PUBLICATION

Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination

Authors
Sinha, D.K., Neveu, P., Gagey, N., Aujard, I., Benbrahim-Bouzidi, C., Le Saux, T., Rampon, C., Gauron, C., Goetz, B., Dubruille, S., Baaden, M., Volovitch, M., Bensimon, D., Vriz, S., and Jullien, L.
ID
ZDB-PUB-100302-18
Date
2010
Source
Chembiochem : a European journal of chemical biology   11(5): 653-663 (Journal)
Registered Authors
Bensimon, David, Vriz, Sophie
Keywords
cage compounds, cells, gene expression, photochemical methods, protein modifications
MeSH Terms
  • Animals
  • Cell Line
  • Chlorocebus aethiops
  • Cyclofenil/chemistry
  • Embryo, Nonmammalian/metabolism
  • Green Fluorescent Proteins/analysis
  • Green Fluorescent Proteins/metabolism
  • Molecular Chaperones/metabolism
  • Molecular Dynamics Simulation
  • Photochemical Processes
  • Photons
  • Receptors, Estrogen/genetics
  • Receptors, Estrogen/metabolism*
  • Recombinant Fusion Proteins/analysis
  • Recombinant Fusion Proteins/metabolism*
  • Tamoxifen/analogs & derivatives
  • Tamoxifen/chemistry
  • Tamoxifen/pharmacology
  • Ultraviolet Rays
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish/metabolism
PubMed
20187057 Full text @ Chembiochem
Abstract
We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping