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：拟议工作的目的是使用光遗传学研究成年神经干细胞（NSC）和胚胎干细胞（ESC）中的关键信号通路-Wnt和Rho-。干细胞因其潜在的组织再生而被广泛探索，包括操纵驻留在患者组织（例如成年神经干细胞）以及植入外源培养的干细胞（例如胚胎或诱导的多能干细胞）或分化的进展的内源性干细胞的策略。但是，目前对控制干细胞自我更新和分化的信号传导机制的基本知识是不完整的，这限制了我们对它们在成人功能中的作用的理解，并使精力控制再生医学应用的干细胞行为变得复杂。光是研究细胞功能的强大工具。特别是，在空间和临时地调节Wnt和Rho信号通路的能力将使我们能够阐明它们在控制干细胞命运中的作用，这对于生物医学应用至关重要。 o这端，我们最近开发了光遗传学方法，使光输入能够通过纳米级信号传导复合物的组装转移到几个定义的信号通路中。具体而言，我们设计了一个典型的Wnt信号通路和RhoA途径的可相连的激动剂。我们还具有使用显微镜技术在纳米级分辨率下进行单分子荧光成像，并使用基于荧光共振能量传递（FRET）的生物传感器来实时表征Rho GTPase活性。拟议工作的第一个目的是确定NSC和ESC中Wnt/� -Catenin信号的光学遗传激活是否可以阐明干细胞调节的机制。第二个目的是使用光遗传学在NSC中探测Rho GTPase信号传导和串扰。第三个目的是确定CRY2是否可以用于细胞表面受体激活和多重刺激细胞信号传导。我们预计，应用新型的光遗传学方法将阐明Wnt和Rho途径在NSC和ESC中的调节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增强细胞和干细胞生物学关键信号通路的未来投资。最终对干细胞生物学和工程的机械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。