Optogenetic Characterization and Control of Stem Cell Signaling

干细胞信号传导的光遗传学表征和控制

• 批准号: 8674874
• 负责人: Ravi S. Kane
• 金额: $ 35.17万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8674874
• 关键词: Adult; Agonist; Biochemical; Biosensor; Cell Adhesion; Cell Surface Receptors; Cell physiology; Cells; Cellular biology; Complex; Coupling; Dependency; Development; Disease model; Electrophysiology (science); Embryo; Engineering; Enzymes; Ephrin-B2; Ephrins; Fluorescence Resonance Energy Transfer; Future; Image; Implant; Investigation; Ion Channel; Knowledge; Learning; Life; Ligands; Light; Mediating; Memory; Methods; Microscopy; Names; Nature; Neuroglia; Neurologic; Neuronal Differentiation; Neurons; Organ; Pathway interactions; Patients; Photoreceptors; Process; Property; Proteins; Public Health; Receptor Activation; Regenerative Medicine; Regulation; Replacement Therapy; Research; Resolution; Role; Signal Induction; Signal Pathway; Signal Transduction; Stem cells; System; Techniques; Time; Tissues; Work; base; cell behavior; cell motility; cellular engineering; cellular imaging; cryptochrome 2; design; embryonic stem cell; human disease; induced pluripotent stem cell; insight; mood regulation; nanoscale; nerve stem cell; neurogenesis; novel; optogenetics; public health relevance; rho; rho GTP-Binding Proteins; self-renewal; sensor; single molecule; stem cell biology; stem cell differentiation; stem cell division; stem cell fate; tissue regeneration; tool

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.

描述：拟议工作的目的是利用光遗传学研究成体神经干细胞 (NSC) 和胚胎干细胞 (ESC) 中的关键信号通路 - Wnt 和 Rho。目前正在广泛探索其潜在的组织再生能力。 ，包括操纵患者组织内的内源干细胞（例如成体神经干细胞）以及植入外源培养的干细胞（例如胚胎或诱导胚胎干细胞）的策略然而，控制干细胞自我更新和分化的信号机制的基本知识目前还不完整，这限制了我们对其在成体功能中的作用的理解，并使精确控制干细胞行为的努力变得复杂。光是细胞功能的强大工具，在空间和时间上调节 Wnt 和 Rho 信号通路的能力将使我们能够阐明它们在研究干细胞命运中的作用，这对于生物医学至关重要。为此，我们最近开发了光遗传学方法，通过组装纳米级信号复合物，将光输入引导到几个确定的信号通路中。具体来说，我们设计了经典 Wnt 信号通路和 RhoA 的光激活激动剂。我们还能够使用显微镜技术以纳米级分辨率进行单分子荧光成像，并使用基于荧光共振能量转移（FRET）的生物传感器来表征 Rho GTPase 活性。这项工作的首要目的是确定 Wnt/¿ 的光遗传学激活是否有效。 NSC 和 ESC 中的连环蛋白信号传导可以阐明干细胞调节机制，第三个目标是确定 Cry2 是否可以被改造以激活细胞表面受体。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 NSC 和 ESC 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对细胞和干细胞生物学关键信号通路的未来研究，由此产生的对干细胞生物学和工程的机制见解将极大地影响基于细胞替代和再生的器官功能恢复方法。药品。