Regulation of Epithelial Plasticity

 描述(由申请人提供)：我们的建议旨在确定细胞内pH(Phi)动态如何调节上皮可塑性，重点放在不同类型的细胞分化上。上皮细胞的命运或形态状态的转变是后生动物发育、动态平衡和组织修复的核心。我们的初步数据表明，三种类型的上皮分化程序都需要增加PHI，这三种程序是上皮向间充质细胞(EMT)的转分化、成体上皮干细胞分化和胚胎干细胞(ES)分化。EMT是正常发育所必需的，有助于损伤后的器官修复，包括导致纤维化的异常修复，并促进癌症转移。成体干细胞自我更新和分化的遗传过程是成体动态平衡和组织修复程序的基本部分。ES细胞的分化模拟了胚胎发育过程中的谱系指定和扩增过程。因此，解决这三种类型的上皮分化是如何被调控的，对于正常和病理的细胞行为都具有广泛的意义。我们的数据支持这一中心假设，即增加phi对于不同类型的上皮细胞分化是必要的。在目标1中，我们将基于我们的发现，即增加phi对于肺和乳腺上皮细胞的EMT是必要的，从而确定Phi调节EMT的分子机制。我们将通过在2D和3D培养中使用克隆细胞模型的遗传编码生物传感器的实时成像以及斑马鱼神经脊发育的体内分析来揭示EMT过程中特定阶段的PHI动力学及其对肌动蛋白细丝重塑和转录事件的调节。我们还将通过测试利用蛋白质结构、癌症突变数据库和新开发的生物信息学程序识别的可滴定蛋白质中可电离残基的分析组合来确定已建立和预测的pH敏感蛋白质，从而确定介导依赖于pH的EMT的分子机制。在目标2中，我们将根据我们的发现来确定Phi如何调节成体和胚胎干细胞系的分化，这一发现是提高Phi对果蝇卵巢卵泡干细胞系的体内分化和小鼠ES细胞的自发分化所必需的。我们将解决Phi动力学如何调节已建立的细胞谱系标记，以及pH传感器在果蝇毛囊细胞和小鼠ES细胞分化中所起的作用，包括研究依赖Phi的刺猬和无翼信号，正如我们的初步数据所建议的那样。我们从我们在结构和细胞生物学、定量活细胞成像和德罗索菲亚遗传学方面的专业知识为这些研究在分子水平上带来了关于信号机制的新观点。虽然phi在组织培养细胞中的测量是常规的，但很少有研究研究phi在体内的动力学。这些研究的成功完成将为调节上皮可塑性的重要和未被研究的机制提供实质性的洞察力，揭示新的针对疾病相关分化计划的治疗靶向的调节因子。

 DESCRIPTION (provided by applicant): Our proposal is designed to determine how intracellular pH (pHi) dynamics regulates epithelial plasticity, with a focus on distinct types of ell differentiation. Transitions in the fate or morphological state of epithelial cells are central to metazoan development, homeostasis, and tissue repair. Our preliminary data indicate that increased pHi is necessary for three types of epithelial differentiation programs, the transdifferentiation of epithelial to mesenchymal cells (EMT), adult epithelial stem cell differentiation, and embryonic stem (ES) cell differentiation. EMT is necessary for normal development, contributes to organ repair after injury, including aberrant repair leading to fibrosis, and promotes cancer metastasis. The genetically distinct process of adult stem cell self-renewal and differentiation is a fundamental part of the program of adult homeostasis and tissue repair. The differentiation of ES cells mimics the process of lineage specification and expansion during embryonic development. Hence, resolving how these three types of epithelial differentiation are regulated has broad significance for both normal and pathological cell behaviors. Our data support testing the central hypothesis that increased pHi is necessary for different types of epithelial cell differentiation. In Aim 1 we will identify molecular mechanisms or pHi-regulated EMT based on our findings that increased pHi is necessary for EMT of lung and mammary epithelial cells. We will reveal stage-specific pHi dynamics and its regulation of actin filament remodeling and transcriptional events during EMT by using real-time imaging of genetically encoded biosensors with clonal cell models in 2D and 3D cultures as well as in vivo analysis of zebrafish neural crest development. We also will identify molecular mechanisms mediating pHi-dependent EMT by testing established and predicted pH-sensing proteins identified using an analytical combination of protein structures, cancer mutation databases, and a newly developed bioinformatics program that identifies titrating ionizable residues in proteins. In Aim 2 we will determine how pHi regulates differentiation of adult and embryonic stem cell lineages based on our findings that increased pHi is necessary for in vivo differentiation of the Drosophila ovarian follicle stem cell lineage, and for spontaneous differentiation of mouse ES cells. We will resolve how pHi dynamics regulates established cell lineage markers and the role that pH sensors play in Drosophila follicle cell and mouse ES cell differentiation, including investigating pHi-dependent hedgehog and wingless signaling, as suggested by our preliminary data. We bring to these studies new views on signaling mechanisms at the molecular level from our expertise in bridging structural and cell biology, quantitative live cell imaging, and Drosophia genetics. Although pHi is routinely measured in tissue culture cells, few studies have investigated pHi dynamics in vivo. Successful completion of these studies will provide substantial insight into a significant and unstudied mechanism for the regulation of epithelial plasticity, revealing new regulators for therapeutic targeting of disease-associated differentiatio programs.