决定的影响 微轨。我们对新陈代谢的理解正在迅速发展，因此，靶向治疗 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一个潜在的新观点 对疾病的干预。