Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon illumination.
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.
Sinha
Deepak Kumar
DK
Ecole Normale Supérieure, Département de Physique, Laboratoire de Physique Statistique UMR CNRS-ENS 8550, 24 rue Lhomond, 75231 Paris, France.
Neveu
Pierre
P
Gagey
Nathalie
N
Aujard
Isabelle
I
Benbrahim-Bouzidi
Chouaha
C
Le Saux
Thomas
T
Rampon
Christine
C
Gauron
Carole
C
Goetz
Bernard
B
Dubruille
Sylvie
S
Baaden
Marc
M
Volovitch
Michel
M
Bensimon
David
D
Vriz
Sophie
S
Jullien
Ludovic
L
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003506:Cyclofenil / Q000737:chemistry; D004625:Embryo, Nonmammalian / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D055668:Photochemical Processes; D017785:Photons; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
D018832:Molecular Chaperones; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; D003506:Cyclofenil