Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13688, 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 is largely in native state at 26°C but predominantly nonnative state at 37°C. 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 protein does not co-localize with lysosomes. A companion image in this group shows that CD4tl, however, does co-localize with endosomes, a property shared with other membrane receptors.

此图像为Apaja等人于2010年发表在《JCB》杂志的第6图中的六幅图像之一，展示了多种模型膜蛋白在质膜处的折叠或非折叠状态，这些蛋白在稳定的四环素诱导型Flp-In T-Rex HEK293细胞系中表达。这三种模型膜蛋白是CD4表面受体的嵌合体，包括：1.一个C端截断的CD4（包含一个灵活的细胞质连接器，CD4tl）；2.一个与野生型噬菌体λ抑制剂的N端DNA结合域融合的CD4tl（CD4tl-lambda）；3.一个与L57C突变型λ抑制剂的CD4tl融合（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。为了考察CD4嵌合体的后内吞分布，将膜蛋白在37°C的活细胞中用CD4抗体捕获标记20分钟，然后在无细胞外抗体的条件下追踪1小时，之后固定并使用第二抗体进行标记以定位内化的CD4嵌合体（绿色）。溶酶体通过LAMP2抗体（红色）进行标记。荧光显微照片是通过配备有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的。这幅单一光学切片显示内化的CD4tl蛋白不与溶酶体共定位。本组图像中的另一幅图像显示，尽管如此，CD4tl却与内体共定位，这一特性与其他膜受体相似。