MEMBRANE LIPID TRAFFIC IN LIVING CELLS

活细胞中的膜脂质运输

• 批准号: 3292717
• 负责人: RICHARD E PAGANO
• 金额: $ 5.78万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292717
• 关键词: Golgi apparatus; cell biology; ceramides; endoplasmic reticulum; fluorescence microscopy; fluorescent dye /probe; hamsters; image processing; intracellular membranes; lipid transport; liposomes; membrane lipids; membrane permeability; membrane proteins; microinjections; online computer; phosphatidylcholines; phosphatidylinositols; pinocytosis; radiotracer; sphingomyelins; tissue /cell culture

We wish to make continuous and quantitative measurements of fluorescence in living cells to study the intracellular traffic and dynamics of membrane lipids. To do so we will construct a digitized fluorescence microscopy system which incorporates low-light-level detection, digital imaging, and the ability to visually detect resonance energy transfer. We will use this instrument in conjunction with a series of fluorescence lipid analogs which this laboratory has developed to study fundamental questions in cell and membrane biology which can only be examined by continuous observation of living cells at low light levels, where photobleaching of the probes and photodamage to cells is effectively eliminated. We will (i) study the translocation of fluorescent lipids to the region of the Golgi apparatus and other intracellular destinations as internalization of fluorescent lipids from the plasma membrane occurs; (ii) use resonance energy transfer as a visual microscopic tool to study the co-localization of appropriately labeled lipids and proteins, and to examine the sidedness of fluorescent lipids within various intracellular membranes; (iii) attempt three-dimensional reconstruction of the Golgi apparatus and endoplasmic reticulum in living cells after treatment with appropriate fluorescent lipids which specifically label these organelles; and (iv) examine the intracellular translocation of fluorescently labeled liposomes following microinjection into living cells and their interactions with cytoplasmic membranes.

我们希望对荧光进行连续和定量测量 活细胞研究膜的细胞内交通和动力学 脂质。 为此，我们将构建一个数字化的荧光显微镜 结合低光级检测，数字成像和 视觉检测共振能量传递的能力。 我们将使用这个 仪器与一系列荧光脂质类似物结合 该实验室已经发展为研究细胞和细胞中的基本问题 膜生物学只能通过连续观察来检查 在低光层的活细胞，探针的光漂白和 有效消除对细胞的光损伤。 我们将（i）研究 荧光脂质转移到高尔基体的区域 和其他细胞内目的地作为荧光的内在化 质膜的脂质发生。 （ii）使用共振能量传递 作为研究适当共定位的视觉微观工具 标记的脂质和蛋白质，并检查荧光的侧面 各种细胞内膜内的脂质； （iii）尝试 高尔基体和内质的三维重建 用适当的荧光处理后活细胞网中的网状 专门标记这些细胞器的脂质； （iv）检查 荧光标记的脂质体的细胞内易位 对活细胞的显微注射及其与细胞质的相互作用 膜。