Optical control of protein activity and gene expression by photoactivation of caged cyclofen

Article abstract of DOI:10.1016/bs.mie.2019.04.009

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.

Full text of DOI:10.1016/bs.mie.2019.04.009

ARTICLE IN PRESS
Optical control of protein
activity and gene expression
by photoactivation of caged
cyclofen
Fatima Hamouria,b,*, Weiting Zhanga,b, Isabelle Aujardc,d,
Thomas Le Sauxc,d, Bertrand Ducosa,b, Sophie Vrize,f,
Ludovic Jullienc,d, David Bensimona,b,g,
*
aLaboratoire de Physique de l’ENS, CNRS-UMR8023, PSL Research University, Paris, France
bInstitut de Biologie de l’ENS, CNRS-UMR8197, INSERM-U1024, PSL Research University, Paris, France
cPASTEUR, Dꢀepartement de Chimie de l’ENS, CNRS, PSL Research University, Paris, France
dSorbonne Universitꢀes, UPMC Univ Paris 06, ENS, CNRS, PASTEUR, Paris, France
eCenter for Interdisciplinary Research in Biology (CIRB), College de France, CNRS UMR 7241,
INSERM U1050, Paris, France
fDepartment of Life Sciences, Paris-Diderot University, Sorbonne-Paris-Citꢀe, Paris, France
gDepartment of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles,
CA, United States
*Corresponding author: e-mail address: bensimon@chem.ucla.edu
Contents
1. Introduction
2
5
6
7
7
2. The caged-cyclofen-OH/ERT system
3. Synthesis of caged cyclofen-OH
4. Protocol for the synthesis of caged cyclofen-OH (cCyc)
4.1 Equipment
4.2 Chemicals
7
4.3 Protocol
8
5. Calibration of caged-cyclofen photo-activation
5.1 The wavelength range of the light source
5.2 The power range of the light source
5.3 Tuning the illumination for uncaging experiments
6. One- and two-photon activation in zebrafish
6.1 Equipment
10
11
11
11
14
15
15
15
15
16
16
18
6.2 Chemicals
6.3 Biological materials
6.4 Protocol
7. Transient and permanent gene activation
7.1 Equipment
7.2 Chemicals
#
Methods in Enzymology
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2019.04.009
2019 Elsevier Inc.
All rights reserved.
1

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Fatima Hamouri et al.
7.3 Biological materials
7.4 Protocol
8. Conclusions
18
18
20
20
References
Abstract
The use of light to control the expression of genes and the activity of proteins is a rapidly
expanding field. While many of these approaches use a fusion between a light
activatable protein and the protein of interest to control the activity of the latter, it is
also possible to control the activity of a protein by uncaging a specific ligand. In that
context, controlling the activation of a protein fused to the modified estrogen receptor
(ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile
method to control the activation of proteins quantitatively, quickly and locally in a live
organism. Here, we present the experimental details behind this approach.
1. Introduction
The study of physiological processes at the single cell level is a bloom-
ing field that is driving the development of many tools and approaches, from
super-resolution (Gao et al., 2012) and light-sheet microscopy (Mickoleit
et al., 2014) to optogenetics (Kralj, Douglass, Hochbaum, Maclaurin, &
Cohen, 2011; Miesenbo€ck, 2009). To investigate physiological processes
non-invasively, various methods have been developed that use light to
spatially and temporally perturb these processes, while monitoring the
cells’ response on a fast (sub-minute) timescale and single cell resolution
(Hochbaum et al., 2014). In particular to photocontrol the activity of proteins
(and the expression of genes), two main approaches have been adopted. One
is based on light-sensitive proteins and the other on light-sensitive protein
ligands.
The first light-sensitive proteins to be used in an optogenetic context
were rhodopsins (Sakmar, 2002; Zemelman, Lee, Ng, & Miesenbo€ck,
2002). These light sensitive ion channels were first used as actuators
of neuronal networks, i.e., to depolarize (Lo´renz-Fonfr´ıa & Heberle,
2014; Nagel et al., 2002) (activate) or hyperpolarize (Bamberg, Tittor,
& Oesterhelt, 1993) (inhibit) neuronal activity. These rhodopsins allow
for control of neuronal networks in vivo with unprecedented spatio-
temporal resolution (Fenno, Yizhar, & Deisseroth, 2011; Hegemann &

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Optical control of protein activity
3
Nagel, 2013; Zhang et al., 2011). More recently these channels have been
modified to be used as sensors of the transmembrane voltage (Hochbaum
et al., 2014).
Following on these pioneering studies, various light-sensitive proteins
were adapted as a means to control the activity of fused proteins. These
photo-sensitive proteins undergo a conformational change via the photo-
isomerization of a bound chromophore naturally present in most cells, such
as riboflavin (Conrad, Manahan, & Crane, 2014; Crosson, Rajagopal, &
Moffat, 2003; Hemmerich, Nagelschneider, & Veeger, 1970; Kim & Lin,
2013; Masuda, 2013), flavin adenine dinucleotide (FAD), flavin mononu-
cleotide (FMN) or cryptochromes (Chaves et al., 2011) (CRY), phyto-
chromes (PHY; Rockwell, Su, & Lagarias, 2006). This conformational
change can induce the dimerization of the photo-sensitive protein with
its partner (and consequently the coupling of appropriately fused proteins).
Alternatively, the conformational change can directly control the activity of
the fused protein.
These approaches based on the fusion between a protein of interest and a
light-sensitive one have the advantage of reversibility and genomic design.
However, reducing leakage and improving signal-to-noise ratios often
requires extensive optimization of the fused complex. Moreover, the acti-
vation of the fused protein sometimes requires prolonged illumination,
which defeats the purpose of the use of light as a means to improve spatio-
temporal resolution (Gautier et al., 2014).
To address some of these issues, another approach based on the use of
caged ligands has been pursued by various groups. There exists a large variety
of small molecules that bind to specific proteins, which are often transcrip-
tional activators. These small ligands (ecdysone, doxycycline, IPTG,
rapamycin, tamoxifen-OH, cyclofen-OH) can be caged and released upon
illumination at an appropriate wavelength (often in the near UV—around
375nm—but also in the visible (see below) or infrared via two-photon exci-
tation). Thus, a caged ecdysone (Lin, Albanese, Pestell, & Lawrence, 2002)
was developed to create a photo-inducible gene expression system. Upon
illumination, the caged ecdysteroid is rapidly converted into active ecdy-
sone, which binds and activates the ecdysone receptor, promoting its
association to a responsive element and inducing the expression of the
gene under its control. Similar systems were developed based on caged-
IPTG (Young & Deiters, 2007) allowing for photo-induction of genes

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Fatima Hamouri et al.
under control of the Lac operator and caged-doxycycline (Cambridge,
Geissler, Keller, & Cu€rten, 2006) photo-inducing the expression of genes
under the Tet operator.
Other systems have been developed to control the activity of cyto-
plasmic proteins when it depends on dimerization. Thus, caged rapamycin
(Karginov et al., 2011; Umeda, Ueno, Pohlmeyer, Nagano, & Inoue, 2011)
was designed to promote the light-induced heterodimerization of two pro-
teins fused to FK-506 binding protein (FKBP) and to the FKBP-rapamycin
binding protein (FRB), enabling the photocontrol in live cells of signaling
proteins (such as the small GTPase Rac involved in membrane ruffling) and
the regulation of the activity of protein kinases.
In the context of the control of cytoplasmic proteins, steroid hormones
and their receptors (Beato, Herrlich, & Schu€tz, 1995; Norman, Mizwicki, &
Norman, 2004) have long been used as tissue-specific inducible systems.
In absence of ligand, a protein fused to the hormone binding domain
(and expressed under a tissue-specific promoter) is sequestered by cytoplas-
mic chaperones and therefore usually inactive. Introduction of appropriate
steroids releases the fused protein from its chaperone complex and activates
its function. That approach was used to induce the activity of a large variety
of proteins: transcription factors (Braselmann, Graninger, & Busslinger, 1993;
Briegel et al., 1993; Hyder, Nawaz, Chiappetta, Yokoyama, & Stancel, 1995;
Kulessa, Frampton, & Graf, 1995) such as Gal4, GATA or c-Jun, recombinases
(Metzger, Clifford, Chiba, & Chambon, 1995; Nichols, Rientjes, Logie, &
Stewart, 1997) such as Flp or Cre, kinases (Picard, 2000) such as Erb1 or
Src, oncogenes (Gandarillas & Watt, 1997; McMahon, 2001) such as B-Raf
or cMyc, tumor suppressors ( Jia et al., 2013; Roemer & Friedmann, 1993)
suchasp53andenzymes(Wangetal.,1995)suchasβ-galactosidase.Thecaging
and subsequent photo-release of steroid hormones thus offers a versatile,
generic and proven approach to the quick photo-control of the activity of
many proteins at a cellular level.
The advantage of caged ligands is that they build on existing inducible
systems and are often characterized by a quick localized release of the ligand.
However, they have to satisfy demanding physico-chemical constraints:
– As any drug, the caged compound has to be soluble and permeate the tis-
sues and the cells of the target organism;
– the active, uncaged molecule has to be stable when illuminated;
– the illumination characteristics (intensity, wavelength, time-lapse) and
both the caged inducer and the uncaged products have to be non-toxic
to the cells.

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Optical control of protein activity
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A drawback of caged ligand systems is that uncaging is irreversible, though the
effect of the released ligand can be limited in time via diffusion and degrada-
tion. Moreover, issues of leakage and background activity have to be addressed
separately by controlling the concentrations of ligand and/or receptor.
2. The caged-cyclofen-OH/ERT system
Within the steroid class of activators, tamoxifen-OH has long been
used as the ligand of the estrogen receptor (ER) binding domain or more
precisely, ERT2 (hereafter shorten as ERT) peptide: a binding domain
engineered to interact specifically with tamoxifen-OH in order to reduce
possible interference with the endogenous estrogen pathway (Feil et al.,
1996; Feil, Wagner, Metzger, & Chambon, 1997). Tamoxifen-OH has been
caged (Link, Shi, & Koh, 2005; Shi & Koh, 2004; Wong et al., 2017) but
its instability under illumination hinders its use in physiological conditions
(Inlay et al., 2013). To address that issue, caged-cyclofen-OH (Sinha, Neveu,
Gagey, Aujard, Benbrahim-Bouzidi, et al., 2010a) has been developed as
a stable alternative to caged-tamoxifen-OH, see Fig. 1. Cyclofen-OH has
similar affinity to the ERT binding domain as tamoxifen-OH and is stable
when illuminated.
Fig. 1 The caged-cyclofen-OH/ERT system. Proteins fused to the estrogen receptor
engineered to bind specifically to tamoxifen-OH (ERT) are sequestered by cytoplasmic
chaperones (CC). Illumination of caged 4,5-dimethoxy-2-nitrobenzyl cyclofen-OH (cCyc)
with UV (375nm) light or multiphoton excitation (at 750 and 1064nm with two- and
three-photon excitation, respectively) releases the hormone-like steroid cyclofen-OH
(Cyc). Its binding to the ERT domain frees the fused protein from its cytoplasmic com-
plex with chaperones.

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Fatima Hamouri et al.
Cyclofen-OH has been caged with a 4,5-dimethoxy-2-nitrobenzyl moi-
ety (Sinha, Neveu, Gagey, Aujard, Benbrahim-Bouzidi, et al., 2010a; Sinha,
Neveu, Gagey, Aujard, Le Saux, Rampon, et al., 2010b) and has been used
in various biological contexts to activate proteins fused to ERT with one-,
two-, and three-photon excitations or to modify the spectral properties of
fluorescent proteins (Sinha, Neveu, Gagey, Aujard, Benbrahim-Bouzidi,
et al., 2010a; Sinha, Neveu, Gagey, Aujard, Le Saux, Rampon, et al.,
2010b; Jullien & Aujard, 2017; Rampon et al., 2015). Since its original
synthetic pathway was not compatible with production at large scales, we
subsequently modified its synthesis ( Jullien & Aujard, 2017). The present
alternative preparative method (see below) is now compatible with the
production of caged precursors of cyclofen-OH up to the 10g scale.
Cyclofen-OH has been subsequently caged with coumarin-based caging
moieties absorbing up to 500nm (Fournier et al., 2013). The resulting
blue light absorbing caged cyclofen-OH enabled us to achieve in live
zebrafish embryos chromatic orthogonal photoactivation of two biologically
active species controlling biological development with UV and blue-cyan
light sources, respectively (Fournier et al., 2013).
In the following we shall first describe how caged cyclofen-OH is syn-
thesized, how its decaging can be calibrated against illumination duration
and intensity and how it is used in the context of zebrafish physiology.
3. Synthesis of caged cyclofen-OH
Since the original synthesis reported in (Fournier, Aujard, et al., 2013)
was not compatible with large scale production and required a final careful
purification step to isolate the caged compound from its active precursor, we
developed an alternative preparative method, which is compatible with the
production of caged precursors of cyclofen-OH up to the 10-g scale.
The new synthetic pathway starts with a McMurry coupling on 4,40-
dihydroxybenzophenone, enabling to introduce the cyclohexyl ring. The
second step introduces the caging arylalkyl moiety by phenol mono-
substitution. The second phenol group is then alkylated (in the original pro-
tocol, the order of the latter steps was reversed). The overall yield for the
synthesis of compound cCyc from 4,40-dihydroxybenzophenone is 47%
and intermediate purifications now rely on precipitations only. This route
of synthesis is summarized in Scheme 1.

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7
O
a
b
c
OH
HO
N
OH
HO
OH
O
O
O
MeO
MeO
MeO
MeO
1
2
NO2
NO2
caged-Cyclofen-OH (cCyc)
Scheme 1 Improved synthesis of the caged cyclofen-OH cCyc. (a) cyclohexanone, TiCl4,
Zn, THF, reflux, 2h (yield: 81–85%); (b) 4,5-dimethoxy-2-nitrobenzyl bromide, K2CO3,
acetonitrile/H2O, r.t., 16h; (c) 2-(dimethylamino)ethyl chloride hydrochloride, Cs2CO3,
acetone, reflux, 18h (yield: 70%).
4. Protocol for the synthesis of caged cyclofen-OH (cCyc)
4.1 Equipment
1. Three-necked, round-bottomed flask (500mL) equipped with a mag-
netic stirring bar, a thermometer, a condenser connected to an argon
inlet, a septum/an addition funnel (100mL); 5, 10, 50mL syringes, fun-
nel (100mL) containing a plug of glass wool; separating funnel, fritted
glass; round-bottomed flask (250mL)
2. Stirring hotplate
3. Analytical balance
4. Ice bath; oil bath
5. Vacuum pump
6. Rotary evaporator
7. Merck silica gel 60F254 precoated plates for thin layer chromatography.;
UV lamp (254/365nm)
8. Aluminum foil
9. NMR spectrometer
4.2 Chemicals
General procedures: Commercially available reagents were used as obtained.
1
The H and 13C NMR spectra were recorded at room temperature on
Bruker AM 250, 300 or 400 spectrometers; chemical shifts are reported
in ppm with protonated solvent as internal reference (1H, CHCl3 in CDCl3
7.26ppm, CHD2SOCD3 in CD3SOCD3 2.49ppm; 13C, 13CDCl3 in
CDCl3 77.0, 13CD3SOCD3 in CD3SOCD3 39.7ppm); coupling constants
(J) are given in Hz. Microanalyses were performed by the Service de

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Fatima Hamouri et al.
Microanalyses de Gif sur Yvette. Mass spectra (chemical ionization and
high resolution with NH3 or CH4) were performed by the Service de
Spectromꢀetrie de Masse de Chimie ParisTech (MS) and by the Service de
Spectromꢀetrie de Masse de l’ICOA (HRMS, Orlꢀeans).
1. Dry tetrahydrofuran (Sigma-Aldrich)
2. Titanium chloride (Sigma-Aldrich)
3. Zinc powder (Sigma-Aldrich)
4. Argon gas
5. 4,40-Dihydroxybenzophenone (Alfa Aesar)
6. Cyclohexanone (Sigma-Aldrich)
7. Potassium carbonate (K2CO3) (VWR)
8. Ethyl acetate (VWR)
9. Brine (saturated solution of NaCl in water)
10. Magnesium sulfate (MgSO4) (VWR)
11. Dichloromethane or cyclohexane/ethyl acetate: 3/1, v/v; (VWR)
12. Silica gel 60 (0.040–0.063nm; Merck) for column chromatography
(Sigma-Aldrich)
13. DMSO-d6 and CDCl3 for NMR experiments (Sigma-Aldrich)
14. Acetonitrile (ACN)/H2O (2/1, v/v).(VWR)
15. 4,5-Dimethoxy-2-nitrobenzyl bromide (Sigma-Aldrich)
16. Phosphorous pentoxide (P2O5) (VWR)
17. 2-(Dimethylamino)ethyl chloride hydrochloride (Alfa Aesar)
18. Acetone (VWR)
19. Cesium carbonate (Cs2CO3) (VWR)
20. Methanol (VWR)
4.3 Protocol
Synthesis of 1: 4-(cyclohexylidene(4-hydroxyphenyl)methyl)
phenol
1.1 Working at ꢀ10°C, add titanium chloride (6.2mL, 56mmol; 6 equiv-
alents in 4,40-dihydroxybenzophenone) dropwise under argon to a
stirred suspension of zinc powder (8.20g, 126mmol; 14 equivalents
in 4,40-dihydroxybenzophenone) in 80mL of dry tetrahydrofuran.
1.2 Warm the mixture to room temperature and reflux for 2h.
1.3 Working at 0°C, add a solution of 4,40-dihydroxybenzophenone
(2.0g, 9mmol) and cyclohexanone (4mL, 38mmol; 4 equivalents in
4,40-dihydroxybenzophenone) in 120mL of dry tetrahydrofuran to
the cooled mixture and reflux for 2h.

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1.4 Bring the reaction mixture to room temperature and add 10% (w/v)
aqueouspotassiumcarbonate(30mL). Filterandextractwithethylacetate.
1.5 Wash the organic layer with brine, dry over MgSO4, and concentrate
in vacuo.
1.6 Load the concentrate on a column chromatography (cyclohexane/
ethyl acetate, 3:1, v/v) to yield 1 as a white powder (2.15g, 85%).
Alternatively, precipitation of the concentrate in dichloromethane
affords 1 (2.1g, 81%). Precipitation can also be carried out in cyclohex-
ane or in a cyclohexane/ethyl acetate mixture.
1
1.7 Perform NMR analysis of the white powder 1 to obtain: H NMR
(250MHz, DMSO-d6, δ): 9.28 (s, 2H), 6.82 (d, 4H, J¼8Hz), 6.65
(d, 4H, J¼8Hz), 2.14 (m, 4H), 1.52 (m, 6H); 13C NMR (100MHz,
DMSO-d6, δ): 155.5 (2C), 136.2, 133.8, 133.6 (2C), 130.3 (4C),
114.6 (4C), 31.9 (2C), 28.1(2C), 26.3. These data are in line with the
reported values for the same compound (Sinha, Neveu, Gagey, Aujard,
Benbrahim-Bouzidi, et al., 2010a; Sinha, Neveu, Gagey, Aujard,
Le Saux, Rampon, et al., 2010b).
Synthesis of 2: 4-((4-(4,5-dimethoxy-2-nitrobenzyloxy)phenyl)
(cyclohexylidene)methyl)phenol
2.1 Prepare a mixture of 1 (1.0g, 3.5mmol) and potassium carbonate
(470mg, 3.4mmol, 0.95eq.) in acetonitrile/H2O (60mL, 2/1, v/v).
2.2 Add portion-wise 4,5-dimethoxy-2-nitrobenzyl bromide (940mg,
3.4mmol, 0.95eq.) over a period of 1h (about 150mg every 10min).
2.3 Stir the resulting mixture for 16h at room temperature in the dark.
2.4 Filter the precipitate, wash with acetonitrile and then dry in vacuo over
P2O5.
2.5 Analyze the white powder obtained 2 (1.4g containing 96% of 2, 4% of
the product of 1 dialkylation, and traces of 1; this mixture was used
1
without further purification for the next step) by NMR to yield: H
NMR (ppm, 300MHz, CDCl3, δ): 7.77 (s, 1H), 7.35 (s, 1H), 7.04
(AA0XX0, 2H, J¼9Hz), 6.97 (AA0XX0, 2H, J¼9Hz), 6.91
(AA0XX0, 2H, J¼9Hz), 6.73 (AA0XX0, 2H, J¼9Hz), 5.48 (s, 2H),
4.68 (s, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 2.23 (m, 4H), 1.57 (m, 6H).
13C NMR (75MHz, DMSO-d6, δ): 156.4, 155.9, 153.3, 148.0,
139.9, 137.1, 136.2, 133.7, 133.5, 130.7 (2C), 130.5 (2C), 122.5,
114.9 (2C), 114.5 (2C), 111.5, 108.4, 66.7, 56.3, 56.2, 32.1 (2C),
28.3 (2C), 26.4; MS (CI, NH3): m/z 493.30 (calculated average mass
for [C28H29NO6 +NH4]+: 493.23; HRMS (TOF MS ES+): m/z
476.2077 (calculated mass for [C28H29NO6 +H]+: 476.2073.

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Fatima Hamouri et al.
Synthesis of cCyc: 2-(4-((4-(4,5-dimethoxy-2-nitrobenzyloxy)
phenyl)cyclohexylidene)methyl)phenoxy-N,N-dimethyl-
ethanamine
3.1 Prepare a solution of 2 (800mg, 1.68mmol) in acetone (50mL) and
Cs2CO3 (1.64g, 5mmol, 3eq.).
3.2 Add 2-(dimethylamino)ethyl chloride hydrochloride (484mg,
3.36mmol, 2eq.) portion-wise over a period of 30min and stir the
resulting suspension at reflux temperature in the dark for 18h.
3.3 Cool the reaction mixture to room temperature and filter it through
a funnel containing a plug of glass wool or cotton.
3.4 Wash the precipitate with dichloromethane and evaporate the solvent.
3.5 Load the concentrate on a column chromatography (CH2Cl2/Methanol:
9/1) to yield cCyc as a light brown powder (640mg, 70% yield). cCyc
can be obtained as yellowish crystals after recrystallization from iso-
propyl ether (m.p.: 116–117°C) (Sinha, Neveu, Gagey, Aujard,
Benbrahim-Bouzidi, et al., 2010a; Sinha, Neveu, Gagey, Aujard, Le
Saux, Rampon, et al., 2010b).
3.6 Analyze product with NMR to yield: 1H NMR (400MHz, CDCl3, δ):
7.76 (s, 1H), 7.35 (s, 1H), 7.04 (AA0XX0, 2H, J¼9Hz), 7.00 (AA0XX0,
2H, J¼9Hz), 6.90 (AA0XX0, 2H, J¼9Hz), 6.82 (AA0XX0, 2H,
J¼9Hz), 5.47 (s, 2H), 4.03 (t, 2H, J¼6Hz), 3.96 (s, 3H), 3.94
(s, 3H), 2.70 (t, 2H, J¼6Hz), 2.32 (s, 6 H), 2.23 (m, 4H), 1.58
(m, 6H); 13C NMR (100MHz, CDCl3, δ): 157.0, 156.2, 153.8,
147.7, 138.9, 138.5, 136.8, 137.7, 133.2, 131.0 (2C), 130.7 (2C),
129.6, 114.3 (2C), 113.8 (2C), 109.4, 107.9, 67.0, 65.8, 58.3, 56.3,
56.3, 45.8 (2C), 32.4 (2C), 28.6 (2C), 26.8. elemental analysis calcd
(%) for C32H38N2O6 (546.66): C 70.32, H 7.00, N 5.12; found:
C 70.11, H 7.24, N 5.02.
5. Calibration of caged-cyclofen photo-activation
The liberation of the cyclofen-OH inducer necessitates photo-
activation, which involves light absorption by the caged precursor cCyc
followed by effective bond breakage leading to cyclofen-OH and the
nitrosobenzaldehyde by-product from the caging moiety (Zhang et al.,
2018). The corresponding process can be written as the chemical reaction (1).
cCyc + nhν ! cyclofen-OH + by-product
(1)

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where n hν (n¼1 or 2 with one- and two-photon excitation) designates the
photon(s) effectively driving the reaction. These photons are provided
by a light source and the experimentalist should provide enough photons
to exhaust the conversion of the caged precursor but without generating
detrimental side-effects on the biological sample. Therefore one should
always characterize a light source to achieve and optimize any photo-
activation experiment. Several criteria should be taken into account.
5.1 The wavelength range of the light source
To be effective, the photoactivation wavelength should be within the
absorption range of the caged precursor or more precisely of the caging moi-
ety. With respect to cCyc, this information has been provided in (Aujard
et al., 2006) for one- and two-photon excitation. The wavelength of the
light source should thus be comprised between 325 and 425nm with
one-photon excitation and between 700 and 800nm with two-photon exci-
tation. Possible relevant light sources are benchtop UV lamps (generally used
to read out thin layer chromatography plates in the chemical laboratory; to
photoactivate biological samples over large areas), or light sources installed
on microscopes. With one-photon excitation, light sources delivering pho-
tons at 405nm are usually available but some setups provide access to exci-
tation wavelengths down to 365nm (as used for DAPI imaging). With two-
photon excitation, the microscope should be equipped with a femtosecond
pulsed laser source delivering 150–200fs laser pulses around 750nm.
5.2 The power range of the light source
Once the light source has been identified, it needs to be calibrated for
the photon flux it generates at the biological sample. A light source is gen-
erally characterized by its power P. This power can be directly measured
with a power-meter when it is available (e.g., from Thorlabs). The sensi-
tive element of the power-meter should be located at the position of
the biological sample (e.g., at the front of a microscope objective). Alter-
natively, one can rely on a calibration exploiting the photoconversion
of a reference (see below).
5.3 Tuning the illumination for uncaging experiments
With one-photon excitation, the power P (in Watt or Joulesꢀ1) first has to
be transformed into a photon flux J (in molmꢀ2 sꢀ1). Knowing the

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Fatima Hamouri et al.
frequency ν (expressed in Hz) of the light source (or its wavelength: λ ¼c/ν)
and the surface S (expressed in m2) of the illuminated zone, one has
J ¼ P=NAhνS
(2)
In Eq.(2), the denominator is the energy of 1mol of photon and
it involves the Avogadro number NA (¼ 6ꢁ1023 photons/mole) the
Planck constant h, and the light frequency ν (for λ ¼350nm: h ν ¼h
c/λ ¼5.68ꢁ10ꢀ19 J). Then the rate constant k of the uncaging reaction
(1) (expressed in sꢀ1) can be calculated using Eq.(3)
k ¼ εðλÞϕJ
(3)
where ε(λ) is the molar absorption coefficient of the caging moiety at wave-
length λꢀ1(expressed in m2 molꢀ1; it can be calculated by writing ε(λ)
[m2 mol ]¼0.1 ε0(λ) [Mꢀ1 cmꢀ1]) and ϕ is the quantum yield of uncaging,
which measures the probability of releasing the caged substrate once light has
been absorbed by the caged precursor. For cCyc, one has ε0(350nm)¼
6000 Mꢀ1 cmꢀ1, ε0(400nm)¼2000 Mꢀ1 cmꢀ1, and ϕ¼6ꢁ10ꢀ3. To fully
achieve uncaging of cCyc, one should illuminate the sample for a typical
duration of about 5/k (in seconds). With a typical laser excitation power
P¼10μW (at the focal plane of the microscope objective) focused on
a spot of diameter 10μm (the minimal spot size should be commensurate
with the typical size of the cell cross-section): k(350nm) ꢂ1sꢀ1. Hence
illumination of a few seconds is enough to achieve full uncaging of cCyc
in the few cells along the optical path.
Eq.(3) can also be used to calibrate the light source when no power-
meter is available. One should use a photoactive reference (characterized
by εref (λ) and ϕ ref) the fluorescence of which strongly changes upon
illumination. In a first step, the light source is applied on a sample containing
the photoactive reference while recording its fluorescence decay. The
fluorescence decay is then fitted to an exponential form:
ꢂ
ꢀ
ꢁꢃ
IFðtÞ ¼ IFð0Þ + ½IFð∞ÞꢀIFð0Þꢃ 1ꢀ exp ꢀkref t
(4)
in order to retrieve kref. In Eq.(4), IF(t), IF(0), and IF(∞) designate the
recorded fluorescence intensity at time t, time zero, or at steady-state (infinite
time). The photon flux is then deduced from Eq. (3):
kref
J ¼
(5)
εref ðλÞϕref

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Optical control of protein activity
13
In particular, Quꢀerard et al. (2017) describe how fixed cells expressing
genetically encoded reversibly photo-switching proteins can be used as ref-
erences for calibrating light fluxes at 405nm at the focal plane of microscope
objectives.
With two-photon excitation, the uncaging rate constant within the exci-
tation volume Vexc can be expressed (Sinha, Neveu, Gagey, Aujard,
Benbrahim-Bouzidi, et al., 2010a) as:
!
2
T
P
kexc ¼ 0:737δ
(6)
u τP πhνω2xy
where δu is the two-photon uncaging cross section of the caging group at the
excitation wavelength λ (for the 4,5-dimethoxy-2-nitrobenzyl caging group
δu(750nm)¼2ꢁ10ꢀ52 cm4 sphotonꢀ1 ¼2 10ꢀ60 m4 sphotonꢀ1). T is the
period of the laser pulses (typically 10ns for a pulse rate of 100MHz) of inci-
dent power at the focal point P, duration τP (typically 200fs) and beam waist
ωxy. P can be measured with a power-meter, T and τP are specific to the laser
source and are provided by the manufacturer. ωxy can be measured by fluo-
rescence correlation spectroscopy (using a reference fluorophore) or alterna-
tively estimated (Zipfel, Williams, & Webb, 2003) using the numerical
aperture NA of the microscope objective (typically NA ꢂ1):
0:23λ
ωxy ꢄ
(7)
NA
For a typical power P ¼10mW, kexc ꢄ2 104 sꢀ1. When uncaging with
two-photon excitation is performed within a single cell of volume V, the
rate constant for uncaging within the cell is given by:
Vexc
k ¼
kexc
(8)
V
where Vexc ꢄ5.57 ω2xy ωz can be measured by FCS or alternatively estimated
from the wavelength λ, the numerical aperture NA and the refractive index n
of the medium, (Zipfel et al., 2003; n¼ 1.33 for water) with:
ꢄ
ꢅ
1
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ωz ꢄ 0:38λ
ꢄ 0:8λ
(9)
nꢀ n2 ꢀNA2
For the values quoted above Vexc ꢄ0.1 μm3. Thus for a typical cell of
volume V¼100–1000 μm3, the uncaging rate k¼ 2–20sꢀ1. To achieve full
uncaging of cCyc in one cell, one should illuminate the sample for a dura-
tion of about 5/k, typically about 1s.

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Fatima Hamouri et al.
6. One- and two-photon activation in zebrafish
Based on the previous considerations and estimates one should first test
the uncaging of cCyc in an appropriate line of zebrafish, such as Tg(ef1a:
loxP-GFP-loxP-dsRed2), available from Zfin (line ne2466tg) where gene
activation is readily observed as a change in fluorescence (from the green
fluorescence of GFP to the red fluorescence of dsRed). Injecting mRNA
of Cre-ERT (about 3pg) at the one cell stage (or crossing with a Cre-
ERT expressing transgenic line such as cmr1tg from Zfin) allows for activa-
tion of the recombinase upon uncaging of cCyc (at a typical concentration
of 4–6 μM), followed by excision of the GFP gene and expression of dsRed.
Measuring the intensity of the illumination source (UV lamp, UV or two-
photon laser) at the position of the embryo allows for a quick estimate of the
time required to uncage cCyc and observe the appearance of dsRed in the
illuminated embryos, see Fig. 2. Illumination with various time-spans
around that estimate allows one to determine the optimal illumination
parameters for uncaging of cCyc and gene activation which may then be
used in other contexts (see below).
dsRed
IoxP
ef1a
GFP
IoxP
STOP
A
B
C
D
Fig. 2 Global and local activation of Cre-ERT by cCyc uncaging. 3ng of Cre-ERT mRNA
was injected at the one cell stage in a transgenic line Tg(ef1a:loxP-GFP-loxP-dsRed2) and
incubated 1h in 3 μM of cCyc. (A) image of the embryo in the GFP channel prior to illu-
mination. (B) Expression of dsRed following transfer of the embryos into fresh embryo
medium (no cCyc), followed by global 5min uncaging with a UV lamp (intensity:
J ¼2.4ꢁ10–3 mol/m2 s). (C) same as in (B) but with 30s local illumination using the
Hg-lamp of the microscope to uncage cCyc in the embryo head and turn on expression
of dsRed in the head only. (D) same as before but illuminating at a diffraction-limited
fixed spot for 10s with a two-photon laser (200fs, 76MHz, 750nm; Mira-Coherent) to
uncage cCyc in a single cell of an eye at 10hpf (and observing the embryo at 48hpf,
where the initial activated cell divided into two cells).

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Optical control of protein activity
15
6.1 Equipment
UV lamp (365nm, 6W, Fischer-Bioblock) for global activation.
Microscope with a 25ꢁ (or 40ꢁ) water immersion objective (Leica or
Olympus) equipped for local activation with a Hg lamp or a UV laser line
(from a 365nm CW; Crystal Laser).
Two-photon laser source (200fs, 76MHz, 750nm, provided by a mode-
locked Ti-Sapphire laser, Mira-Coherent) illuminating the embryo via a
25ꢁ (or 40ꢁ) microscope objective for two-photon local activation.
Power meter (NOVA II Laser Measurement Instruments or Thorlabs
S170C Microscope Slide Power Sensor) for measurement of the
illumination power.
6.2 Chemicals
Embryo medium (EM): NaCl (29.4g/100mL), KCl (1.27g/100mL),
CaCl2ꢅ2H2O (4.85g/100mL), and MgSO4ꢅ7H2O (8.13g/100mL)
(The Zebrafish Book, (Westerfield, 2007)).
Embryo medium as above supplemented with 3–6 μM of cCyc.
6.3 Biological materials
Transgenic zebrafish embryos, allowing for expression of a fluorescent pro-
tein marker upon photo-activation (see below the protocols for the engi-
neering of such embryos).
6.4 Protocol
Using a power-meter measure the illumination intensity of the source (UV
lamp or laser) at the position where the embryos will be placed. Estimate, with
Eqs. (5) or (8), the uncaging rate and illumination time required for the exper-
iment. Incubate (preferably dechorionated) transgenic embryos (at few hpf
up to 2dpf ) in EM supplied with 3–6 μM cCyc for 1–2h away from light. Just
before photo-activation transfer the embryo to fresh EM and illuminate them
witha UV or two-photonlightsource. If global activationissought, illuminate
the embryos for about 5min. With a UV lamp positioned a few cm above a
Petri dish holding them. If local photo-activation is sought place the embryo
under a water immersion microscope objective and illuminate the desired
area with a UV laser or the UV lamp (ꢂ365nm) of a microscope for a
timespan estimated from Eq.(5) (typically a few seconds). If activation in a
single cell is sought illuminate the desired cell with a focused two-photon laser
(at ꢂ750nm) for a timespan estimated from Eq.(8) (about 1s).
Let the embryo grow for a few hours to a few days before observing the acti-
vated fluorescent protein marker using a fluorescent microscope or binocular.

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Fatima Hamouri et al.
7. Transient and permanent gene activation
A quantitative description of the molecular networks that sustain mor-
phogenesis is one of the challenges of developmental biology. In addition
the induction of tumors (by turning on some oncogenes and/or turning
off tumor suppressor genes) in specific cells of a growing animal could illu-
minate the mechanisms of oncogenesis. These challenges require the precise
spatial and temporal control over the expression of the target gene(s). The
caged-cyclofen-OH approach described above allows one to do that in
transgenic lines expressing the appropriate plasmid construct, see Fig. 3.
To achieve transient genetic activation, we use a fusion between the ERT
domain and a transcription factor (Gal4) which is activated by cyclofen-OH
uncaging (Feng et al., 2017). A transgenic line is engineered which expresses
that fusion protein ubiquitously. A second line is engineered which expresses
the desired gene under control of a UAS promoter. By crossing those lines, an
embryo is generated in which local photo-activation of cCyc, leads to release
of Gal-ERT from its chaperone complex and activation of the gene under
the UAS promoter. As the released cyclofen-OH diffuses out of the cell,
the Gal4-ERT is re-sequestered by cytoplasmic chaperones and the tran-
scription activity is shut off (unless cCyc is globally uncaged and left in the
medium in which case permanent global activation can be achieved).
To achieve local and permanent genetic activation, we fuse the ERT
domain with a Cre recombinase. A transgenic line is engineered which
expresses that fusion protein ubiquitously. A second line is engineered
which expresses the desired gene downstream of an insertion marker and
a stop codon flanked by loxP sites and under a ubiquitous promoter. By
crossing those lines, an embryo is generated in which local photo-activation
of cCyc, leads to the release of Cre-ERT from its chaperone complex, the
excision of the DNA fragment flanked by the loxP sites and the permanent
activation of the desired gene by the ubiquitous promoter. In contrast with
the cyclofen-induced activation of Gal-ERT which is reversible, out-
diffusion of the released cyclofen-OH does not reverse the Cre-induced
excision of the DNA fragment between loxP sites.
In the following we will briefly describe how the required transgenic
zebrafish lines can be engineered. More details can be found in the available
literature (Westerfield, 2007).
7.1 Equipment
Micro injection machine (FemtoJet, Eppendorf )

A
B
C
Sequestered
Gal-ERT
caged
cyclofen
20X
uncaging
laser activation
UV/
2-photon
Gal-ERT
Active
t
transient activation of MyGene
Eos ubi INS UAS
T2A CFP
MyGene
D
E
F
Cre-ERT Sequestered
caged
uncaging
20X
UV/
2-photon
cyclofen
laser activation
t
Cre-ERT
Active
permanent activation of MyGene
Ubi
T2A CFP
MyGene
loxp
loxp
T2A CFP
Eos fp stop MyGene
Ubi
Fig. 3 See legend on next page.

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Fatima Hamouri et al.
7.2 Chemicals
Embryo medium: NaCl (29.4g/100mL), KCl (1.27g/100mL),
CaCl2ꢅ2H2O (4.85g/100mL), and MgSO4ꢅ7H2O (8.13g/100mL)
(The Zebrafish Book, (Westerfield, 2007))
7.3 Biological materials
Zebrafish embryos
The host Tol2 (Kawakami, 2007) plasmid pT24-uas:Prot-T2A-cfp-ubi:
Eos prepared with standard molecular biology protocols (not detailed
here) which contains the homologous cDNA sequence of the gene of
interest (Prot) from Danio rerio.
The tol2 transposase mRNA synthesized by in vitro transcription from
the linearized template pCS2tol2 digested with NotI using the
mMESSAGE mMACHINE SP6 Kit (Ambion).
7.4 Protocol
Generation of the double transgenic line ubi:Gal4-ERT (or ubi:Cre-ERT);
uas:Prot-T2A-cfp (or ubi:loxP-Eos-loxP-Prot-T2A-cfp). Prot stands for
the gene of interest (in the example below Fgf8).
For the injection method please refer to (Rosen, Sweeney, & Mably,
2009). For injection, approximately1 nL of a solution (10ng/μL) of the plas-
mid pT24-uas:Prot-T2A-cfp; ubi:Eos (or ubi:loxP-Eos-loxP-Prot-T2A-cfp)
was injected into the cytoplasm of one-cell stage eggs together with the tol2
transposase mRNA (100ng/μL) in 0.2M KCl. Potential F0 fish were iden-
tified via the mosaic expression of EosFP at 3–5 days post fertilization and
were raised to sexual maturity (3 months). The F1-progeny obtained from
the cross of the putative F0 carrier with WT animals were screened for
germ-lineage transmission via the fluorescence of the EosFP marker.
Fig. 3 Transient and permanent activation of a gene of interest (MyGene). (A) Using UV
or two-photon illumination, a Gal4-ERT fusion is released (B) and transiently activates a
gene of interest under a UAS promoter. A fluorescent CFP protein is used as a photo-
activation marker while an EosFP serves as an insertion marker. The genetic construct
shown in (B) is injected as a plasmid at one cell stage to generate a mosaic embryo
and/or engineer a transgenic line. (C) Due to diffusion of the released cyclofen-OH,
the activation of transcription lasts a few hours, but the translated protein may linger
on for a few days. (D) Using similar illumination on a Cre-ERT fusion may permanently
label a single cell (shown here in red) since out-diffusion of the released cyclofen-OH will
not reverse the Cre-induced excision of a stop codon and its insertion marker, putting
the expression of MyGene under a ubiquitous promoter (E). This approach can be used
to permanently activate a gene of interest (F).

ARTICLE IN PRESS
Optical control of protein activity
19
The ubi:Gal4-ERT (or ubi:Cre-ERT) fish were obtained in the same
way using CFP as a marker expressed in the eye of the embryos 3 days after
their birth.
A double transgenic line ubi:Gal4ERT and uas:Prot-T2A-cfp;ubi:Eos
(or ubi:Cre-ERT and ubi:loxP-Eos-loxP-Prot-T2A-cfp) was created by
crossing the ubi:Gal4-ERT (ubi:Cre-ERT) fish and the uas:Prot-T2A-
cfp;ubi:Eos (ubi:loxP-Eos-loxP-Prot-T2A-cfp) fish, and screened for blue
eyes and green body, see Fig. 4.
Fig. 4 Generation of a double transgenic line: the plasmid of interest (here uas:fgf8-T2A-
cfp; ubi:Eos) is injected with the mRNA of the tol2 transposase at the one cell stage. F0
embryos are identified by the mosaic expression of EosFP at 3–5dpf, they are raised to
sexual maturity, crossed with WT embryos and the F1 progeny selected by the uniform
expression of EosFP. The F1 progeny is crossed with Gal4-ERT embryos expressing CFP
under a crystallin promoter. Double transgenic embryos are identified by their uniform
EosFP expression and CFP expressing crystalline.

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Fatima Hamouri et al.
8. Conclusions
The photo-activation of caged-cyclofen-OH (cCyc) has been used to
control the activity of a variety of proteins (the Gal4 transcription factor,
Cre-recombinase, caspase, GFP, etc.) fused to the ERT domain of the estro-
gen receptor. Activating a transcription factor has been used to transiently
turn on a target gene, while the activation of a Cre-ERT recombinase
has been used to permanently turn on a target gene (behind a stop codon
flanked by loxP sites). The uncaging of cyclofen and the consequent activa-
tion of the protein fused to the ERT receptor can be performed within a
time-span of seconds to minutes globally (using a UV lamp) or locally
(within a few cells using one or two-photon excitation). This approach is
versatile and easy to implement in various organisms (zebrafish, mice, etc.).
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