CAREER: Mechanobiology of Planar Cell Polarity

上皮细胞排列在体内许多器官的表面，包括中空器官的内表面。因此，它们通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制品在组织工程和再生医学中具有广泛的应用。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。细胞片或平面内的上皮细胞极性受到细胞内和细胞间信号传导的严格调节。这种信号似乎不会在培养的细胞中维持，因此，在制造的组织中重建PCP从未实现。此外，一些先天性畸形，如脊柱裂，是由于上皮细胞在胚胎发育过程中无法正常发挥功能。这个教师早期职业发展计划（CAREER）项目将研究体外平面细胞极性的机械和生化调节机制。该项目将系统地研究几何约束、基质刚度、机械应变和化学梯度对PCP的启动和维持的影响，并确定将外部机械信号传递给细胞以建立PCP的分子。该项目的教育活动将向广大受众提供基于项目的实践经验，重点是妇女和代表性不足的少数群体。本科生和研究生将接受机械生物学项目课程的培训。此外，一个夏季项目为计算科学家和生物学家提供通过该项目开发的先进生物工程工具的培训，将促进跨学科交流。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，所获得的基础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有关上皮细胞（平面极化）介导基于应变的信号传导的基本机制的知识。该项目的重点是神经上皮细胞，这些细胞在胚胎发育期间负责形成神经管，破坏这些细胞可能导致神经管缺陷（如脊柱裂）。这将通过三个研究目标来实现。第一个是研究界面几何形状和基质刚度对PCP信号复合物在单细胞水平上的不对称分布的影响。该目标将使用新的图案化技术来控制细胞可以生长的区域，以及可调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）在组织水平上对齐PCP中的作用。将通过分子测定、活细胞成像和组织表型评估在分子、细胞和组织水平上评价这种对齐。最后的目标是确定机械传感器中继机械信号的PCP途径。这将通过选择性敲低培养细胞中的各种关键机械感受器来完成，以确定这些变化如何影响PCP的应变介导的对齐。从这些实验中获得的知识将推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。除了回答对组织生长和发育至关重要的基本问题外，该研究还将支持包括上皮细胞层在内的生物制造和组织工程系统的进步，这对正常组织功能至关重要。该奖项反映了NSF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.