CIL:24815

Intracellular localization of GFP-GOLPH3 in BY4742 sac1∆ vps74∆. SAC1 encodes an integral membrane phosphoinositide phosphatase that is localized to the ER and Golgi. In the sac1∆ vps74∆ mutant, GFP-GOLPH3 localizes, in addition to the Golgi apparatus, to nuclear ER and cortical ER and/or PM. Thus, correct Golgi localization of Vps74 (see CIL# 24812 for GFP-Vps74 in sac1∆ vps74∆ mutant) and GOLPH3 requires synthesis of PtdIns4P in the Golgi by Pik1-Frq1 and maintenance of low PtdIns4P levels in non-Golgi membranes by the Sac1 lipid phosphatase. Cells grown in liquid medium were mounted in growth medium and 3D image stacks were collected at 0.4-µm z increments on a DeltaVision workstation (Applied Precision) based on an inverted microscope (IX-70; Olympus) using a 100× NA 1.4 oil immersion lens. Images were captured at 23C with a 12-bit CCD camera (CoolSnap HQ; Photometrics) and deconvolved using the iterative-constrained algorithm (Agard, 1984) and the measured point spread function. One image from the approximate center of z stack is shown in Fig1B GOLPH3/sac1∆ panel in J Cell Biol. 187: 967-975. 2009. Images in Fig 1B include CIL#s 24815, 24812, 24813.

绿色荧光蛋白标记的GOLPH3（GFP-GOLPH3）在酿酒酵母BY4742菌株sac1缺失（sac1∆）与vps74缺失（vps74∆）双突变体中的胞内定位。SAC1基因编码一种定位于内质网（Endoplasmic Reticulum, ER）和高尔基体的整合膜磷酸肌醇磷酸酶。在sac1∆ vps74∆突变体中，GFP-GOLPH3除定位于高尔基体外，还可定位至核内质网、皮层内质网及/或质膜（Plasma Membrane, PM）。由此可见，Vps74（该蛋白在sac1∆ vps74∆突变体中的定位可参见细胞图像库（Cell Image Library, CIL）编号24812的GFP-Vps74图像）与GOLPH3的正确高尔基体定位，依赖于Pik1-Frq1复合物在高尔基体中合成磷脂酰肌醇4-磷酸（PtdIns4P），以及Sac1脂质磷酸酶维持非高尔基体膜内的PtdIns4P处于低水平。
本研究以液体培养基培养细胞，采用生长培养基进行封片后，在搭载奥林巴斯IX-70倒置显微镜的DeltaVision工作站（Applied Precision）上，使用100×数值孔径（Numerical Aperture, NA）1.4的油浸物镜，以0.4μm的z轴步长采集三维图像堆栈。于23℃下通过12位电荷耦合器件（Charge-Coupled Device, CCD）相机（CoolSnap HQ；Photometrics）捕获图像，并采用迭代约束算法（Agard, 1984）与实测点扩散函数完成反卷积处理。本次展示的图像为《Journal of Cell Biology》187卷：967-975（2009年）中图1B的GOLPH3/sac1∆组图面板中，z堆栈近似中心处的单张图像；该图1B中的图像包含细胞图像库编号24815、24812、24813。