The role of intracellular pH in the specification of cell fate

细胞内 pH 在细胞命运规范中的作用

• 批准号: 1933240
• 负责人: Todd Nystul
• 金额: $ 89万
• 依托单位: University of California-San Francisco
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1933240&HistoricalAwards=false
• 关键词: role; intracellular; pH; specification; cell

Non-Technical Paragraph Stem cells in embryos and adults have the capacity to change to specific cell types, such as pancreatic cells that make insulin, intestinal cells that absorb nutrients, and master blood cells that can become an immune cell or a cell that carries oxygen. How stem cells change their identity is important for understanding human development, developmental defects, and approaches for regenerative medicine. For development, research is aimed at understanding how an embryonic stem cell becomes all the diverse cell types that make organs, like the heart and liver. Development “gone wrong” is often the cause of defects such as cleft-lip palate and heart malformations, as well as diseases such as cancer and diabetes. And regenerative medicine holds promise to repair damaged organs or treat diseases like Alzheimer’s. Hence, how stem cells change their identity or “fate” is a highly significant question to resolve. One new answer to this question is that when stem cells change their identity they change their internal acid-base balance. The current proposal tests predictions on how these changes in acid-base balance ensure a correct cell fate. Predictions will be tested by using new tools to accurately measure acid-base balance in live isolated stem cells and in stem cells in whole animals, and by using new computational and experimental approaches to understand how changes in acid-base balance change the shape and function of proteins as well as the expression of genes previously recognized for controlling how a cell fate is specified. Technical Paragraph Studies on how cell fate is specified from naïve stem cells mostly focus on regulation by signaling circuits, transcriptional programs, and epigenetic changes. New findings reveal that intracellular pH (pHi) dynamics is a previously unrecognized critical regulator of stem cell fates in three stem cell models: mouse embryonic stem cells (mESCs), adult Drosophila follicle stem cells (FSCs), and adult mouse intestinal stem cells (ISCs). Building on these findings will generate a mechanistic understanding of how pHi dynamics specifies cell fate. Aim 1 addresses how pHi dynamics regulates mESC pluripotency and FSC differentiation by testing the hypothesis that pHi regulates distinct stem cell states through pHi-dependent effects on the activity or ligand-binding affinity of selective endogenous proteins with established roles in regulating cell fate decisions. Roles for known (-catenin, DIDO3, phosphofructokinase-1) and predicted (BCL9 and FOXM1) pH sensitive proteins will be resolved at molecular, cellular and animal scales. Aim 2 determines the conservation of pHi dynamics in cell fate decisions through studies using ISC organoids. Preliminary data support testing the hypothesis that pHi-dependent differentiation of ISCs occurs at two steps; crypt budding and lineage specification. Crypt budding will be determined by focusing on pHi-regulated actinomyosin contractility and Wnt signaling, and lineage specification to secretory cells will be resolved by using lineage-specific fluorescent reporters and reporters for Wnt and Notch pathway activity. Outcomes will generate mechanistic insights on how pHi dynamics regulates cell fate decisions that will be significant to understand human development, developmental defects, and approaches for regenerative medicine.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.

胚胎和成人中的干细胞有能力转变为特定的细胞类型，如制造胰岛素的胰腺细胞，吸收营养的肠道细胞，以及能够成为免疫细胞或携带氧气的细胞的主宰血细胞。干细胞如何改变其身份对于理解人类发育、发育缺陷和再生医学的方法是重要的。对于发育，研究的目的是了解胚胎干细胞如何成为构成器官的所有不同类型的细胞，如心脏和肝脏。发育“出错”往往是唇腭裂和心脏畸形等缺陷的原因，也是癌症和糖尿病等疾病的原因。再生医学有望修复受损的器官或治疗阿尔茨海默氏症等疾病。因此，干细胞如何改变自己的身份或“命运”是一个非常重要的问题需要解决。这个问题的一个新答案是，当干细胞改变其身份时，它们会改变内部的酸碱平衡。目前的提案测试了关于酸碱平衡的这些变化如何确保正确的细胞命运的预测。通过使用新的工具来准确测量活的分离干细胞和整个动物的干细胞中的酸碱平衡，并使用新的计算和实验方法来了解酸碱平衡的变化如何改变蛋白质的形状和功能，以及先前被认为控制细胞命运的基因的表达，这些预测将得到检验。关于幼稚干细胞如何决定细胞命运的研究主要集中在信号通路、转录程序和表观遗传学变化的调控上。新的发现表明，在三种干细胞模型中，细胞内pH(Phi)动态是先前未知的干细胞命运的关键调节因素：小鼠胚胎干细胞(MESCs)、成年果蝇毛囊干细胞(FSCS)和成年小鼠肠道干细胞(ISCs)。建立在这些发现的基础上，将产生对phi动力学如何指定细胞命运的机械性理解。目的1通过检验Phi对选择性内源性蛋白的活性或配体结合亲和力的依赖作用来调节不同的干细胞状态这一假说，阐明Phi动力学如何调控mESC的多能性和FSC的分化，这些蛋白在调节细胞命运决定中具有既定的作用。已知的(-连环蛋白、DIDO_3、磷酸果糖激酶-1)和预测的(BCL_9和FOXM_1)pH敏感蛋白的作用将在分子、细胞和动物范围内得到解决。目的2通过使用ISC有机化合物的研究，确定PHI动力学在细胞命运决定中的保守性。初步数据支持这一假设，即ISCs的Phi依赖分化发生在两个步骤：隐窝萌发和谱系指定。隐窝的萌发将通过关注Phi调节的放线肌球蛋白收缩和Wnt信号来确定，而分泌细胞的谱系指定将通过使用谱系特异的荧光报告程序和Wnt和Notch途径活性的报告程序来解决。结果将产生关于PHI动力学如何调节细胞命运决定的机械性见解，这对于理解人类发育、发育缺陷和再生医学的方法将具有重要意义。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Intracellular pH dynamics regulates intestinal stem cell lineage specification.

• DOI: 10.1038/s41467-023-39312-9
• 发表时间: 2023-06-23
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Liu, Yi;Reyes, Efren;Castillo-Azofeifa, David;Klein, Ophir D.;Nystul, Todd;Barber, Diane L.
• 通讯作者: Barber, Diane L.