MEMBRANE LIPID TRAFFIC IN LIVING CELLS

我们希望进行连续和定量的荧光测量 活细胞研究膜的胞内运输和动力学 脂类。为了做到这一点，我们将构建一个数字化荧光显微镜 一种集微光检测、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 与一系列荧光类脂类似物相结合的仪器 这个实验室已经发展成研究细胞和细胞中的基本问题 只有通过连续观察才能检测到的膜生物学 在弱光下的活细胞，其中探针和光漂白 有效地消除了对细胞的光损伤。我们将(I)研究 荧光脂类向高尔基体区域的移位 和其他细胞内的目的地作为荧光的内化 脂类从质膜发生；(Ii)利用共振能量转移 作为一种视觉微观工具来研究适当的共本地化 标记的脂类和蛋白质，并检查荧光的侧向性 各种细胞膜中的脂类；(Iii)尝试 高尔基体与内质网的三维重建 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类；以及(Iv)检查 荧光标记脂质体胞内转运的实验研究 活细胞内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.