ABSTRACT
When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of ATF6α by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human ATF6α N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-hATF6α deletion variant was cleaved to liberate active transcription activator encompassing GV-hATF6α fragment which could translocate into the nucleus. The translocated GV-hATF6α fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-hATF6α(333) represents an innovative tool to investigate regulated intramembrane proteolysis of ATF6α. It can substitute active pATF6(N) binding motif-based reporter cell lines.
Subject(s)
Activating Transcription Factor 6/metabolism , Endoplasmic Reticulum/metabolism , Luciferases/metabolism , Membrane Proteins/metabolism , Unfolded Protein Response , eIF-2 Kinase/metabolism , Activating Transcription Factor 6/genetics , Cell Line, Tumor , Dithiothreitol/pharmacology , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/genetics , Endoribonucleases/metabolism , Gene Expression Regulation/drug effects , Genes, Reporter/genetics , HeLa Cells , Humans , Luciferases/genetics , Membrane Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Signal Transduction/drug effects , Signal Transduction/genetics , eIF-2 Kinase/geneticsABSTRACT
We propose an efficient bioimaging strategy using Yb3+,Er3+,Eu3+-triplet doped YVO4 nanoparticles which were synthesized with polymer as a template. The obtained particles possess nanoscale, uniform, and flexible excitation. The effect of Eu3+ ions on the luminescence properties of YVO4:Yb3+,Er3+,Eu3+ was investigated. The upconversion mechanism of the prepared material was also discussed. The structure and optical properties of the prepared material were characterized by using X-ray diffraction (XRD), Fourier-transform IR spectroscopy (FTIR), scanning electron microscopy (SEM), Transmission electron microscopy (TEM) upconversion and photoluminescence spectra. The Commission International de I'Eclairage (CIE) chromaticity coordinates was investigated to confirm the performance of color luminescent emission. The prepared YVO4:Yb3+,Er3+,Eu3+ nanoparticles could be easily dispersed in water by surface modification with cysteine (Cys) and glutathione (GSH). The aqueous dispersion of the modified YVO4:Yb3+,Er3+,Eu3+ exhibits bright upconversion and downconversion luminescence and has been applied for bioimaging of HeLa cells. Our developed material with dual excitation offers a promising advance in bioimaging.
