powered by the
caged Cyclofen-OH technology
"Once upon a time, a physicist (David Bensimon) asked a chemist (Ludovic Jullien) whether he could design a caged inducer to photocontrol protein activity in living organisms. For sure! However we also needed a biologist (Sophie Vriz) to accept the challenge to validate the caged Cyclofen-OH technology. It has been a long but so nice adventure, which has involved the tight integration of the work from many talented students, postdocs, and collaborators... Thanks to all of them!"
ACTIFLASH & EXAMPLES OF RESULTS
Actiflash is the stable Tamoxifen-like photoactivable inducer to perform a spatial and temporal control of your favorite proteins under illumination.
A protein fused to the ERT2 receptor is inactivated by its assembly with a chaperone complex (CC). Upon photoactivation of caged Cyclofen-OH (cCyc), Cyclofen-OH (Cyc) is released and binds to the ERT2 moiety, which causes assembly disruption and activation of the ERT2-fused protein.
Credit: Reproduced with permission from D. K. Sinha et al, Photocontrol of Protein Activity in Cultured Cells and Zebrash with One- and Two-Photon Illumination, Chem. Bio. Chem., 2010, 11, 653-663. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.
a–d: Photocontrol of nuclear GFP-nls-ERT translocation in CV1 cells transfected with gfp-nls-ERT plasmid and incubated in a medium containing 6 μM of caged Cyclofen-OH (cCyc). In absence of UV illumination (a), the cells exhibit weak cytoplasmic fluorescence and occasional nuclear one, as do cells incubated without ligand. In contrast, upon UV illumination, the cytoplasmic fluorescence disappears and the nuclear one increases (b), as in cells incubated with Cyclofen-OH. With two photon illumination (750 nm, 10 mW for 10 s), nuclear GFP-nls-ERT translocation can be selectively performed in one CV1 cell (indicated with an arrow in c) as evidenced by comparing the fluorescence levels in the cytoplasm and in the nucleus of the targeted cell 0 (c) and 60 (d) min after illumination, using the nearby non-illuminated cell as a reference. Same display range in a–b and in c–d; e–f: Photocontrol of Cre-ERT activity in HEK derivatives cells. The cells expressing the lox-rfp-nls-lox-gfp-mb were transfected with the cre-ERT plasmid and incubated in 5 μM cCyc. Upon UV illumination, the initially nuclear localized RFP expressing cells (e) were converted into cells expressing a membrane bound GFP (f), as expected from releasing Cyclofen-OH which disrupts the recombinase – chaperone complex. The epifluorescence images superpose the red and green emission associated to the expressions of RFP and GFP respectively. Scale bar: 25 μm.
Credit: Experiments performed by Pierre Neveu, Michel Volovitch, and Sophie Vriz.
a-c: Photocontrol of nuclear translocation of GFP-nls-ERT in wild type embryos injected with gfp-nls-ERT mRNA at the one-cell stage and further incubated in 3 μM cCyc. Fluorescence confocal images at 4 to 6 hpf (GFP-ERT fluorescence) show that the GFPnls-ERT nuclear translocation is governed by the illumination. Absent without illumination (a), it can be observed in the whole embryos 30 min after illumination with UV light (b). The dependence of the extent of nuclear translocation of GFP-nls-ERT 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 μM Cyclofen-OH; d-f: Photocontrol of Cre-ERT recombinase activity in ef1fi:loxP-egfp-loxP-dsRed zebrafish embryos injected with cre-ERT mRNA at one-cell stage and further incubated with 3 μM cCyc. In epifluorescence microscopy at 48 hpf, the non-UV illuminated embryos do not express dsRed (d) whereas they express dsRed either globally (with UV illumination; e) or at the single cell level (with two-photon excitation, 750 nm, 20 mW for 10 s; 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 μm for b and f, 200 μm for d and e.
Credit: Reproduced with permission from D. K. Sinha et al, Photocontrol of Protein Activity in Cultured Cells and Zebrash with One- and Two-Photon Illumination, Chem. Bio. Chem., 2010, 11, 653-663. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. et Experiments performed by Pierre Neveu, Michel Volovitch, and Sophie Vriz.
One-time activation of kRASG12V expression was induced by transient cyclofen-OH incubation or photo-activation of caged cyclofen-OH at 1dpf (A). Periodic kRASG12V expression was achieved by 1 day incubation in 2 μM cyclofen-OH every 5 days for a course of two months (E). (B–D) Transient induction of kRASG12V did not affect normal development (as shown by the histopathological staining) with no CFP expression in one-year old fish. (E–H) Tumors were observed (albeit rarely) in fish periodically treated with cyclofen-OH. A representative one-year old fish displayed a clear tumor (E) revealed by histopathological staining (H). Interestingly, the cells in the tumor still expressed EosFP (F), but lost CFP expression (G). (I–L) Constitutive kRASG12V expression following light-activation of caged cyclofen-OH induced tumor (I) in 5 monthold fish as revealed by both fluorescent markers (J,K) and H&E staining (L). Scale bar: B, D, F, H, L 200 μm; J, 1 cm.
Credit: Reproduced with permission from Z. P. Feng, S. Nam, F. Hamouri, I. Aujard, B. Ducos, S. Vriz, M. Volovitch, L. Jullien, S. Lin, S. Weiss, D. Bensimon, Optical Control of Tumor Induction in the Zebrafish, Sci. Rep., 2017, 7, 9195.
Simple conditioning: Caged Cyclofen-OH is cell-permeant and can be added either in the external medium or directly injected for conditioning.
Excellent chemical stability: Caged Cyclofen-OH does not generate any basal activation of protein function and it benefits from an excellent temporal resolution upon uncaging
Favorable wavelength ranges for uncaging: Uncaging requires either UV-A light or a strong IR laser. Visible light is inactive, which facilitates the experiments with biological samples.
Photochemical stability: Caged Cyclofen-OH liberates Cyclofen-OH, which is photostable in contrast to Tamoxifen-OH encountering photodegradation under illumination
HOW TO USE ACTIFLASH