Optogenetic Characterization and Control of Stem Cell Signaling

描述：这项拟议工作的目标是利用光遗传学来研究成人神经干细胞(NSCs)和胚胎干细胞(ESCs)中的关键信号通路-Wnt和Rho。干细胞在组织再生方面的潜力正在被广泛探索，包括操纵患者组织内的内源性干细胞(例如成人神经干细胞)以及植入外源性培养的干细胞(例如胚胎或诱导的多能干细胞)或其分化的后代。然而，目前对控制干细胞自我更新和分化的信号机制的基本知识还不完全，这限制了我们对它们在成人功能中作用的了解，并使精确控制干细胞行为应用于再生医学的努力复杂化。光是研究细胞功能的有力工具。特别是，能够在空间和时间上调控Wnt和Rho信号通路将使我们能够阐明它们在控制干细胞命运中的作用，这对生物医学应用是至关重要的。为此，我们最近开发了光遗传学方法，使光输入能够通过组装纳米级信号复合体进入几个定义的信号通路。具体地说，我们设计了一种典型的Wnt信号通路和RhoA通路的光激活激动剂。我们还有能力使用显微镜技术在纳米级进行单分子荧光成像，并使用基于荧光共振能量转移(FRET)的生物传感器实时表征活细胞中Rho GTP酶的活性。这项拟议工作的第一个目的是确定神经干细胞和胚胎干细胞中Wnt/？-catenin信号的光基因激活是否可以阐明干细胞调节的机制。第二个目标是利用光遗传学来探索神经干细胞中的Rho GTP酶信号和串扰。第三个目标是确定是否可以设计Cry2来激活细胞表面受体，并对细胞信号进行多重刺激。我们预计，应用我们的新型光遗传学方法将阐明Wnt和Rho通路在调节NSCs和ESCs细胞命运选择中的动态作用。此外，我们将开发的新型光遗传学方法将进一步加强未来细胞和干细胞生物学中关键信号通路的研究。由此产生的对干细胞生物学和工程学的机械论见解将极大地影响基于细胞替代和再生医学恢复器官功能的方法。

DESCRIPTION: The objective of the proposed work is to use optogenetics to investigate key signaling pathways - Wnt and Rho - in adult neural stem cells (NSCs) and embryonic stem cells (ESCs). Stem cells are being broadly explored for their potential for tissue regeneration, including strategies to manipulate endogenous stem cells residing within a patient's tissues (e.g., adult neural stem cells) as well as to implant exogenously cultured stem cells (e.g., embryonic or induced pluripotent stem cells) or their differentiated progeny. However, basic knowledge of the signaling mechanisms that control stem cell self-renewal and differentiation is currently incomplete, which limits our understanding of their role in adult function and complicates efforts to precisely control stem cell behavior for regenerative medicine applications. Light is a powerful tool to investigate cellular function. In particular, the abilityto modulate the Wnt and Rho signaling pathways spatially and temporally would allow us to elucidate their role in controlling stem cell fate, which is critical for biomedical applications. o this end, we recently developed optogenetic methods that enable a light input to be channeled into several defined signaling pathways through the assembly of nanoscale signaling complexes. Specifically, we engineered a photo-activatable agonist of the canonical Wnt signaling pathway as well as the RhoA pathway. We also have the ability to use microscopy techniques to conduct single molecule fluorescent imaging at nanoscale resolutions and to use biosensors based on fluorescence resonance energy transfer (FRET) to characterize Rho GTPase activities in live cells in real time. The first aim of the proposed work is to determine whether optogenetic activation of Wnt/¿-catenin signaling in NSCs and ESCs can elucidate mechanisms of stem cell regulation. The second aim is to use optogenetics to probe Rho GTPase signaling and crosstalk in NSCs. The third aim is to determine whether Cry2 can be engineered for cell surface receptor activation and for multiplexed stimulation of cellular signaling. We anticipate that applying our novel optogenetic approaches will elucidate the dynamic roles of the Wnt and Rho pathways in regulating cell fate choices in NSCs and ESCs. Moreover, the novel optogenetic methods that we will develop will further enhance future investigations of key signaling pathways in cell and stem cell biology. The resulting mechanistic insights into stem cell biology and engineering will greatly impact approaches to restore organ function based on cell replacement and regenerative medicine.