Photocontrol of protein activity in a single cell of a live organism
D. K. Sinha,a P. Neveu,a,b,j N. Gagey,b I. Aujard,b C. Benbrahim-Bouzidi,b T. Le Saux,b C. Rampon,c C. Gauron,c
B. Goetz,d S. Dubruille,e C. Leucht,f L. Bally-Cuif,f M. Volovitch,g D. Bensimon,h,i L. Jullien,b S. Vrizc
a) Ecole Normale Supérieure, Laboratoire de Physique Statistique, UMR 8550 CNRS, 24, rue Lhomond, 75231 Paris Cedex
05, France; b) Ecole Normale Supérieure, Département de Chimie, UMR 8640 CNRS ENS UPMC-Paris 6 PASTEUR, 24,
rue Lhomond, 75231 Paris Cedex 05, France; c) Université Paris Diderot - Paris 7, U770 INSERM, 80, rue du Général
Leclerc, Bat G. Pincus, 94276 Le Kremlin-Bicêtre Cedex, France; d) Ecole Normale Supérieure, Département de Chimie,
UMR 8642 CNRS ENS UPMC-Paris 6 BIOSYMA, 24, rue Lhomond, 75231 Paris Cedex 05, France; e) Institut Curie, UMR
176 Institut Curie-CNRS, 26, rue d'Ulm, 75005 Paris, France; f) Helmholtz Zentrum Muenchen, German Research Center
for Environmental Health, Department Zebrafish Neurogenetics, Institute of Developmental Genetics, Ingolstaedter
Landstrasse 1, D-85764 Neuherberg Germany; g) Ecole Normale Supérieure, Département de Biologie, UMR 8542, 46, rue
d'Ulm, F-75231 Paris Cedex 5, France; h) Ecole Normale Supérieure, Département de Biologie, 46, rue d'Ulm, F-75231
Paris Cedex 5, France; i) Department of Chemistry and Biochemistry, UCLA, Los Angeles, USA; j) Kavli Institute for
Theoretical Physics, University of California at Santa Barbara, Santa Barbara CA 93106, USA
Cells respond to external signals by modifying their internal state and their environment. In
multicellular organisms in particular, cellular differentiation and intra-cellular signaling are essential
for the coordinated development of the organism. While some of the major players of these complex
interaction networks have been identified, much less is known of the quantitative rules that govern
their interaction with one another and with other cellular components (affinities, rate constants,
strength of non linearities such as feedback or feedforward loops, etc.). To investigate these
interactions (a prerequisite before understanding or modeling them), one needs to develop means to
control or interfere spatially and temporally with these processes.
In the preceding context, we have retained the principle of a small lipophilic molecule to
photo-activate several properly engineered proteins in vivo. We have adopted a steroid-related inducer
as various proteins (e.g. Engrailed, Otx2, Gal4, p53, kinases such as Raf-1, Cre and Flp recombinases)
fused to a steroid receptor were shown to be activated by binding of an appropriate ligand.[1] In its
absence, the receptor forms a cytoplasmic assembly with a chaperone complex: the fusion-protein is
inactivated. Its function is restored in the presence of the steroid ligand which binds to the receptor and
disrupts the complex, see Fig.1.
Fig. 1 A protein fused to the ERT receptor is inactivated by the assembly formed with a chaperone complex. Upon photoactivation of a caged
precursor (cInd), a non-endogeneous inducer (Ind, 4-hydroxy-cyclofen) is released, binds to the ERT receptor and sets the protein fusion free
from its assembly with the chaperone complex.
The present non-invasive optical method has been implemented for the fast control of protein
activity in a single cell of a live zebrafish embryo.[2] In particular, we labeled single cells transiently
(by activating a fluorescent protein) or irreversibly (by activating a Cre recombinase in an appropriate
transgenic animal). The present method could be used more generally to investigate important
physiological processes (for example in embryogenesis, organ regeneration and carcinogenesis) with
high spatio-temporal resolution (single cell and faster than minute scales).
References
1. D. Picard, Curr. Op. Biotech., 5, 511-515 (1994).
2. D. K. Sinha et al., submitted.