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MEMBRANE LIPID TRAFFIC IN LIVING CELLS

活细胞中的膜脂质运输

基本信息

批准号：
3292718
负责人：
RICHARD E PAGANO
金额：
$ 5.82万
依托单位：
CARNEGIE INSTITUTION OF WASHINGTON, D.C.
依托单位国家：
美国
项目类别：
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

项目摘要

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的测量活细胞研究细胞内交通和膜动力学脂质。为此，我们将构建数字化荧光显微镜该系统结合了微光检测、数字成像和视觉检测共振能量转移的能力。我们将使用这个仪器与一系列荧光脂质类似物结合使用，该实验室旨在研究细胞和膜生物学只能通过连续观察来检查活细胞在低光照条件下，探针发生光漂白有效消除对细胞的光损伤。我们将 (i) 研究荧光脂质易位至高尔基体区域和其他细胞内目的地作为荧光的内化质膜中的脂质发生； (ii) 使用共振能量转移作为一种视觉显微镜工具来研究适当的共定位标记脂质和蛋白质，并检查荧光的单面性各种细胞内膜内的脂质； (三) 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中的网状结构专门标记这些细胞器的脂质； (iv) 检查荧光标记脂质体的细胞内易位如下活细胞显微注射及其与细胞质的相互作用膜。