Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13693, Homo sapiens. CIL. Dataset

This is one of six images in Figure 6 in Apaja et al., JCB 2010 that shows the fate of various model membrane proteins, that are either folded or in the unfolded state at the plasma membrane, expressed in stable tetracycline-inducible Flp-In T-Rex HEK293 cell lines. The three model membrane proteins were chimeras of the CD4 surface receptor consisting of 1.) a C-terminally truncated CD4 that contained a flexible cytoplasmic linker (CD4tl), 2.) a CD4tl fused to the N-terminal DNA-binding domain of the wild type bacteriophage lambda repressor (CD4tl-lambda), and 3.) a CD4tl fused to a L57C mutant lambda repressor (CD4tl-lambdaC). The CD4tl-lambdaC cytosolic domain was largely in native state at 26°C but predominantly nonnative state at 37°C thus allowing the use of thermal shifts to follow the fate of unfolded proteins. To examine the post-endocytic distribution of CD4 chimeras, membrane proteins were labeled by CD4 Ab capture for 20 mins in live cells at 37°C and chased for 1 h in the absence of extracellular Ab before fixation and labeling with secondary antibody to localize the internalized CD4 chimeras (green). Endosomes were labeled with 10 µg/ml Alexa Fluor 594-labeled transferrin uptake for 1 h at 37°C (red). Fluorescence micrographs were obtained by a confocal microscope (LSM510 or LSM710; Carl Zeiss, Inc.) equipped with a Plan-Apochromat 63×/NA 1.4 objective in multitrack mode. This single optical section shows that internalized CD4tl-lambda protein at least partly co-localizes with endosomes, in contrast to internalized CD4tl-lambdaC, the unfolded mutant, seen in a companion image in this group.

本图系 Apaja 等人在 2010 年发表于《JCB》杂志的图 6 中的六幅图像之一，展示了各种模型膜蛋白在质膜中的折叠或非折叠状态下的命运。这些模型膜蛋白在稳定的四环素诱导型 Flp-In T-Rex HEK293 细胞系中表达，其中三种模型膜蛋白是 CD4 表面受体的嵌合体，包括：1. 终端截短的 CD4（CD4tl），其中包含一个灵活的细胞质连接域；2. 与野生型噬菌体 λ 抑制子的 N 端 DNA 结合域融合的 CD4tl（CD4tl-lambda）；3. 与 L57C 突变型 λ 抑制子融合的 CD4tl（CD4tl-lambdaC）。在 26°C 时，CD4tl-lambdaC 的细胞质域主要处于天然状态，而在 37°C 时则主要处于非天然状态，从而允许利用温度变化追踪非折叠蛋白的命运。为了检测 CD4 嵌合体的内吞后分布，细胞质膜蛋白在 37°C 的活细胞中通过 CD4 抗体捕获进行标记 20 分钟，然后在无细胞外抗体的条件下追踪 1 小时，随后进行固定和用次级抗体进行标记以定位内化的 CD4 嵌合体（绿色）。内吞体通过 10 µg/ml 的 Alexa Fluor 594 标记的转铁蛋白摄取在 37°C 下进行标记 1 小时（红色）。荧光显微镜图像是通过配备有 Plan-Apochromat 63×/NA 1.4 物镜的多道模式下的共聚焦显微镜（LSM510 或 LSM710；卡尔·蔡司公司）获得的。这张单光切片图像显示，内化的 CD4tl-lambda 蛋白至少部分与内吞体共定位，这与在同一组图像中的另一张图像中观察到的内化的非折叠突变体 CD4tl-lambdaC 形成对比。