Regulation of Epithelial Plasticity

 描述（由申请人提供）：我们的提案旨在确定细胞内pH（pHi）动态如何调节上皮可塑性，重点是不同类型的细胞分化。上皮细胞的命运或形态状态的转变对后生动物的发育、稳态和组织修复至关重要。我们的初步数据表明，增加的pHi对于三种类型的上皮分化程序是必要的，即上皮细胞向间充质细胞（EMT）的转分化、成人上皮干细胞分化和胚胎干（ES）细胞分化。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独特过程是成体稳态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特化和扩增过程。因此，解决这三种类型的上皮分化是如何调节的，对正常和病理细胞行为都具有广泛的意义。我们的数据支持测试的中心假设，增加的pHi是必要的不同类型的上皮细胞分化。在目标1中，我们将根据我们的发现来确定分子机制或pHi调节的EMT，即增加的pHi对于肺和乳腺上皮细胞的EMT是必要的。我们将揭示特定阶段的pHi动力学及其调节肌动蛋白丝重塑和转录事件EMT期间通过使用实时成像的遗传编码的生物传感器与克隆细胞模型在2D和3D文化，以及在体内分析斑马鱼神经嵴发育。我们还将通过测试已建立和预测的pH敏感蛋白来确定介导pH依赖性EMT的分子机制，该蛋白质使用蛋白质结构，癌症突变数据库和新开发的生物信息学程序的分析组合来识别，该程序识别蛋白质中的滴定可电离残基。在目标2中，我们将确定pHi如何调节成体和胚胎干细胞谱系的分化，这是基于我们的发现，即增加的pHi对于果蝇卵泡干细胞谱系的体内分化和小鼠ES细胞的自发分化是必要的。我们将解决pHi动力学如何调节已建立的细胞谱系标志物和pH传感器在果蝇卵泡细胞和小鼠ES细胞分化中发挥的作用，包括研究pHi依赖的刺猬和无翅信号，正如我们的初步数据所建议的那样。我们为这些研究带来了分子水平上信号机制的新观点，这些新观点来自我们在桥接结构和细胞生物学，定量活细胞成像和果蝇遗传学方面的专业知识。虽然pHi通常在组织培养细胞中测量，但很少有研究研究体内pHi动力学。这些研究的成功完成将为上皮可塑性调节的重要和未研究的机制提供实质性的见解，揭示疾病相关分化程序的治疗靶向的新调节剂。

 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.