Multiscale coordination of planar cell polarity

项目摘要 细胞具有在体外自组装成器官样结构的显著能力。然而，在这方面， 目前的体外衍生器官缺乏适当的大小和更高层次的模式，这些特征需要 培养皿中不存在的生物体水平信息。值得注意的是，集体极化和 细胞在整个组织中的单向排列，这种现象称为平面细胞极性 (PCP)缺乏体外器官，但对器官的正常形成和功能至关重要。从而 要实现组织工程在体外产生功能器官的承诺， 细胞如何建立长距离集体极化。 我们建立了小鼠皮肤作为模型系统来研究多尺度 PCP在扩张和再生组织中的协调。通过开发方法来执行 离体培养、长期活体成像、生物物理扰动和器官型重建 在表皮中，我们有了关于PCP在组织中建立的关键新发现， 细胞和分子尺度。我们发现单轴组织变形 打破线索，定义了PCP在表皮中排列的主轴。我们进一步研究发现 在缺乏整体线索情况下生长的原代角质形成细胞自发地、局部地 平面极性从头排列的域。通过超分辨率成像和测绘 PCP成分的粘附相互作用，我们确定了钙粘蛋白介导的顺式- 非对称PCP复合物的聚类和排序中的相互作用。 这项工作的广泛目标是建立在这些以前的发现和技术 目前，科学家们正在研究五氯苯酚在不同生物长度尺度上的组织方式。使用 哺乳动物的皮肤表皮作为一个模型系统，具体目标1将确定如何远程 机械性信号使表皮上的平面细胞极性偏向并对齐。具体目标2将 研究细胞、皮肤细胞通过自身代谢自发产生PCP的机制。 organization.具体目标3将破译纳米结构和生化 PCP复合物的相互作用。利用我实验室最近开发的技术创新 对内源性标记的PCP蛋白进行实时和超分辨率成像， 和器官型组织，这项工作将提供基本的新见解多尺度 协调PCP。

PROJECT SUMMARY Cells have a remarkable capacity to self-assemble into organ-like structures in vitro. However, current in vitro-derived organs lack proper size and higher-order patterning, features that require organism-level information not present in a dish. Notably, the collective polarization and unidirectional alignment of cells across a tissue, a phenomenon known as planar cell polarity (PCP), is lacking in vitro organs, yet is essential for proper organ formation and function. Thus, to fulfill the promise of tissue engineering to generate functional organs in vitro we must understand how cells establish long-range collective polarization. We have established the murine skin as a model system to investigate the multiscale coordination of PCP in an expansive and regenerative tissue. By developing methods to perform ex vivo culturing, long-term live imaging, biophysical perturbations, and organotypic reconstitution of the epidermis, we have made key new discoveries about PCP establishment at the tissue, cellular and molecular scales. We discovered that uniaxial tissue deformation acts as a symmetry breaking cue that defines the major axis of PCP alignment in the epidermis. We further showed that that primary keratinocytes grown in the absence of global cues establish spontaneous, locally aligned domains of planar polarity de novo. Through super-resolution imaging and mapping the adhesive interactions of PCP components, we identified a role for cadherin-mediated cis- interactions in the clustering and sorting of asymmetric PCP complexes. The broad goal of this work is to build on these previous discoveries and technological developments to decipher how PCP is organized across different biological length scales. Using the mammalian skin epidermis as a model system, Specific Aim 1 will determine how long-range mechanical cues bias and align planar cell polarity across the epidermis. Specific Aim 2 will investigate the mechanisms by which cells skin cells spontaneously generate PCP through self- organization. Specific Aim 3 will decipher the nanoscale architecture and biochemical interactions of PCP complexes. Using technical innovations recently developed in my laboratory to perform live and super-resolution imaging of endogenously-tagged PCP proteins in both native and organotypic tissues, this work will provide fundamental new insights into the multiscale coordination of PCP.