Tracking the 3-D intracellular spatial trajectory of an individual IgE molecule

追踪单个 IgE 分子的 3D 细胞内空间轨迹

• 批准号: 7385715
• 负责人: JAMES H WERNER
• 金额: $ 24.14万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-15 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7385715
• 关键词: 3-Dimensional; Actins; Adhesions; Advanced Development; Affinity; Algorithms; Allergic; Anaphylaxis; Antigens; Bacteria; Benchmarking; Binding; Biological Models; Biological Process; Cell Line; Cell division; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular Morphology; Clathrin; Clathrin-Coated Vesicles; Complex; DNA Repair; Data; Detection; Development; Diffusion; Dimensions; Disruption; Early Endosome; Endocytosis; Engineering; Enzymes; Event; Fluorescence; Glycerol; Growth; Growth Factor; Hand; Histamine Release; Human; Hypersensitivity; IgE; IgE Receptors; Image; Individual; Intracellular Transport; Invasive; Investigation; Kinetics; Label; Lasers; Lead; Learning; Life; Lipid Bilayers; Location; Malignant Neoplasms; Measurement; Membrane; Methods; Microscope; Microscopic; Microscopy; Microtubules; Molecular; Molecular Conformation; Molecular Motors; Motion; Motor; Movement; Multivesicular Body; Noise; Pathway interactions; Phosphorylation; Polymers; Positioning Attribute; Process; Protein Tyrosine Kinase; Proteins; Quantum Dots; Rate; Rattus; Recording of previous events; Recruitment Activity; Rest; Running; Signal Pathway; Signal Transduction; Speed; Structure; System; Technology; Testing; Time; Translating; Validation; Virus; Water; Work; antigen binding; base; coated pit; crosslink; improved; insight; instrument; instrumentation; intracellular protein transport; knowledge base; late endosome; lipid transport; mast cell; novel; protein protein interaction; protein transport; receptor; receptor mediated endocytosis; research study; response; single molecule; size; therapeutic target; tool; trafficking; two-photon

DESCRIPTION (provided by applicant): We will be following the 3-D spatial trajectory taken by individual IgE molecules as they journey through a rat mast cell. The studies will be made on a novel confocal microscope under development in our lab that is capable of following the motion of a single fluorescent molecule in 3 dimensions. We will study the receptor mediated endocytosis of FceRI receptors on the rat mast cell line RBL-2H3. The IgE-FceRI recognition system is a vital part of the human allergic response and a good model system for following the spatial and kinetic details of intricate, multi-component signal transduction cascades. Complex, multi-component signaling pathways such as this are very important not only in mounting an allergic response, but also in the cell division and growth that is stimulated by external growth factors. Signal transduction cascades that have run haywire cause not only hypersensitive allergies and anaphylaxis, but often cancer. The disruption of these pathways is a clear target for therapeutics for treatment. Proper and controlled disruption of these pathways, however, demands a better knowledge-base concerning the exact proteins involved, and their kinetic and spatial relation to one another. The work proposed herein has the potential to add substantively to this knowledge base by following individual IgE movements in 3-D inside a mast cell. In these studies, IgE will be fluorescently labeled with a highly photostable quantum dot and bound to its high affinity receptor on mast cells. Adding a polyvalent antigen will lead to IgE cross linking, starting a phosphorylation cascade. As a means of down-regulating this cascade, some of the IgE receptor complexes are recruited to clathrin coated pits and internalized, where they are transported to a succession of early and late endosomal compartments. We will follow the spatial and temporal dynamics of this internalization in 3 dimensions with single molecule sensitivity. The specific aims of this work include: Aim 1. Experimentally determine the rates of motion we can follow and the spatial accuracy for simple test systems and verify we can follow individual quantum dots in a cell. Aim 2. We hypothesize that there will be distinctly different and classifiable transport modes for IgE along its internalization path that depend on spatial location. This work advances the development of instrumentation to follow the motion of individual fluorescently labeled molecules in three dimensions. This instrument enables one to follow the 3 dimensional position of a selected protein in real time to see where it goes, how long it spends there, how it gets from A to B, and potentially the conformation of the molecule at its current location. This technology will eventually be used to lead to new insights on protein-protein interactions and help better elucidate complex signal transduction cascades, such as those pathways corrupted in certain cancers, the human allergic response, or host cell functions that are "hi-jacked" by invasive bacteria and viruses.

描述（由申请人提供）：我们将遵循单个IgE分子穿过大鼠肥大细胞的3-D空间轨迹。这些研究将对我们实验室中正在开发的新共聚焦显微镜进行，该显微镜能够遵循3个维度的单个荧光分子运动。我们将研究大鼠肥大细胞系RBL-2H3上FCERI受体的受体介导的内吞作用。 IgE-FCERI识别系统是人类过敏反应的重要组成部分，也是遵循复杂的多组分信号转导级联的空间和动力学细节的良好模型系统。诸如此类的复杂，多组分信号通路不仅在安装过敏反应方面非常重要，而且在外部生长因子刺激的细胞分裂和生长中也非常重要。发行干草的信号转导级联反应不仅会导致过敏性过敏和过敏反应，而且会导致癌症。这些途径的破坏是治疗治疗的明确靶标。但是，这些途径的适当和控制的破坏需要更好地了解所涉及的确切蛋白质以及它们相互关系和空间关系的更好的知识基础。本文提出的工作有可能通过遵循肥大细胞内3-D中的单个IgE运动来实质性地增加此知识库。在这些研究中，IgE将用高度光稳定的量子点荧光标记，并与其在肥大细胞上的高亲和力受体结合。添加多价抗原将导致IgE交叉链接，开始磷酸化级联反应。作为下调该级联的一种手段，将一些IgE受体复合物募集到网状蛋白涂层坑中并内化，并将其运输到一系列早期和晚期内体隔室。我们将遵循具有单分子敏感性的3个维度的这种内在化的空间和时间动力学。这项工作的具体目的包括：目标1。通过实验确定我们可以遵循的运动速率以及简单测试系统的空间精度，并验证我们可以跟随单元格中的单个量子点。 AIM 2。我们假设IgE沿其内在化路径的IGE将有明显不同的，可分类的传输模式，该路径取决于空间位置。这项工作推进了仪器的发展，以遵循三个维度的单个荧光标记的分子的运动。该仪器使人们能够实时遵循所选蛋白质的3维位置，以查看它的去向，它在那里花费了多长时间，如何从A到B，以及分子在当前位置的构象。该技术最终将用于导致对蛋白质 - 蛋白质相互作用的新见解，并有助于更好地阐明复杂的信号转导级联反应，例如在某些癌症中损坏的那些途径，人类过敏反应或被侵入性细菌和病毒“ hi-jack”的宿主细胞功能。