Question

You are analyzing a protein that you believe affects endocytosis, but you are not sure which stage. You create cells with your gene of interest mutated in the genome, and a tet-off system driving a wild-type (WT) copy of your gene (tet-off allows a specific gene to be turned off by adding a tetracycline family drug). After confirming that the system works, you culture the cells with and without tetracycline and in the presence of very high levels of EGF. You then use a gold-labelled antibody to the EGFR receptor and electron microscopy to view localization during endocytosis. Assume that your protein is essential for multivesicular body formation. Where would EGFR be located in the late endosome with and without tetracycline?

Answer 1

During formation of multi vesicular body ( MVB) formation, early endosome pinched off and contain receptor along with cargo molecules in to late endosomes. This late endosomes is also known as miltivesicular body pathway. So in case of with out tetracyclin, protein resposible fir MVB formation will function normally and lastly EGFR will be located in to late endosomes fused with lysosome.

In presence of tetracyclin-- deletion of the particular gene which involve in MVB formation, so the EGFR will be present in early endosome and gold particles staining will be present in early endosome.

Explanation-

In multivesicular body (MVB) pathway, as ligand ( EGFR) binds to its receptor leads to endocytosed of surface proteins and ligands. After endocytosis receptor and ligand are delivered into the endosomal system. During endocytosis some receptor recycle back to the plasma membrane for example Tfr through recycling endosomes. While cargo proteins destined to lysosome for degradation such as EGFR, . After enter in to the cell , first it present in early endosome. Lster they are sorted into internal lumenal vesicles of MVBs ( late endosomes ) . And lastly these Mature MVBs fuse with the lysosome and these internal vesicles are delivered to the lysosomal lumen where cargo and vesicles both are degraded by the lysosomal enzymes.