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Description

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.

Biological Sources

NCBI Organism Classification
Homo sapiens
(human)
Cell Line
293
Cellular Component
plasma membrane
lysosome

Biological Context

Biological Process
receptor internalization
response to unfolded protein

Attribution

Names
Pirjo M. Apaja
Haijin Xu
Gergely L. Lukacs
Published
JCB 2010, 191:553-570
Pubmed
20974815

Grouping

This image is part of a group.

Imaging

Image Type
recorded image
Imaging Mode
confocal microscopy
Parameters Imaged
fluorescence emission
Source of Contrast
distribution of a specific protein
distribution of epitope
Visualization Methods
Alexa Fluor 488
Alexa Fluor 555
Processing History
unprocessed raw data
Data Qualifiers
raw, unprocessed data

Sample Preparation

Methods
crosslinking-fixative fixed tissue
detergent permeabilized
Relation To Intact Cell
dispersed cells in vitro

Dimensions

Spatial Axis Image Size Pixel Size
X 512px 56nm
Y 512px 56nm
*CIL – Cell Image Library accession number. Please use this to reference an image.