Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13690, 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). Lysosomes were marked by by LAMP2 antibody (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-lambdaC protein co-localizes with lysosomes, indicative of the cellular response to the nonnative folding of this model membrane receptor.

此图6中的六幅图像之一，收录于Apaja等人在2010年发表于《Journal of Cell Biology》的论文中，展示了各种模型膜蛋白在质膜上的折叠状态或非折叠状态下的命运，这些蛋白表达于稳定的四环素诱导型Flp-In T-Rex HEK293细胞系中。三种模型膜蛋白为CD4表面受体的嵌合体，包括：1.) 端部截断的CD4，其中包含一个灵活的细胞质连接域（CD4tl）；2.) 与野生型噬菌体λ抑制剂的N端DNA结合域融合的CD4tl（CD4tl-lambda）；3.) 与L57C突变型λ抑制剂融合的CD4tl（CD4tl-lambdaC）。在26°C下，CD4tl-lambdaC的细胞质域主要处于天然状态，但在37°C下则主要处于非天然状态，从而允许利用热变性追踪非折叠蛋白的命运。为了检测CD4嵌合体的后内吞分布，膜蛋白在37°C下活细胞中通过CD4抗体捕获进行标记20分钟，并在无细胞外抗体的条件下追踪1小时后进行固定和用次级抗体标记，以定位内化的CD4嵌合体（绿色）。溶酶体通过LAMP2抗体（红色）进行标记。荧光显微镜图像是通过配备多道模式下的Plan-Apochromat 63×/NA 1.4物镜的共聚焦显微镜（LSM510或LSM710；卡尔蔡司公司）获得的。此单光切图像显示，内化的CD4tl-lambdaC蛋白与溶酶体共定位，这表明细胞对这种模型膜受体的非天然折叠状态的反应。