Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13689, 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 protein co-localizes with endosomes, like other plasma membrane receptors. This contrasts with the behavior of the internalized, unfolded mutant CD4tl-lambdaC, seen in a companion image in this group.

本图系Apaja等人在2010年发表于《细胞生物学杂志》（JCB）的图6中六幅图像之一，展示了多种模型质膜蛋白的命运，这些蛋白在质膜中可能处于折叠或非折叠状态，并表达于稳定的四环素诱导型Flp-In T-Rex HEK293细胞系中。这三种模型质膜蛋白为CD4表面受体的嵌合体，包括：1.) C端截短并含有柔性细胞质连接体的CD4（CD4tl）；2.) 与野生型噬菌体λ阻遏蛋白N端DNA结合域融合的CD4tl（CD4tl-lambda）；3.) 与L57C突变型λ阻遏蛋白融合的CD4tl（CD4tl-lambdaC）。在26°C时，CD4tl-lambdaC的细胞质结构域主要处于天然状态，而在37°C时则主要处于非天然状态，从而允许通过热变性追踪非折叠蛋白的命运。为了考察CD4嵌合体的后内吞分布，在37°C的活细胞中，通过CD4抗体捕获标记膜蛋白20分钟，并在无细胞外抗体的条件下追踪1小时后固定并使用二抗进行标记，以定位内化的CD4嵌合体（绿色）。内吞体通过10 µg/ml的Alexa Fluor 594标记转铁蛋白摄取在37°C下标记1小时（红色）。荧光显微镜图像由配备有Plan-Apochromat 63×/NA 1.4物镜的共聚焦显微镜（LSM510或LSM710；卡尔·蔡司公司）在多通道模式下获得。该单光切图像显示，内化的CD4tl蛋白与内吞体共定位，类似于其他质膜受体。这与该组另一图像中观察到的内化、非折叠突变型CD4tl-lambdaC的行为形成对比。