Novel TIRF microscopy analyzing trafficking & signaling at the cell cortex

新型 TIRF 显微镜分析贩运

• 批准号: 7432044
• 负责人: Derek K. Toomre
• 金额: $ 248.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7432044
• 关键词: 1-Phosphatidylinositol 3-Kinase; Abbreviations; Accounting; Acoustics; Address; Adipocytes; Affect; Algorithms; Area; Arts; Attenuated; Automobile Driving; Award; Back; Binding; Biochemical; Biochemistry; Biological; Biology; Boxing; Buffers; Caliber; Calibration; Cell Line; Cell membrane; Cell surface; Cells; Cellular biology; Clathrin; Cluster Analysis; Collaborations; Collection; Collimator; Color; Coma; Communities; Complex; Computational Biology; Computer Vision Systems; Computers; Conflict (Psychology); Confocal Microscopy; Coupled; Coupling; Cues; Cytoskeleton; Data; Data Set; Defect; Depth; Development; Diabetes Mellitus; Diffusion; Dimensions; Dimerization; Disadvantaged; Discipline; Docking; Down-Regulation; Drops; Dyes; Employee Strikes; Endocytosis; Engineering; Ensure; Environment; Event; Exocytosis; Eye; Face; Feedback; Fiber; Figs - dietary; Flare; Fluorescein-5-isothiocyanate; Fluorescence; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Functional disorder; Funding; Genetic Screening; Germany; Glass; Glucose Transporter; Glycerol; Goals; Grant; Green Fluorescent Proteins; Image; Image Analysis; Imagery; Incidence; Insulin; Interdisciplinary Study; Investments; Joints; Kinetics; Knowledge; Label; Laboratories; Lasers; Learning; Legal patent; Length; Life; Light; Lighting; Link; Lipids; Localized; Location; Macromolecular Complexes; Malignant Neoplasms; Maps; Masks; Measures; Mediating; Membrane; Membrane Microdomains; Membrane Protein Traffic; Methodology; Methods; Microscope; Microscopy; Microtubules; Modeling; Molecular; Monitor; Morphologic artifacts; Motivation; Motor; Nature; Non-Insulin-Dependent Diabetes Mellitus; Optics; Organelles; PTEN gene; Paper; Parasites; Pathway interactions; Penetration; Performance; Personal Satisfaction; Phosphatidylinositols; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phosphotransferases; Photobleaching; Physiologic pulse; Planet Mars; Play; Pliability; Positioning Attribute; Postdoctoral Fellow; Private Sector; Probability; Process; Proteins; Publications; Pulse taking; Pupil; Quantum Dots; RNA Interference; Radial; Randomized; Range; Reagent; Recruitment Activity; Refractive Indices; Regulation; Relative (related person); Reporter; Research; Research Infrastructure; Research Personnel; Resolution; Risk; Role; Sampling; Scanning; Science; Scientist; Seminal; Series; Side; Signal Pathway; Signal Transduction; Silicon Dioxide; Simulate; Site; Small Interfering RNA; Solid; Solutions; Sorting - Cell Movement; Source; Spain; Spatial Distribution; Specific qualifier value; Specimen; Speed; Spottings; Standards of Weights and Measures; Structure; Support of Research; Surface; System; Techniques; Technology; Testing; Text; Thick; Time; Total Internal Reflection Fluorescent; Touch sensation; Training; Transfection; Tubulin; Vesicle; Visual; Wolves; Work; analytical method; basal insulin; base; blood glucose regulation; cell cortex; cell motility; cell type; cellular imaging; concept; day; density; design; desire; extracellular; flotillin; fluorescence imaging; fluorescence microscope; fluorophore; handbook; human wyatt protein; image processing; improved; innovation; insight; instrument; instrumentation; insulin signaling; interest; lens; medical schools; micromanipulator; migration; millisecond; mouse wyatt protein; nanometer; novel; object shape; photoactivation; prototype; radius bone structure; receptor; research study; response; scaffold; simulation; single molecule; success; tool; trafficking; trans-Golgi Network; trend; user-friendly; virtual

My major goal is to advance knowledge about events on or near the plasma membrane. This region directly controls membrane traffic to and from the cell surface (exo- and endocytosis) and is where extracellular signals are amplified and modulated by assembly of signaling scaffolds. The introduction of total internal reflection fluorescence (TIRF) microscopy, a technique that allows unprecedented axial resolution, has revolutionized studies of dynamic processes at the cell cortex. I propose 1) to develop two highly innovative multi-angle TIRF microscopes and 2) to apply these instruments towards the elucidation of mechanisms that regulate exo- and endocytosis. These microscopes will allow the penetration depth of the light beam to be varied rapidly and avoid traditional imaging artifacts. Together with new analytical methods, they will permit high-resolution 3D imaging of a ~50- 1000 nanometer cortical region of living cells. Additionally, a highly innovative FRAP implementation will allow us to `pulse' photoactivate single vesicles and track their fate. I will use this novel instrumentation to expand our ongoing studies on exo- and endocytic traffic. A main new goal will be to elucidate mechanisms in the vesicular trafficking pathways that regulate levels of glucose transporters (Glut4) at the cell surface, a process whose dysfunction leads to type 2 diabetes. I will test the hypothesis that the exocyst complex participates in the spatial regulation of the insulin responsiveness of Glut4 vesicle exocytosis. Using photoactivatable Glut4-Dendra I will determine whether insulin signaling triggers a switch from lipid raft to clathrin-mediated endocytic pathways. To address where PI3K signaling acts, I will implement inducible dimerization technology to rapidly turn on/off PI(3,4,5)P3 at the plasma membrane. The innovative approaches of this proposal capitalize on my unique expertise in interdisciplinary research spanning instrumentation, cell biology, and quantitative biology and will fundamentally impact biology and a medically important field.

我的主要目标是推进有关质膜上或附近事件的知识。这一地区 直接控制进出细胞表面的膜运输（胞吞和胞吞）， 细胞外信号通过信号支架的组装而被放大和调节。引入 全内反射荧光（TIRF）显微镜，一种允许前所未有的技术， 轴向分辨率，彻底改变了在细胞皮层的动态过程的研究。我建议1） 开发两种高度创新的多角度TIRF显微镜，2）应用这些仪器 对阐明机制，调节外吞和内吞。这些显微镜将 允许光束的穿透深度快速变化 藏物再加上新的分析方法，它们将允许对约50- 500微米的物体进行高分辨率3D成像。 1000纳米的活细胞皮层区域。此外，高度创新的FRAP实施 将允许我们“脉冲”光激活单个囊泡并跟踪它们的命运。我会用这本小说 仪器，以扩大我们正在进行的研究外吞和内吞交通。一个主要的新目标是 阐明调节葡萄糖水平的囊泡运输途径的机制 2型糖尿病是由细胞表面的谷氨酸转运蛋白（Glut 4）引起的，这一过程的功能障碍导致2型糖尿病。我会 检验外囊复合物参与胰岛素空间调节的假设 Glut 4囊泡胞吐反应。使用可光活化的Glut 4-Dendra I将确定 胰岛素信号传导是否触发从脂筏到网格蛋白介导的内吞途径的转换。到 为了解决PI 3 K信号传导作用的问题，我将实施诱导型二聚化技术， 在质膜上开/关PI（3，4，5）P3。本提案的创新方法利用了 我在跨学科研究方面的独特专业知识，涵盖仪器、细胞生物学和 定量生物学，并将从根本上影响生物学和医学重要领域。