Related Experiment Video
Updated: Jan 20, 2026

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation
Published on: October 4, 2024
A fluorescent protein-readout for transcriptional activity reveals regulation of APP nuclear signaling by
Uwe Konietzko1, Manuel T Gersbacher2, Jeremy Streuli2
1Institute for Regenerative Medicine (IREM), University of Zurich, Wagistrasse 12, 8952 Schlieren, Switzerland.
Abstract:
Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cell-based drug screening methods for nuclear translocation.
More Related Videos
06:09Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription
Published on: August 11, 2021
12:47Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
Published on: December 27, 2016
Related Concept Videos
Master Transcription Regulators
Regulated Protein Degradation
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
Eukaryotic Transcription Activators
The binding domains are capable of recognizing and interacting with regulatory sequences on the DNA. These...
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein....
Cooperative Binding of Transcription Regulators
Regulation of Nuclear Protein Sorting