Spatially and Temporally Resolved Perturbation of Cells

描述（由申请人提供）：尽管典型的细胞直径仅为数十微米，但它是高度有序和空间异质的结构。细胞膜受体和蛋白质接收细胞外刺激并将其转化为细胞内信号，这些信号决定细胞的行为，并且这些信号传播中的错误是多种疾病的基础。受体和蛋白质在细胞膜上的分布决定并影响这些细胞内信号传播的速率和模式。膜蛋白通常在质膜的不同形态区域表达，并且在局部区域内，蛋白倾向于形成簇和斑块，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到达其膜表面的特定信号的机制和信号传导途径，我们的目标是开发一种技术，通过该技术，可以将精确定时的刺激或一组刺激以高空间（亚微米）和时间（亚微秒）分辨率传递到细胞。这样的工具将发现用于细胞表面蛋白质的功能映射和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。该工具基于合成纳米级胶囊的能力以及用单个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，我们有以下目标：（1）用近红外染料或具有高双光子吸收截面的发色团光敏化纳米胶囊的壳，因此我们可以使用近红外或双光子激光脉冲来触发释放;（2）开发具有均匀尺寸的新型纳米胶囊，其在~ 20 nm至~ 100 nm之间可调谐;这些纳米胶囊还应该能够以高浓度包封大范围的分子（从小的神经递质到肽和蛋白质），并且应该表现出长期的可储存性，和（3）映射由物理上被笼住的淀粉样蛋白42（A-42）的释放引起的神经元反应。A？-42被认为是阿尔茨海默病的致病因素，尽管其作用机制知之甚少;在这里，我们建议研究A <$-42神经元激活的时空动力学。细胞通过一系列复杂且相互依赖的信号转导途径对它们的环境做出响应，这些信号转导途径通常开始于具有高空间和时间分辨率的细胞膜（例如，胞吐作用、胞吞作用、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，我们在这里提出开发一种方法，通过该方法可以将精确定时的刺激以高时空分辨率传递到细胞。这种方法将有助于详细研究疾病过程的信号通路，例如阿尔茨海默病，这是一种与衰老相关的破坏性神经系统疾病。

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.