CAREER: Mechanobiology of Planar Cell Polarity

上皮细胞排列在身体许多器官的表面，包括中空器官的内表面。因此，它们经常通过细胞的厚度表现出不同的功能特性和能力——这被称为平面细胞极性（PCP）。全功能上皮组织的生物构建在组织工程和再生医学中有着广泛的应用。实现这一目标的主要障碍之一是，在培养皿中生长的上皮细胞通常不表现出这种极性。上皮细胞的极性在一层或一层细胞内受到细胞内和细胞间信号的严格调控。这种信号似乎在培养细胞中无法维持，因此，在人造组织中重建PCP从未实现过。此外，一些先天性异常，如脊柱裂，是由于上皮细胞在胚胎发育过程中功能失常所致。本学院早期职业发展计划（Career）项目将在体外研究平面细胞极性的机械和生化调节机制。本项目将系统研究几何约束、基质刚度、机械应变和化学梯度对PCP发生和维持的影响，并确定将外部机械信号传递给细胞以建立PCP的分子。这个项目的教育活动将向广大受众提供实际的、基于项目的经验，重点是妇女和代表性不足的少数民族。本科生和研究生将接受机械生物学项目课程的培训。此外，一个为计算科学家和生物学家提供先进生物工程工具培训的暑期项目将促进跨学科的交流。通过提高对内皮细胞如何建立这种必要的功能变异的理解，该项目将支持生物制造和组织工程系统的发展，以产生正常器官功能所必需的上皮细胞层。此外，所获得的基本知识将促进对正常和病理组织生长和发育的理解。该项目的总体研究目标是扩大对平面极化上皮细胞介导菌株信号传导的基本机制的了解。这个项目的重点是在胚胎发育过程中负责神经管形成的神经上皮细胞，其破坏可能导致神经管缺陷（如脊柱裂）。这将通过三个研究目标来完成。首先是研究界面几何形状和基质刚度对单细胞水平PCP信号复合物不对称分布的影响。该目标将使用新颖的模式技术来控制细胞可以生长的区域，以及可调的水凝胶来模拟细胞外基质刚度的变化。第二个目标是阐明菌株和Wnt梯度（一组信号转导途径，其中蛋白质通过细胞表面受体将信号传递到细胞中）在组织水平上对PCP的定位中的作用。这种排列将通过分子分析、活细胞成像和组织表型评估在分子、细胞和组织水平上进行评估。最后的目标是确定将机械信号传递到PCP通路的机械传感器。这将通过选择性敲除培养细胞中的各种关键机械受体来确定这些变化如何影响菌株介导的PCP排列。从这些实验中获得的知识将促进对神经上皮细胞中平面细胞极性发展的基本理解，并将更广泛地适用于一般上皮细胞。除了回答组织生长和发育的关键基本问题外，这项研究还将支持生物制造和组织工程系统的进步，包括上皮细胞层，这对正常组织功能很重要。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Epithelial cells line the surfaces of many organs in the body, including the inner surface of hollow organs. As such, they often exhibit different functional properties and abilities through the thickness of the cell - something that is called planar cellular polarity (PCP). The biofabrication of fully functional epithelial tissues has a broad application in tissue engineering and regenerative medicine. One of the main obstacles for achieving this goal is that epithelial cells grown in culture dishes typically do not demonstrate this polarity. The epithelial cell polarity within a sheet or plane of cells is tightly regulated by signaling within and between cells. This signaling does not appear to be maintained in cultured cells and, as a result, reestablishing PCP in manufactured tissues has never been achieved. In addition, some congenital abnormalities, such as spina bifida, are due to the failure of epithelial cells to function properly during embryonic development. This Faculty Early Career Development Program (CAREER) project will study the mechanical and biochemical regulatory mechanisms of planar cell polarity in vitro. The project will systematically study the effects of geometrical confinement, matrix stiffness, mechanical strains, and chemical gradients on the initiation and maintenance of PCP, as well as identify the molecules that relay external mechanical signals to the cells for establishing PCP. The educational activities in this project will provide hands-on, project-based experience to a broad audience, with an emphasis on women and underrepresented minorities. Undergraduate and graduate students will be trained in a project-based course in mechanobiology. In addition, a summer program that provides computational scientists and biologists with training in advanced bioengineering tools that are developed through this project will facilitate interdisciplinary communication. By improving understanding of how endothelial cells establish this necessary functional variation, this project will support the development of biomanufacturing and tissue engineering systems to produce layers of epithelial cells that are necessary for normal organ function. In addition, the fundamental knowledge gained will advance understanding with respect to normal and pathological tissue growth and development. The overall research goal of this project is to expand knowledge about the fundamental mechanisms through which epithelial cells, which are planar polarized, mediate strain-based signaling. This project is focused on neuroepithelial cells that are responsible for the formation of the neural tube during embryonic development, disruption of which can result in neural tube defects (such as spina bifida). This will be accomplished through three research objectives. The first is to investigate the effects of interfacial geometry and matrix stiffness on the asymmetrical distribution of PCP signaling complexes at the single cell level. This objective will use novel patterning techniques to control the areas in which the cells can grow as well as tunable hydrogels to simulate variations in extracellular matrix stiffness. The second objective is to elucidate the role of strains and the Wnt gradient (a set of signal transduction pathways in which proteins pass signals into a cell through cell surface receptors) in the alignment of PCP at the tissue level. This alignment will be evaluated at the molecular, cellular, and tissue-level through molecular assays, live-cell imaging, and tissue phenotype assessment. The final objective is to identify the mechanosensors that relay mechanical signals to the PCP pathway. This will be done through selective knock-down of various key mechanoreceptors in the cultured cells to determine how these changes affect strain-mediated alignment of PCP. The knowledge gained from these experiments will advance fundamental understanding of the development of planar cell polarity in neuroepithelial cells and will be more broadly applicable to epithelial cells in general. In addition to answering fundamental questions that are key to tissue growth and development, this research will support the advancement of biomanufacturing and tissue engineering systems that include layers of epithelial cells, which are important for normal tissue function.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.