| Affiliation: | 1. Ecole Normale Supérieure, Département de Physique and Département de Biologie, Laboratoire de Physique Statistique UMR CNRS‐ENS 8550, 24 rue Lhomond, 75231 Paris (France);2. Ecole Normale Supérieure, Département de Chimie, UMR CNRS‐ENS‐UPMC Paris 06 8640 PASTEUR, 24 rue Lhomond, 75231 Paris (France);3. Kavli Institute for Theoretical Physics, University of California at Santa Barbara, Santa Barbara CA 93106 (USA);4. Collège de France, UMR 8542 CNRS‐ENS‐CDF, 11 place M. Berthelot, 75231 Paris Cedex 05 (France);5. Université Paris Diderot‐Paris 7, 80, rue du Général Leclerc Bat G. Pincus, 94276 Le Kremlin‐Bicêtre Cedex (France);6. Institut Curie, UMR 176 Institut Curie–CNRS, 26, rue d'Ulm, 75005 Paris (France);7. Institut de Biologie Physico‐Chimique, Laboratoire de Biochimie Théorique, CNRS UPR 9080, 13, rue Pierre et Marie Curie, 75005 Paris (France);8. Ecole Normale Supérieure, Département de Biologie, 46, rue d'Ulm, 75231 Paris Cedex 05 (France);9. Department of Chemistry and Biochemistry, UCLA, Los Angeles (USA) |
| 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 (ERT2). Cyclofen‐OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ERT2 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. |
