MEMBRANE LIPID TRAFFIC IN LIVING CELLS

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，我们将构建一个数字化荧光显微镜， 系统集成了微光探测、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 仪器结合一系列荧光脂质类似物， 这个实验室已经发展到研究细胞中的基本问题， 膜生物学，只能通过连续观察 在低光照水平下的活细胞，其中探针的光漂白和 有效地消除了对细胞的光损伤。 （一）研究 荧光脂质移位到高尔基体区域 和其他细胞内的目的地，如荧光的内化， 脂质从质膜发生;（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 标记的脂质和蛋白质，并检查荧光 各种细胞内膜内的脂质;（iii）尝试 高尔基体和内质网的三维重建 在活细胞中的网状物在用适当的荧光 脂质特异性标记这些细胞器;和（iv）检查 荧光标记的脂质体的细胞内易位 活细胞的显微注射及其与细胞质的相互作用 膜。

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.