Molecular Determinants of Confined Migration

项目摘要：在包括发育和转移在内的许多过程中，细胞可以移入 3D微环境中的微轨迹。这些微轨迹是由细胞本身通过使用 降解基质的基质金属蛋白酶，或微轨迹可以作为自然结构的产物而存在 器官的。虽然MicroTrack迁移发生在体内，但对细胞 被用来在微轨上移动。我们已经开发了一种独特的平台，使用微制造来重建这些 通过微模塑胶原蛋白在体外进行微跟踪。微轨可以制作成各种尺寸，它们可以是 设计成多种不同的形状，包括锥形通道和分叉通道。我们的微细加工 微轨迹在结构上与在体外和体内发现的轨迹难以区分。此外，它们还提供了一种独特的 与其他基于PDMS的平台相比具有优势，因为胶原蛋白可在所有4个壁上进行细胞黏附 在轨道上，MicroTrack的纤维壁可以被细胞变形，并且轨道更接近于 在体内发现的机械和化学特性。重要的是，我们到目前为止的工作已经表明， 微轨道中的驱动运动与2D衬底上或在 未成型的胶原蛋白。在这里，我们建议建立在我们之前的两个主要发现的基础上，这两个发现是：1.纽蛋白 是微轨运动所必需的，2.细胞限制改变了迁移并与细胞新陈代谢相关。 使用这种新的MicroTrack平台与工程探测器相结合来监测粘附力和细胞能量， 改变细胞收缩和细胞突起的光遗传探针，以及新的力测量技术， 我们将研究驱动细胞迁移的分子机制和迁移过程中的决策 专注于粘着动力学和细胞能量学的微轨道。在目标1中，我们研究了焦点的作用 黏附动力学和张力，基于我们的初步展示，重点关注纽蛋白-塔林-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、Talin和肌动蛋白之间的联系，我们 将探索细胞-基质黏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获得性影响有限空间中的迁移和迁移决策。 根据我们之前的工作表明细胞外基质结构改变了ATP的利用，我们假设 这种增加的限制将增加细胞的能量需求。在目标3中，我们将调查 控制细胞迁移决定的分子和机械机制。旨在扰乱武力的构造 将使用细胞和基质之间的传递和药物干预来评估效果 细胞伸缩性和细胞硬度对细胞能量利用、黏附和迁移方向的影响 微音轨。我们对新陈代谢的了解正在迅速发展，因此，治疗靶向 新陈代谢途径正在形成。将迁移行为与新陈代谢联系起来提供了一个潜在的新点 对疾病的干预。

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.