Spatially and Temporally Resolved Perturbation of Cells

细胞的空间和时间扰动

• 批准号: 7279475
• 负责人: Daniel T Chiu
• 金额: $ 16.57万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7279475
• 关键词: Affect; Affinity; Age; Alzheimer' s Disease; Amyloid; Animal Model; Area; Binding; Biocompatible; Biological; Brain; Calcium; Caliber; Cell Surface Proteins; Cell membrane; Cell physiology; Cells; Chemicals; Chemistry; Chromosome Pairing; Complex; Condition; Cultured Cells; Dependence; Development; Disease; Dyes; Encapsulated; Endocytosis; Ensure; Environment; Exclusion; Exhibits; Exocytosis; Extravasation; Feasibility Studies; Glutamate Receptor; Glutamates; Height; Hydrolysis; Image; In Vitro; Individual; Ion Channel; Ions; Kinetics; Lasers; Lateral; Life; Lipids; Localized; Maps; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Mitogen-Activated Protein Kinases; Molecular; Molecular Weight; Movement; N-Methylaspartate; Neurons; Neurotransmitters; Nicotinic Receptors; Nucleic Acids; Numbers; Optics; Pathway interactions; Pattern; Peptides; Photosensitizing Agents; Physiologic pulse; Physiological; Pliability; Positioning Attribute; Process; Property; Proteins; Pulse taking; Quantum Dots; Range; Rate; Receptor Activation; Resolution; Rhodamine B; Sampling; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Slice; Small Interfering RNA; Spatial Distribution; Stimulus; Structure; Surface; Synapses; Synaptic Transmission; System; Techniques; Time; Vesicle; Water; Width; Work; absorption; base; caged molecule; capsule; cell behavior; chemical group; chemical reaction; chromophore; concept; cytokine; design; desire; experience; extracellular; in vivo; interest; millisecond; nanometer; nanoscale; nervous system disorder; neurotransmission; photolysis; prevent; quantum; receptor; receptor density; release of sequestered calcium ion into cytoplasm; research study; response; size; small molecule; spatiotemporal; tool; two-photon

DESCRIPTION (provided by applicant): Although a typical cell measures only tens of micrometers in diameter, it is a highly organized and spatially heterogeneous structure. Cell membrane receptors and proteins receive extracellular stimulations and transform them into intracellular signals, signals that dictate the behavior of the cell and errors in the propagation of these signals underlie a wide range of diseases. The distribution of receptors and proteins on the cell membrane determines and affects the rate and pattern of the propagation of these intracellular signals. Membrane proteins are often expressed in different morphological regions of the plasma membrane, and within a local area, the proteins tend to form clusters and patches, rather than distribute homogeneously and continuously on the membrane. To study and dissect the mechanism and signaling pathways by which a single neuronal cell processes the arrival of a particular signal at its membrane surface, we aim to develop a technique by which a precisely timed stimulus or set of stimuli can be delivered to the cell with high spatial (sub-micrometers) and temporal (sub- microseconds) resolution. Such a tool will find use for functional mapping of cell surface proteins and for probing the dynamics of signal transduction and synaptic transmission triggered by the localized activation of receptors. This tool is based both on the ability to synthesize nanoscale capsules and to release the confined molecules from select capsules with a single laser pulse. To develop and demonstrate this method, we have the following aims: (1) Photosensitize the shell of nanocapsules with near-IR dyes or with chromophores that have high two-photon absorption cross sections, so we may use near-IR or two-photon laser pulses to trigger release, (2) Develop new nanocapsules having homogeneous sizes, and which are tunable from ~20nm to ~100nm; these nanocapsules also should be able to encapsulate a wide range of molecules (from small neurotransmitters to peptides and proteins) at high concentrations and should exhibit long-term storability, and (3) Map neuronal response caused by release of physically caged amyloid-¿ 42 (A¿-42). A¿-42 is believed to be the causative factor in Alzheimer disease, although its mechanism of action is poorly understood; here we propose to study the spatial-temporal dynamics of neuronal activation by A¿-42. Cells respond to their environment through a complex and interdependent series of signal transduction pathways that frequently begin at the cell membrane with high spatial and temporal resolutions (e.g. exocytosis, endocytosis, synaptic transmission). To study and dissect the mechanism and signaling pathways by which a cell processes the arrival of a particular signal at its membrane surface, we propose here to develop a method by which a precisely timed stimulus can be delivered to the cell with high spatiotemporal resolution. This method will facilitate the detailed study of signaling pathways that underlie disease processes, such as Alzheimer that is a devastating neurological disease associated with aging.

描述(申请人提供)：虽然一个典型的电池直径只有几十微米，但它是一个高度组织和空间异质的结构。细胞膜受体和蛋白质接受细胞外刺激，并将其转化为细胞内信号，这些信号决定了细胞的行为，这些信号的传播错误导致了一系列疾病。细胞膜上受体和蛋白质的分布决定并影响这些细胞内信号的传播速度和模式。膜蛋白通常表达在质膜的不同形态区域，在一个局部区域内，蛋白往往形成簇和斑块，而不是均匀和连续地分布在膜上。为了研究和剖析单个神经元细胞处理特定信号到达其膜表面的机制和信号通路，我们的目标是开发一种技术，通过它可以以高空间(亚微米)和时间(亚微秒)分辨率将精确定时的刺激或一组刺激传递到细胞。这样的工具将被用于细胞表面蛋白的功能图谱，并用于探测由受体的局部激活触发的信号转导和突触传递的动力学。该工具既基于合成纳米级胶囊的能力，也基于使用单一激光脉冲从选定胶囊中释放受限分子的能力。为了开发和验证这种方法，我们的目标是：(1)用近红外染料或具有高双光子吸收截面的生色团对纳米胶囊的壳层进行光敏化，这样我们就可以使用近红外或双光子激光脉冲来触发释放；(2)开发尺寸均匀的新的纳米胶囊，其可调范围从~20 nm到~100 nm；这些纳米胶囊还应该能够在高浓度下包裹各种分子(从小的神经递质到多肽和蛋白质)，并且应该表现出长期的储存性，以及(3)由于释放物理笼子中的淀粉样蛋白-42(A？-42)而引起的MAP神经元反应。A？-42被认为是阿尔茨海默病的致病因素，尽管其作用机制尚不清楚；在此，我们建议研究A？-42激活神经元的时空动力学。细胞通过一系列复杂和相互依赖的信号转导途径对环境做出反应，这些信号转导途径通常始于细胞膜，具有高空间和时间分辨率(如胞吐、内吞、突触传递)。为了研究和剖析细胞处理特定信号到达其膜表面的机制和信号通路，我们在这里提出了一种方法，通过它可以以高时空分辨率将精确的定时刺激传递到细胞。这种方法将有助于详细研究疾病过程背后的信号通路，例如阿尔茨海默氏症，这是一种与衰老相关的毁灭性神经疾病。