Dynamic multicolor protein labeling in living cells

Article abstract of DOI:10.1039/c7sc01364g

Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14 kDa protein tag giving a bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene rhodanine (HMBR). H

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DOI: 10.1039/C7SC01364G.
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Dynamic multicolor protein labeling in living cells
Chenge Lia,b, Marie-Aude Plamonta,b, Hanna L. Sladitschekc, Vanessa Rodriguesa,b, Isabelle
Aujarda,b, Pierre Neveuc, Thomas Le Sauxa,b, Ludovic Julliena,b,* and Arnaud Gautiera,b,*
Received 00th January 20xx,
Accepted 00th January 20xx
Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14-kDa protein tag giving a
bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene
rhodanine (HMBR). Here, we report a collection of fluorogens enabling to tune the fluorescence color of FAST from green-
yellow to orange and red. Beyond allowing multicolor imaging of FAST-tagged proteins in live cells, these fluorogens
enable dynamic color switching because of FAST’s reversible labeling. This unprecedented behavior allows selective
detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color cross-
correlation, opening exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.
DOI: 10.1039/x0xx00000x
www.rsc.org/
particular by a high off-rate constant enabling rapid complex
dissociation upon fluorogen removal. FAST is a 14-kDa variant
of the photoactive yellow protein (PYP) that was engineered to
Introduction
Imaging the dynamics of proteins in living cells is essential for
deciphering biological processes. A common strategy for
imaging proteins is to fuse them to peptide or protein
sequences that provide fluorescence, such as autofluorescent
proteins1,2 or self-labeling tags3-5 (such as SNAP-tag6,7/CLIP-tag8
and Halo-tag9) that can be labeled specifically with chemical
probes. A related strategy is to use protein tags that can
generate fluorescence upon interaction and activation of
fluorogenic compounds (so-called fluorogens)10,11. Fluorogen-
activating proteins (FAPs) able to specifically bind fluorogens
such as malachite green and thiazole orange and activate their
fluorescence have been developed from single-chain
specifically bind and activate the fluorescence of the
fluorogenic dye HMBR. HMBR undergoes two spectroscopic
changes upon binding insuring high labelling selectivity. While
unbound HMBR mainly deexcites from the excited state
through non-radiative processes, bound fluorogen undergoes
a very large increase of fluorescence quantum yield because of
immobilization within FAST cavity. Moreover, FAST binds
HMBR in its anionic form, minor form in solution at pH 7.4,
leading to an apparent 80 nm absorption red shift. FAST is thus
an acronym that echoes back to its fast exchange dynamics
and stands for Fluorescence-Activating and absorption Shifting
Tag, highlighting the two spectroscopic changes ensuring
labeling selectivity.
antibodies12,13
.
Because fluorogens exhibit very low
fluorescence background in cells, proteins can be imaged with
high contrast without the need for washing the fluorogen
excess.
The complex FAST:HMBR fluoresces green-yellow light (λem
= 540 nm) upon blue light excitation. In order to provide
investigators with the possibility to tune the spectral
properties of FAST in function of the imaging conditions, we
sought for alternative fluorogens able to bind FAST and to
form complexes with novel fluorescence properties. We
explored if changing the structure of HMBR could shift both
the excitation and the emission of the tag:fluorogen complex
to the red edge of the visible spectrum (Figure 1A). We report
The concept of fluorogenic reporter was recently pushed
one step further with the design of a tunable genetically
encodable reporter called Yellow Fluorescence-Activating and
absorption-Shifting Tag (Y-FAST, here-after called FAST) that
not only can be switched on by addition of a fluorogenic
ligand, but can also be switched off rapidly by removal of the
fluorogen through washing14. This fluorescence on/off switch
behavior was made possible by designing a system displaying
dynamic reversible fluorogen binding, characterized in
a
collection of fluorogen analogs with various spectral
properties. We present in particular HBR-3,5DOM (4-hydroxy-
3,5-dimethoxybenzylidene rhodanine), a new fluorogen that
forms a tight complex with FAST fluorescing red light under
green light excitation. This allows choosing FAST color by
choosing the appropriate fluorogen, providing an experimental
versatility that is not encountered with autofluorescent
proteins. We also show that we could exploit dynamic
fluorogen binding to switch color from red to green or green to
red by simply changing the fluorogen used. The ability to
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Figure 1. Multicolor imaging of FAST-tagged proteins. (A) The fluorescence color of FAST can be adapted by changing fluorogen (POI: protein of interest). (B) Structures of the
fluorogens discussed in this study: HMBR (X = H, Y = Me, Z = H), HBR-3E (X = H, Y = Et, Z = H), HBR-2,5DM (X = Me, Y = H, Z = Me), HBR-3,5DM (X = H, Y = Me, Z = Me), HBR-3OM (X =
H, Y = OMe, Z = H), HBR-3OE (X = H, Y = OEt, Z = H), HBR-3,5DOM (X = H, Y = OMe, Z = OMe). (C-E) Absorption (dashed line, left axis) and emission (solid line, right axis) spectra of
the free fluorogens (grey line) and FAST:fluorogen complexes (colored line) in pH 7.4 phosphate buffer saline at 25°C (C: HMBR, D: HBR-3,5DM, E: HBR-3,5DOM). Note that the
shoulder at 480 nm on the absorption spectra of the free fluorogens correspond to anionic fluorogens present in little amounts at pH 7.4 (See also Figure S1 an Table S1).
Table 1. Physicochemical properties of FAST:fluorogen complexes in PBS pH 7.4. Abbreviations are as follows: λabs, wavelength of maximal absorption; λem, wavelength of
maximal emission; ε, molar absorption coefficient at λabs
; ꢀλabs = λabs,bound – λabs,unbound, absorption red-shift upon FAST binding; φ, fluorescence quantum yield; Fbound/Funbound,
fluorescence activation upon FAST binding; Brightness = ε × φ; KD, thermodynamic dissociation constant. Spectroscopic data of mCherry15 are given for comparison.
Complex
λabs
(nm)
481
481
494
499
494
497
518
ꢀλabs
(nm)
80
81
88
98
91
94
112
λem
(nm)
540
543
552
562
561
562
600
ε
φ
Fbound/Funbound
Brightness
KD
(mM–1cm–1
)
(%)
23
13
29
49
36
17
31
(ꢁM)
0.13
0.08
0.008
0.08
0.31
0.27
0.97
FAST:HMBR
FAST:HBR-3E
45
50
50
48
40
41
39
300
300
430
660
350
230
220
10,300
6,500
14,500
23,500
14,400
7,000
FAST:HBR-2,5DM
FAST:HBR-3,5DM
FAST:HBR-3OM
FAST:HBR-3OE
FAST:HBR-3,5DOM
12,000
mCherry
587
610
72
22
15,800
switch color dynamically allowed us furthermore to develop a major role on the (im)mobility of the fluorogen within FAST
new strategy to selectively image FAST-tagged proteins in cavity. In addition, the spectral properties of push-pull systems
spectrally crowded environment. Spectral overlap between such as HMBR and its analogs are directly related to the
fluorophores limits most experiments to the simultaneous electron donating or withdrawing ability of the donor and
observation of three or four targets, preventing thus highly acceptor groups, as they affect the energy of molecular
multiplexed imaging. Here, we show that one can overcome orbitals. Changing the electron-donating properties of the
spectral crowding using kinetic filtering based on dynamic phenol moiety through adequate substitution can thus affect
color switching and two-color cross-correlation.
spectroscopic properties. In particular, here we can anticipate
that the stronger the electron donating ability of the donor
part, the more red-shifted the absorption and emission. We
thus examined the role of substituents on the aromatic ring.
Figure 1B gives the structure of different analogs obtained by
condensation of the rhodanine heterocycle with appropriate
aromatic aldehydes. The described fluorogens bound tightly to
FAST with dissociation constants below 1 μM, enabling to
anticipate efficient and selective FAST labeling (Table 1). In
addition, all the fluorogenic analogs underwent very large
Results and Discussion
HMBR fluorogen is composed of an electron-donating phenol
ring conjugated to an electron-withdrawing rhodanine
heterocycle. This push-pull structure deexcites non-radiatively
in solution but relax radiatively to the ground state within FAST
cavity. Substituents on the aromatic ring are thus a priori key
determinants of the fluorescence efficiency, as they can play a
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fluorescence increase (from 200 up to 600 fold) and yield of 49 %, and an overall brightness 2.5-fold higher than
absorption red-shift (between 80 and 110 nm) upon FAST the original FAST:HMBR complex. The greatest spectral shift
binding (Table 1). This latter absorption red-shift is due to the was observed with HBR-3,5DOM, which bears two electron-
binding-induced deprotonation of the fluorogens, which are all donating methoxy substituents in ortho position of the
mainly protonated in solution at physiological pH (Table S1 and hydroxyl group. The resulting complex, FAST:HBR-3,5DOM
Figure S1).
displayed a 40-nm red-shifted absorption (λabs = 518 nm) and a
We observed various spectroscopic behaviors. HBR-2,5DM, 60-nm red-shifted emission (λem = 600 nm) (see also Figure 1E).
HBR-3,5DM, HBR-3OM and HBR-3OE formed complexes with These spectral modifications shift the absorption to the green
~15-20 nm red-shifted absorption (λabs from 494 to 499 nm) and the emission to the red, providing thus a new color for
and ~10-20 nm red-shifted emission (λem from 552 to 562 nm) fluorescence imaging. Remarkably, FAST:HBR-3,5DOM is as
relative to FAST:HMBR (see also Figure 1C,D). Of particular bright as the red fluorescent protein mCherry.
interest, FAST:HBR-3,5DM exhibits a fluorescence quantum
We next examined if our new fluorogenic analogs could
efficiently label FAST-tagged proteins in living cells. We first
verified that they were non-toxic for mammalian cells (Figure
S2). Then, we expressed in HeLa cells (i) FAST, (ii) FAST fused to
histone H2B for nuclear localization, and (iii) FAST fused to the
mitochondrial targeting sequence (MTS) from subunit VIII of
human cytochrome c oxidase. Cells were treated with media
containing 5 μM of fluorogen and imaged by confocal
microscopy (Figure 2A). We visualized the various FAST fusions
in their expected localization, in agreement with specific
fluorogenic labeling.
Given that the fluorescence color of FAST can be either
green-yellow (with HMBR) or red (with HBR-3,5DOM), we next
showed that FAST could be imaged in presence of either the
red fluorescent protein mCherry or the green fluorescent
protein EGFP by adding the complementary fluorogen. We
expressed FAST fused to H2B for nuclear targeting together
with either mCherry or EGFP fused to Lyn N-terminal domain
(lyn) for membrane anchoring. Cells co-expressing FAST and
mCherry fusions were treated with media containing 5 μM of
HMBR, while cells co-expressing FAST and EGFP fusions were
treated with media containing 5 μM of HBR-3,5DOM. By using
a dual 488/543 nm excitation and variable spectral detection,
FAST:HMBR could be separated spectrally from mCherry, while
FAST:HBR-3,5DOM could be distinguished from EGFP (Figure
2B), demonstrating the versatility of FAST and the possibility to
easily adapt its color to the spectral conditions of the
experiment. An additional interesting feature of FAST:HBR-
3,5DOM is its high Stokes shift (82 nm), which allowed us to
image FAST-tagged proteins together with EGFP fusions using
a single 488 nm excitation source and two separate spectral
regions (Figure S3), opening interesting prospects for live cell
imaging as single excitation simplifies imaging protocols,
results in
a gain of temporal resolution and reduces
phototoxicity.
A unique feature that distinguishes FAST from other
labeling techniques is the possibility to rapidly reverse the
labeling by washing the fluorogen away. We thus verified that
we kept this property when using the new fluorogenic analogs.
We expressed FAST fused to histone H2B in mammalian cells.
Iterative labeling/washing with the new fluorogens allowed us
to demonstrate that FAST conserved its ability to be switched
on and off at will by fluorogen addition and removal (Figure
3A). Moreover, as HMBR and HBR-3,5DOM give complexes of
Figure 2. Multicolor labeling of FAST-tagged proteins in live cells. (A) Confocal
micrographs of HeLa cells expressing cytoplasmic FAST, nuclear H2B-FAST and
mitochondrial MTS-FAST and labeled with 5 ꢁM of HBR-3,5DM, HBR-3OM and HBR-
3,5DOM (HBR-3OM, HBR-3,5DM: Ex/Em 488/493-797 nm; HBR-3,5DOM: Ex/Em
514/519-797 nm). (B) Confocal micrographs of live HeLa cells co-expressing lyn-
mCherry/H2B-FAST (Top) and lyn-EGFP/H2B-FAST (Bottom) labeled with 5 ꢁM HMBR
or HBR-3,5DOM respectively (Green channel Ex/Em 488/493–575 nm; Red channel
Ex/Em 543/578–797 nm). Scale bars 10 ꢁm.
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Figure 3. Reversible FAST labeling. (A) Confocal micrographs of an HeLa cell expressing nuclear H2B-FAST sequentially labeled with HBR-3,5DOM (red), HBR-3,5DM (cyan), HBR-
3OM (green) and HMBR (yellow) (HBR-3OM, HBR-3,5DM, HMBR: Ex/Em 488/493-797 nm; HBR-3,5DOM: Ex/Em 514/519-797 nm). Labeling was reversed by removing the
fluorogen-containing solutions followed by a wash with pH 7.4 PBS. (B) Confocal micrographs of an HeLa cell expressing nuclear H2B-FAST sequentially labeled with HMBR and
HBR-3,5DOM (Green channel: Ex/Em 488/493-550 nm; Red channel: Ex/Em 543/600-797 nm). The color switch was performed by first washing away the excess of HMBR with pH
7.4 PBS, and then addition of the HBR-3,5DOM solution. (C) Time series of a live HeLa cell expressing H2B-FAST labeled with HBR-3,5DOM upon replacement of the
medium with an HMBR solution (Single excitation at 488 nm; HMBR emission channel 493-538 nm; HBR-3,5-DOM emission channel 649-797 nm). HMBR was added at
t = 0 s. See also Movie S1. (A-C) The concentration of the fluorogen solutions was 5 ꢁM. Cells were grown in a minifluidic channel enabling easy solution replacement
.
Scale bars 10 ꢁm.
Figure 4. Selective imaging. (A) Measuring the degree of anti-correlation of the green and red fluorescence signals upon dynamic color switching by two-color cross-correlation is
an effective method to extract quantitatively the signal of FAST in spectrally crowded environment containing green and red fluorescent species. (B) Micrographs show live HeLa
cells co-expressing three proteins: lyn-EGFP, MTS-mCherry and H2B-FAST. Lyn-EGFP (green) and MTS-mCherry (red) images are confocal micrographs obtained in absence of
fluorogen, while H2B-FAST (blue) image results from two-color cross-correlation upon HBR-3,5DOM-to-HMBR exchange (Green channel Ex/Em 488/493–575 nm; Red channel
Ex/Em 543/578–797 nm). Figure S4 and Movie S2 show HBR-3,5DOM-to-HMBR exchange. Scale bars 10 ꢁm.
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different fluorescence colors, FAST could be switched from
one color to the other by washing away one fluorogen and
adding the other one (Figure 3B). Color switch could be further
done in a dynamic fashion in few seconds simply by changing
the medium content (Figure 3C and Movie S1).
Conclusion
This study presents fluorogenic dyes expanding the spectral
properties of FAST to the red edge of the visible spectrum.
Originally, FAST emitted green-yellow light upon blue light
excitation when using HMBR as fluorogenic partner. Here, we
show that FAST can fluoresce red light upon green light
excitation using HBR-3,5DOM instead of HMBR. With FAST, it is
thus now possible to adapt the tag color to the spectral
conditions of the experiment without changing the protein tag,
providing an experimental versatility that is not encountered
with autofluorescent proteins. In addition, the ability to
change the color by changing the dye renders possible to
switch color in a single experiment20,21. In this work, we show
that the fast exchange dynamics characterizing FAST and the
high permeability of the designed fluorogens allow color
swapping on the second timescale. We used this unique kinetic
signature to develop a method for the selective imaging of
intracellular FAST-tagged proteins in spectrally crowded
environments. A major obstacle to multiple observations is the
spectral overlap between fluorophores, which limits most
experiments to the observation of three or four targets. By
measuring the degree of temporal anti-correlation of the two
fluorescent states of FAST during dynamic color switching, we
successfully imaged FAST-tagged proteins in presence of green
and red fluorescent species despite spectral crowding. Imaging
three proteins with only two observation channels is a
promising step towards overcoming spectral crowding and
opens exciting prospects for pushing the frontiers of
multiplexed imaging.
The ability to switch color dynamically by fluorogen
exchange provides FAST with a unique signature that can be
exploited to push multiplexed imaging. Modern biology needs
to be able to observe multiple targets (> 10-50) to study
biological processes in all their complexity16. Despite the large
collection of probes and biosensors available, distinguishing a
large number of labels remains one of the biggest challenges in
imaging. A major obstacle to multiple observations is spectral
overlap between fluorophores, which limits most experiments
to the observation of three or four targets. New approaches
for highly multiplexed imaging relying on other parameters
than spectral discrimination are thus essential if one wants to
be able to better analyze multiparametric cellular processes.
We thus explored if the unique signature provided by dynamic
color switching could provide a solution for overcoming
spectral crowding. Upon dynamic color switching, HMBR and
HBR-3,5DOM are fluorescent in a temporally anti-correlated
manner, as they both bind the same tag. Kinetic information
has been previously exploited to image selectively reactive
fluorescent species in presence of high fluorescence
background17-19. We thus anticipated that measuring the
degree of anti-correlation of the green and red fluorescence
signals should enable to extract FAST contribution in presence
of green and red fluorescent species – even though spectral
discrimination is impossible in these conditions – exploiting the
fact that only the green and red signals originating from FAST
would interconvert (Figure 4A). To evaluate this idea, we
imaged by time-lapse confocal microscopy HeLa cells co-
expressing FAST (fused to histone H2B), mCherry (fused to a
mitochondrial targeting sequence (MTS)) and EGFP (fused to
lyn for membrane anchoring) upon dynamic fluorogen
exchange (Figure 4B, Figure S4A,B and Movie S2). Cross-
correlation of the temporal evolution of the green and red
fluorescence signals allowed us to extract selectively the
contribution of H2B-FAST and fully eliminate the signals of
mitochondrial mCherry and membrane-targeted EGFP (Figure
Acknowledgements
This work has been supported by the Agence National de la
Recherche (ANR-14-CE09-0002- 01), a PhD grant (C. L.) from
Region Ile-de-France in the framework of DIM NanoK, PSL
Research University (project IMRESOV), France BioImaging and
the Equipex Morphoscope 2.
Materials and Methods
4B). Note that we chose different localizations for FAST, Cherry Chemical synthesis
— general information Commercially
and EGFP to assign proteins without any ambiguity in this available reagents were used as starting materials without
proof-of-principle experiment, but the approach is generic and further purification. NMR spectra were recorded on a AC
1
can of course be expanded to co-localized proteins. This Bruker spectrometer at 300 MHz for H and 75 MHz for 13C;
experiment demonstrates that dynamic color switching and chemical shifts are reported in ppm with protonated solvent as
1
two-color cross-correlation allow unveiling the presence of internal reference H, CHCl3 in CDCl3 7.26 ppm, CHD2SOCD3 in
FAST in spectrally crowded environment containing both green CD3SOCD3 2.50 ppm; 13C, 13CDCl3 in CDCl3 77.0 ppm,
and red fluorescent species.
13CD3SOCD3 in CD3SOCD3 39.5 ppm; coupling constants J are
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given in Hz. Mass spectra (chemical ionization or high General protocol for the synthesis of HBR-3E, HBR-3OE,
resolution) were performed by the Service de Spectrométrie HBR-3,5DM and HBR-3,5DOM To stirred solution of
a
de Masse de Chimie ParisTech and the Institut de Chimie rhodanine (1.5 mmol, 1.0 eq) and the substituted 4-hydroxy-
Organique et Analytique de l’Université d’Orléans. Column benzaldehyde (1.56 mmol, 1.1 eq) in 4.5 mL of absolute
chromatography was performed on silica gel 60 (0.040-0.063 ethanol was added piperidine (1.5 mmol, 1.0 eq). The solution
nm) Merck. Analytical thin-layer chromatography (TLC) was was stirred at the room temperature for 16 h and then
conducted on Merck silica gel 60 F254 precoated plates.
neutralized by an aqueous solution of hydrochloric acid. After
cooling to 4 and standing for 2 h, the precipitate was filtered
℃
Synthesis of 4-hydroxy-3-ethylbenzaldehyde To a solution of through a glass filter and the crude solid was washed with
2-ethylphenol (6.1 g, 50 mmol) in 10% aqueous sodium water, ethanol and dried over P2O5, to give the desired
hydroxide (80 mL, 200 mmol) was added trichloromethane product.
(15.0 g, 125 mmol) dropwise at 60°C over 1 h, and then the
reaction mixture was stirred for 2 h at 60
mixture was neutralized by an aqueous solution of one (HBR-3E) yellow powder (70 %). 1H NMR (300 MHz,
hydrochloric acid and extracted with dichloromethane. The CD3SOCD3, in ppm): 13.67 (s, 1H), 10.35 (s, 1H), 7.54 (s, 1H),
combined organic layers were washed with brine, dried over 7.30 (m, 2H), 6.94 (d, J = 8.1 Hz, 1H), 2.57 (q, J = 7.5 Hz, 2H),
magnesium sulfate, and concentrated under reduced pressure. 1.15 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz, CD3SOCD3,
in ppm):
℃. After cooling, the (Z)-5-(3-ethyl-4-hydroxybenzylidene)-2-thioxothiazolidin-4-
δ
δ
The residue was purified by flash chromatography on silica gel 195.5, 169.4, 158.3, 132.8, 132.0, 131.3, 130.6, 124.0, 120.5,
with cyclohexane/ethylacetate (7.5/2.5, v/v) to yield the 115.8, 22.5, 13.7; MS (ESI): m/z 264.3 [M-H]-, calcd mass for
desired 4-hydroxy-3-ethylbenzaldehyde (1.1 g, 15% yield) as a [C12H10NO2S2]-: 264.0; HRMS (ESI): m/z 264.0157 [M-H]-, calcd
1
grey pink solid. H NMR (300 MHz, CDCl3,
δ
in ppm): 9.84 (s, mass for [C12H10NO2S2]-: 264.0153.
1H), 7.72 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 8.1 Hz,
1H), 6.51 (s, 1H), 2.70 (q, J = 7.5 Hz, 2H), 1.27 (t, J = 7.5 Hz, 3H); (Z)-5-(3-ethoxy-4-hydroxybenzylidene)-2-thioxothiazolidin-4-
13C NMR (75 MHz, CDCl3, in ppm): 192.3, 160.5, 131.5, 131.4, one (HBR-3OE) Orange powder (74 %). 1H NMR (300 MHz,
δ
130.6, 129.4, 115.6, 22.8, 13.5; MS (ESI): m/z 149.2 [M-H]-, CD3SOCD3,
calcd mass for [C9H9O2]-: 149.1; HRMS (ESI): m/z 149.0608 [M- 7.12 (d, J = 1.8 Hz, 1H), 7.07 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 6.95 (d,
H]-, calcd mass for [C9H9O2]-: 149.0603.
J = 8.1 Hz, 1H), 4.08 (q, J = 6.9 Hz, 2H), 1.36 (t, J = 6.9 Hz, 3H);
13C NMR (75 MHz, CD3SOCD3,
in ppm): 195.4, 169.4, 150.2,
δ in ppm): 13.69 (s, 1H), 10.00 (s, 1H), 7.55 (s, 1H),
δ
General protocol for the synthesis of HBR-3OM and HBR- 147.3, 132.8, 125.1, 124.4, 121.0, 116.4, 115.4, 63.9, 14.6; MS
2,5DM A solution containing rhodanine (133 mg, 1.0 mmol) (ESI): m/z 280.1 [M-H]-, calcd mass for [C12H10NO3S2]-: 280.0;
and the substituted 4-hydroxy-benzaldehyde (1.0 mmol) in 40 HRMS (ESI): m/z 280.0108 [M-H]-, calcd mass for
mL of water was stirred at 90°C for 7 days. After cooling to 4°C [C12H10NO3S2]-: 280.0102.
and standing overnight, the precipitate was filtered through a
glass filter and the crude solid was washed with water, ethanol (Z)-5-(4-hydroxy-3,5-dimethylbenzylidene)-2-
and dried over P2O5, to give the desired product.
thioxothiazolidin-4-one (HBR-3,5DM) Yellow powder (73 %).
1H NMR (300 MHz, CD3SOCD3,
in ppm): 13.67 (s, 1H), 9.26 (s,
1H), 7.45 (s, 1H), 7.17 (s, 2H), 2.21 (s, 6H); 13C NMR (75 MHz,
in ppm): 195.6, 169.5, 156.6, 132.6, 131.5(2C),
in ppm): 13.68 (s, 1H), 10.08 (s, 1H), 125.4(2C), 124.0, 120.8, 16.6(2C); MS (ESI): m/z 264.3 [M-H]-,
7.57 (s, 1H), 7.15 (s, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.94 (d, J = 8.1 calcd mass for [C12H10NO2S2]-: 264.0; HRMS (ESI): m/z 264.0158
Hz, 1H), 3.83 (s, 3H); 13C NMR (75 MHz, CD3SOCD3, in ppm): [M-H]-, calcd mass for [C12H10NO2S2]-: 264.0153.
δ
(Z)-5-(4-hydroxy-3-methoxybenzylidene)-2-thioxo-1,3-
1
thiazolidin-4-one (HBR-3OM) Yellow powder (52 %). H NMR CD3SOCD3,
(300 MHz, CD3SOCD3,
δ
δ
δ
195.5, 169.5, 150.0, 148.1, 132.8, 125.1, 124.4, 121.2, 116.4,
114.4, 55.7; MS (ESI): m/z 266.2 [M-H]-, calcd mass for (Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-2-
[C11H8NO3S2]-: 266.0; HRMS (ESI): m/z 265.9951 [M-H]-, calcd thioxothiazolidin-4-one (HBR-3,5DOM) Brown powder (72 %).
mass for [C11H8NO3S2]-: 265.9946.
1H NMR (300 MHz, CD3SOCD3,
1H), 7.57 (s, 1H), 6.88 (s, 2H), 3.83 (s, 6H); 13C NMR (75 MHz,
CD3SOCD3, in ppm): 195.3, 169.3, 148.3(2C), 139.2, 133.0,
δ in ppm): 13.71 (s, 1H), 9.46 (s,
(Z)-5-(4-hydroxy-2,5-dimethylbenzylidene)-2-thioxo-1,3-
δ
thiazolidin-4-one (HBR-2,5DM) Orange powder (38 %). 1H 123.2, 121.4, 108.5(2C), 56.1(2C); MS (ESI): m/z 296.1 [M-H]-,
NMR (300 MHz, CD3SOCD3,
1H), 7.64 (s, 1H), 7.10 (s, 1H), 6.75 (s, 1H), 2.33 (s, 3H), 2.14 (s, [M-H]-, calcd mass for [C12H10NO4S2]-: 296.0051.
3H); 13C NMR (75 MHz, CD3SOCD3,
in ppm): 196.3, 169.8,
δ
in ppm): 13.70 (s, 1H), 10.22 (s, calcd mass for [C12H10NO4S2]-: 296.0; HRMS (ESI): m/z 296.0056
δ
158.9, 140.1, 131.1, 130.0, 123.4, 122.9, 122.0, 117.7, 19.6, Physical Chemistry experiments pH measurements were
16.1; MS (ESI): m/z 264.1 [M-H]-, calcd mass for [C12H10NO2S2]-: performed on a standard pH meter PHM210 Radiometer
264.0; HRMS (ESI): m/z 264.0158 [M-H]-, calcd mass for Analytical (calibrated with aqueous buffers at pH 4 and 7 or
[C12H10NO2S2]-: 264.0153.
10) with a Crison 5208 Electrode (Barcelona, Spain). UV/Vis
absorption spectra were recorded in 1 cm × 1 cm quartz
cuvettes (Hellma) on a diode array UV/Vis spectrophotometer
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C7SC01364G
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ARTICLE
(Evolution array, Thermo Scientific). Corrected fluorescence Mammalian cell culture HeLa cells were cultured in DMEM
spectra upon one-photon excitation were recorded with a supplemented with phenol red, Glutamax I, 10% (vol/vol) fetal
Photon Technology International QuantaMaster QM-1 calf serum (FCS), and 1% penicillin−streptomycin at 37 °C
spectrofluorimeter (PTI, Monmouth Junction, NJ) equipped within a 5% CO2 atmosphere. For microscopic imaging, cells
with
a Peltier cell holder (TLC50, Quantum Northwest, were seeded in μDish or μSlide IBIDI (Biovalley) coated with
Shoreline, WA). The overall emission quantum yields after one- poly-L-lysine. Cells were transiently transfected using
photon excitation
φ
were determined as previously Genejuice (Merck) according to the manufacturer’s protocol.
described14. Affinity constants were determined by Cells were washed with PBS, and treated with DMEM media
spectrofluorometric titration using a Spark 10M plate reader (without serum and phenol red) containing the fluorogens at
(Tecan) following protocols previously described14.
the indicated concentration. Cells were imaged directly
without washing.
Molecular Biology FAST is a variant of the photoactive yellow
protein (PYP) containing the mutations C69G, Y94W, T95M, Cell viability assay HeLa cells were treated with DMEM media
F96I, D97P, Y98T, Q99S, M100R, T101G. The construction of containing the fluorogens at the indicated concentrations for
the plasmid pAG87 for the bacterial expression of FAST fused the indicated times. Cell viability was evaluated by
to a His-tag was previously described14. The constructions of fluorescence
microscopy
using
the
LIVE/DEAD®
the plasmids pAG104, pAG109 and pAG106 for the mammalian viability/cytotoxicity assay kit (Molecular Probes, Life
expression of FAST, FAST fused to zebrafish H2B (H2B-FAST), Technologies) following the manufacturer’s protocol.
and FAST fused to the mitochondrial targeting sequence (MTS)
from subunit VIII of human cytochrome c oxidase (MTS-FAST) Fluorescence microscopy Confocal micrographs were acquired
were previously described14. The plasmid pAG28 for the on a Zeiss LSM 710 Laser Scanning Microscope equipped with
mammalian expression of EGFP fused to Lyn N-terminal a Plan Apochromat 63 ×/1.4 NA oil immersion objective. ZEN
domain (lyn) for inner membrane anchoring was previously software was used to collect the data. Images were analyzed
described18. The plasmids CV2307 and CV2326 for the with Image J.
mammalian expression of mCherry fused to the membrane
Two-color cross-correlation analysis The two-color cross-
correlated images were obtained as follows. A movie was
acquired in the green and red (g: green and r: red)
fluorescence channels over the evolution duration observed
after adding HMBR fluorogen to the cellular medium. The
cross-correlated image was then computed from the movie as
anchoring (lyn) peptide from the mouse tyrosine-protein
kinase Lyn and to the N-terminal 25 amino acid mitochondrial
targeting signal (MTS) of COX4 from S. cerevisiae were
assembled using MXS chaining by sequentially inserting a CMV
promoter,
the
lyn
(MGCIKSKRKDVEN)
or
MTS
(MLSLRQSIRFFKPATRTLCSSRYLL) peptide tags to a plasmid
containing the mCherry coding sequence coupled to the
bovine growth hormone poly A signal.
ꢅ
ꢈ
ℑ ꢀꢁ ꢄꢂꢃꢄ − ꢃꢆꢅꢇꢂꢃꢄ − ꢃꢆꢈꢇꢉ where
of the FAST concentration proportional square root function, i
denotes the frame number, ꢃꢄꢅ (respectively ꢃꢄꢈ
is the
ℑ
takes the imaginary part
∑
)
Bacterial expression and protein purification pAG87 was
transformed in Rosetta(DE3)pLysS E. coli (New England
Biolabs). Cells were grown at 37°C in Lysogeny Broth (LB)
medium complemented with 50 μg/ml kanamycin and 34
fluorescence intensity of pixel p in the green (respectively red)
channel of the image i, and ꢃꢆꢅ (respectively ꢃꢆꢈ) designate the
average fluorescence intensity of pixel p over the whole movie
duration in the green (respectively red) channel. Eventually a
μg/ml chloramphenicol to OD600nm 0.6. Expression was induced median filter (3×3 pixel2) was applied to yield the final image.
for 4 h by adding isopropyl β-D-1-thiogalactopyranoside (IPTG)
1
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to a final concentration of 1 mM. Cells were harvested by
centrifugation (6,000 × g for 15 min at 4°C) and frozen. The cell
pellet was resuspended in lysis buffer (phosphate buffer 50
mM, NaCl 150 mM, MgCl2 2.5 mM, protease inhibitor, DNase,
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3
4
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mM Imidazole. His-tagged proteins were eluted with
5
volumes of PBS complemented with 0.5 M Imidazole, followed
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J. Name., 2013, 00, 1-3 | 7
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DOI: 10.1039/C7SC01364G
ARTICLE
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8 | J. Name., 2012, 00, 1-3
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