Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.
PLoS One. 2012;7(9):e44776. doi: 10.1371/journal.pone.0044776. Epub 2012 Sep 12.
P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.
P-糖蛋白（Pgp；也称为 MDR1、ABCB1）是血脑屏障（BBB）中最重要和研究最充分的外排转运体；然而，其组织形式尚不清楚。本研究旨在采用最近开发的超分辨率荧光显微镜方法——光谱精度距离显微镜/光谱位置确定显微镜（SPDM），来研究 Pgp 在脑毛细血管内皮细胞腔面膜上的空间分布。通过计算机模拟解决了细胞膜曲率对分布分析的潜在干扰影响。永生化人脑血管内皮细胞（hCMEC/D3）被用作人 BBB 的模型。hCMEC/D3 细胞被转导了一种 Pgp-绿色荧光蛋白（GFP）融合蛋白，该融合蛋白被整合到慢病毒衍生载体中。通过 SPDM 可视化 Pgp-GFP 融合蛋白的表达和定位。SPDM 在 z 方向上的分辨率有限，导致在成像过程中产生投影，从而影响超分辨率图像中荧光分子的表观空间分布。因此，对不同弯曲细胞膜上分子分布进行了模拟，并研究了它们的投影空间分布。在用荧光探针 eFluxx-ID Gold 孵育细胞并在有无维拉帕米的情况下进行 FACS 分析，证实了融合蛋白的功能。通过 SPDM 在 hCMEC/D3 细胞中检测到超过 112,000 个单个 Pgp-GFP 分子（每个细胞对应约 5,600 个 Pgp-GFP 分子），平均空间分辨率约为 40nm。我们发现 Pgp-GFP 在 hCMEC/D3 细胞中呈聚集形成分布，而基于模拟结果可以排除当前随机细胞膜曲率的影响。单个形成随机分布在细胞膜上。
