Photocontrol of protein activity

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Photocontrol of protein activity in vivo

Controlling protein activity and gene expression in space and time is a much sought after goal in biology. Inducible gene expression systems have been used but they remain limited by the technology associated to the inducer delivery. Ideally, the latter should be noninvasive, fast, local, and tunable.

In collaboration with David Bensimon (Physics Department, ENS) and Sophie Vriz (CdF), we have implemented a technology fulfilling all those criteria. The method relies on a known method to control the activity of proteins by fusing them with a steroid receptor and activating them by providing the ligand. Here we have achieved single cell release by developing photo-activable variants of a commonly used ligand, tamoxifen, to release proteins fused to a receptor specific for tamoxifen (ERT2). The experimental protocol has proven especially simple in the case of implementation in zebrafish embryos: (i) The embryos are incubated at an early stage of development in an aqueous solution of a caged tamoxifen analog cInd that penetrates the whole organism; (ii) The embryos are washed and transferred to an illumination chamber where release of the active tamoxifen analog Ind down to the single cell level can be accomplished by one- or two-photon excitation with a focused laser beam.

The present protocol has been validated by photocontrolling the targeting of fluorescent proteins fused to the ERT2 ligand binding domain to nuclei in live zebrafish embryos (Figure 1 a-c), or the gene expression in transgenic strains using the Cre recombinase fused to ERT2 (Figure 1 d-f). In collaboration with Michel Volovitch and Alain Prochiantz (CdF), we now wish to apply this protocol to investigate different processes active during development or regeneration. Indeed the method we have developed is particularly suited to investigate morphogenetic problems that require a means to control protein activity with high spatio-temporal resolution (single cell and less than minute timescales). We currently investigate: (i) Cell lineage during tissue regeneration. We are particularly interested in the late stages where our approach (based on photoactivation of Cre-ERT2) should be competitive with respect to existing labeling techniques based on photoactivated fluorescent proteins, such as Kaede (which fade with time); (ii) The role of homeogenes (engrailed, pax6) as morphogens controlling the development of the central nervous system, which imply their diffusion to neighboring cells (if they do then how far and how fast do they diffuse).

 

Figure 1. Phocontrol of protein activity in zebrafish embryos upon uncaging the cyclofen inducer Ind from its caged precursor cInd. a-c: Photocontrol of GFP-nls-ERT2 nuclear translocation in wild type embryos injected with gfp-nls-ERT mRNA at one-cell stage and incubated in 3 mM cInd. Fluorescence confocal images at 4 to 6 hpf (GFP-ERT2 fluorescence) show that the GFP-nls-ERT2 nuclear translocation is governed by the illumination. Absent without illumination (a), it is observed in the whole embryos 30 min after illumination with UV light (b). The dependence of the extent of nuclear translocation of GFP-nls-ERT2 as a function of the duration of the UV illumination observed in b is shown in c. The grey region in c displays the extent of nuclear translocation observed by incubating the embryos in 1 mM Ind; d-f: Photocontrol of Cre-ERT2 recombinase activity in ef1a:loxP-egfp-loxP-dsRed zebrafish embryos injected with cre-ERT mRNA at one-cell stage and incubated with 3 mM cInd. In epifluorescence microscopy at 48 hpf, non-UV illuminated embryos do not express dsRed (d) whereas they express it either globally (with UV illumination; e) or at the single cell level (with two-photon excitation, 750 nm; f) after uncaging at early somitogenesis, as expected if the GFP gene is excised by the recombinase in the original (mother) cell. Scale bar: 50 mm(b,f), 200 mm (d,e).

References

1. D. K. Sinha, P. Neveu, N. Gagey, I. Aujard, C. Benbrahim-Bouzidi, T. Le Saux, C. Rampon, C. Gauron, B. Goetz, S. Dubruille, M. Baaden, M. Volovitch, D. Bensimon, S. Vriz, L. Jullien, Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination, ChemBioChem, 2010, 11, 653-663.

2. D. K. Sinha, P. Neveu, N. Gagey, I. Aujard, T. Le Saux, C. Rampon, C. Gauron, K. Kawakami, C. Leucht, Laure Bally-Cuif, M. Volovitch, D. Bensimon, L Jullien, S. Vriz, Photo-activation of the CreERT2 recombinase for conditional site-specific recombination with high spatio-temporal resolution, Zebrafish, 2010, 7, 199-204.

3. I. Brunet, A. A. Di Nardo, L. Sonnier, M. Beurdeley, A. Prochiantz A, The topological role of homeoproteins in the developing central nervous system, Trends Neurosci., 2007,30, 260-267.