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

Deepak Kumar Sinha (1) , Pierre Neveu (1, 2, 3) , Nathalie Gagey (2) , Isabelle Aujard (2) , Chouaha Benbrahim-Bouzidi , Thomas Le Saux (4) , Christine Rampon (5, 6) , Carole Gauron (6) , Bernard Goetz (7) , Sylvie Dubruille , Marc Baaden (8) , Michel Volovitch (6, 9) , David Bensimon (1) , Sophie Vriz (5, 6) , Ludovic Jullien (2, 4)

1 LPS - Laboratoire de Physique Statistique de l'ENS
2 Département de Chimie - ENS Paris
3 KITP - Kavli Institute for Theoretical Physics [Santa Barbara]
4 PASTEUR - Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640)
5 UPD7 - Université Paris Diderot - Paris 7
6 DESN - Développement et évolution du système nerveux
7 BIOSYMA - Biomolécules : synthèse, structure et mode d'action (UMR 8642)
8 LBT (UPR_9080) - Laboratoire de biochimie théorique [Paris]
9 Département de Biologie - ENS Paris

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.

Domaines: Chimie

Deepak Kumar Sinha, Pierre Neveu, Nathalie Gagey, Isabelle Aujard, Chouaha Benbrahim-Bouzidi, et al.. Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination. ChemBioChem, 2010, 11 (5), pp.653-663. ⟨10.1002/cbic.201000008⟩. ⟨hal-03904748⟩