CAREER: Molecular mechanisms of pH sensitive proteins, pathways, and cell behaviors

维持细胞内pH（pHi）在正常细胞生理学（包括代谢、细胞生长和细胞分裂）中是至关重要的，即使pHi的微小变化也会对细胞过程产生剧烈影响。虽然pHi在调节生物学中的作用是明确的，但感知pHi中这些微小变化的蛋白质和生物分子在很大程度上是未知的。此外，没有良好的工具来操纵和测量单细胞中的pHi，从而限制了该领域的进展。在这个项目中，PI将确定驱动pH敏感蛋白的分子机制，揭示pH动力学如何驱动单细胞行为，并开发下一代工具来改变活细胞中的pHi。该项目将进一步加深我们对pHi如何调节单细胞生物学的理解，并揭示蛋白质如何感知和响应pHi的分子机制。操纵细胞pHi的新工具也将适用于其他领域，包括了解pHi如何驱动病理过程。对于这个项目的更广泛的影响，PI将开发一个变革性的教育计划，探索科学研究作为一种创造性的奋进。这包括为本科生开发基于项目的学习课程，并在中学艺术教室中实施STEAM（科学，技术，工程，艺术和数学）教育的基于活动的学习模块。这些模块将培养创新思维，加强跨学科技能，并建立积极的科学关联作为一种创造性的奋进，这已被证明是鼓励学生追求STEM。人类细胞通常保持细胞内pH值（pHi）在7.0和7.2之间，但pHi增加到约7.6可以信号细胞分裂，移动，或改变他们的新陈代谢。虽然pHi在调节生物学中的作用是明确的，但感知pHi中这些微小变化的蛋白质和生物分子在很大程度上是未知的。因此，该项目将解决pH敏感生物学领域的主要空白。首先，PI将使用最近验证的光遗传学工具来探索时空pH动力学如何调节单细胞行为，包括细胞迁移和极性变化。其次，PI将利用钠质子交换剂（NHE1）作为蛋白质工程的基础，以开发一套下一代光遗传学工具，以提高pHi数小时至数天。第三，PI将通过可电离的残基网络确定pH传感的分子机制，特别是在调节pH敏感生物学的各种含SH 2结构域的信号蛋白中。这项工作将揭示pHi动力学如何驱动单细胞生物学，并支持未来研究pH依赖性细胞通路和pH敏感蛋白质基本设计原理的工作。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。

Maintaining intracellular pH (pHi) is vital in normal cellular physiology including metabolism, cellular growth, and cellular division, with even small changes in pHi having a drastic effect on cellular processes. While the role for pHi in regulating biology is clear, the proteins and biomolecules that sense these small changes in pHi are largely unknown. Furthermore, there are no good tools to manipulate and measure pHi in single cells thereby limiting progress in this field. In this project, the PI will determine the molecular mechanisms driving pH sensitive proteins, reveal how pH dynamics drive single-cell behaviors, and develop next-generation tools for changing pHi in living cells. This project will further our understanding of how pHi regulates single-cell biology and will reveal the molecular mechanisms of how proteins sense and respond to pHi. The new tools to manipulate cell pHi will also be applicable to other fields, including understanding how pHi drives pathological processes. For the Broader Impacts of this project, the PI will develop a transformative education program to explore scientific research as a creative endeavor. This includes developing project-based learning curriculum for undergraduate students and implementing activity-based learning modules for STEAM (Science, Technology, Engineering, Art, and Mathematics) education in middle school art classrooms. These modules will cultivate an innovation mindset, strengthen interdisciplinary skills, and build positive associations of science as a creative endeavor, which has been shown to encourage students to pursue STEM.Human cells normally maintain an intracellular pH (pHi) between 7.0 and 7.2 but increases in pHi to ~7.6 can signal cells to divide, to move, or to change their metabolism. While the role for pHi in regulating biology is clear, the proteins and biomolecules that sense these small changes in pHi are largely unknown. Thus, this project will address major gaps in the field of pH-sensitive biology. First, the PI will use recently validated optogenetic tools to probe how spatiotemporal pH dynamics regulate single-cell behaviors including cell migration and polarity changes. Second, the PI will leverage the sodium-proton exchanger (NHE1) as a foundation for protein engineering in order to develop a suite of next-generation optogenetic tools to raise pHi for hours to days. Third, the PI will determine the molecular mechanisms underlying pH-sensing by ionizable residue networks, specifically in various SH2-domain-containing signaling proteins that regulate pH-sensitive biology. This work will reveal how pHi dynamics drive single cell biology as well as support future work investigating pH-dependent cellular pathways and essential design principles of pH sensitive proteins.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.