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）：我们将跟踪单个IgE分子通过大鼠肥大细胞时的3-D空间轨迹。研究将在我们实验室正在开发的一种新型共聚焦显微镜上进行，该显微镜能够在三维空间中跟踪单个荧光分子的运动。我们将研究受体介导的大鼠肥大细胞系RBL-2 H3上FceRI受体的内吞作用。IgE-FceRI识别系统是人类过敏反应的重要组成部分，也是跟踪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。像这样复杂的多组分信号通路不仅在引起过敏反应中非常重要，而且在由外部生长因子刺激的细胞分裂和生长中也非常重要。失控的信号传导级联不仅会引起过敏性反应，而且常常会导致癌症。这些途径的破坏是治疗剂的明确目标。然而，这些途径的适当和受控的破坏需要更好的知识基础，涉及确切的蛋白质，以及它们彼此的动力学和空间关系。本文提出的工作有可能通过跟踪肥大细胞内3-D中的个体IgE运动来实质性地增加这一知识基础。在这些研究中，IgE将用高度光稳定的量子点进行荧光标记，并与肥大细胞上的高亲和力受体结合。加入多价抗原将导致IgE交联，启动磷酸化级联反应。作为下调该级联的一种手段，一些IgE受体复合物被募集到网格蛋白包被的小凹并内化，在那里它们被转运到一系列早期和晚期内体隔室。我们将遵循这种内在化的空间和时间动力学在3个维度与单分子的灵敏度。这项工作的具体目标包括：目标1。通过实验确定我们可以跟踪的运动速率和简单测试系统的空间精度，并验证我们可以跟踪细胞中的单个量子点。目标二。我们假设，将有明显不同的和可分类的运输模式，IgE沿着其内化路径，取决于空间位置。这项工作推进了仪器的发展，以遵循个别荧光标记分子的三维运动。该仪器使人们能够在真实的时间内跟踪所选蛋白质的三维位置，以查看它去了哪里，在那里停留了多长时间，如何从A到达B，以及分子在其当前位置的构象。这项技术最终将被用于对蛋白质-蛋白质相互作用产生新的见解，并有助于更好地阐明复杂的信号转导级联，例如某些癌症中被破坏的通路，人类过敏反应，或被入侵细菌和病毒“劫持”的宿主细胞功能。