Optical Manipulation of Subcellular Protein Translocation Using a Photoactivatable Covalent Labeling System

Article abstract of DOI:10.1002/anie.202016684

The photoactivatable chemically induced dimerization (photo-CID) technique for tag-fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein–protein interactions. However, light-induced covalent protein dimerization in living cells has yet to be established, despite its various advantages. Herein, we developed a photoactivatable covalent protein-labeling technology by applying a caged ligand to the BL-tag system, a covalent protein labeling system that uses mutant β-lactamase. We further developed CBHD, a caged protein dimerizer, using caged BL-tag and HaloTag ligands, and achieved light-induced protein translocation from the cytoplasm to subcellular regions. In addition, this covalent photo-CID system enabled quick protein translocation to a laser-illuminated microregion. These results indicate that the covalent photo-CID system will expand the scope of CID applications in the optical manipulation of cellular functions.

Full text of DOI:10.1002/anie.202016684

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 11378–11383
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Photochemistry
Hot Paper
Optical Manipulation of Subcellular Protein Translocation Using
a Photoactivatable Covalent Labeling System
Toshiyuki Kowada, Keisuke Arai, Akimasa Yoshimura, Toshitaka Matsui, Kazuya Kikuchi,* and
Shin Mizukami*
Abstract: The photoactivatable chemically induced dimeriza-
tion (photo-CID) technique for tag-fused proteins is one of the
most promising methods for regulating subcellular protein
translocations and protein–protein interactions. However,
light-induced covalent protein dimerization in living cells has
yet to be established, despite its various advantages. Herein, we
developed a photoactivatable covalent protein-labeling tech-
nology by applying a caged ligand to the BL-tag system,
a covalent protein labeling system that uses mutant b-lacta-
mase. We further developed CBHD, a caged protein dimerizer,
using caged BL-tag and HaloTag ligands, and achieved light-
induced protein translocation from the cytoplasm to subcel-
lular regions. In addition, this covalent photo-CID system
enabled quick protein translocation to a laser-illuminated
microregion. These results indicate that the covalent photo-
CID system will expand the scope of CID applications in the
optical manipulation of cellular functions.
intracellular protein translocation using optogenetics has
been developed.[1] This technique can provide essential
information for investigating intracellular protein functions,
as microscopic light irradiation helps control protein translo-
cation spatially and temporally to a considerable extent. An
alternative approach for regulating subcellular protein local-
ization is chemically induced dimerization (CID) of pro-
teins,[2] which can also be used to manipulate protein–protein
interactions in living cells.[3–6] In most cases, the proteins of
interest are fused with a tag protein, and a small-molecule
dimerizer attaches the two tag ligands on both ends. In
addition to optogenetics, a significant effort has been devoted
to the development of photoactivatable CID (photo-CID)
techniques. In a pioneering study, Schreiber et al. demon-
strated the light-control of yeast growth using the light-
induced release of rapamycin immobilized on a bead through
a photo-cleavable linker.[7] After their study, the caged
rapamycin derivatives were used for the photo-regulation of
various biomolecular components such as Rho GTPase,[8]
kinase,[9] and TEV protease.[10] Recently, an eDHFR-caged
trimethoprim system was reported as a suitable non-covalent
photo-CID tool,[11] and various chemical biology methods
have been studied actively using this system.[12–20]
However, the majority of these photo-CID systems utilize
non-covalent protein-labeling technologies to induce protein
dimerization. Since covalent CID systems can maintain stable
protein dimers regardless of ligand and protein concentra-
tions, the development of a covalent photo-CID system that
functions in living cells has been in high demand. Although
the light-induced dissociation of protein dimers in living cells
has been achieved using a covalent CID system with a photo-
cleavable linker tethering two different tags,[21,22] the light-
induced homodimerization of SNAP-tag-fused proteins has
only been demonstrated in vitro.[23] Therefore, the optical
manipulation of protein localization in living cells by covalent
cross-linking using a photo-CID system still requires certain
modifications before it can be implemented successfully. In
this study, we developed a covalent photo-CID system and
demonstrated its application in subcellular protein transloca-
tion with high spatiotemporal resolution in living cells.
Introduction
Since bioactive molecules such as proteins often exhibit
subcellular localization along with other functions, techniques
to regulate subcellular protein localization have attracted
attention as important tools in cell biology. Recently, a more
sophisticated technique that can be used to optically control
[*] Dr. T. Kowada, Dr. T. Matsui, Prof. S. Mizukami
Institute of Multidisciplinary Research for Advanced Materials
Tohoku University
Sendai, Miyagi 980-8577 (Japan)
E-mail: shin.mizukami@tohoku.ac.jp
Dr. T. Kowada, K. Arai, Dr. T. Matsui, Prof. S. Mizukami
Graduate School of Life Sciences
Tohoku University
Sendai, Miyagi 980-8577 (Japan)
Dr. A. Yoshimura, Prof. K. Kikuchi
Graduate School of Engineering, Osaka University
2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)
E-mail: kkikuchi@mls.eng.osaka-u.ac.jp
Prof. K. Kikuchi
Immunology Frontier Research Center
Osaka University
2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)
and
Center for Quantum Information and Quantum Biology
Osaka University
Results and Discussion
We have previously developed a covalent protein-labeling
system, named the BL-tag system, which utilizes a mutant
(E166N) TEM-1 b-lactamase (BL-tag) and b-lactam li-
gands.[24–27] One of the characteristics of the BL-tag system
2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016684.
11378
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is that various b-lactam antibiotics, such as penicillin and
considerably weak, which led us to conclude that the DMNPE
cephalosporin, can be used as specific ligands. The common
structure of the ligands contains a carboxylate residue, and
the esterification of the carboxylate leads to a reduction in the
protein-labeling rate.[28] In addition, the crystal structure of
the complex of the mutant E166N b-lactamase and a b-lactam
antibiotic suggests that the carboxylate residue plays an
important role in complex formation, including the formation
of hydrogen bonds.[29] Accordingly, we constructed a photo-
activatable covalent protein-labeling system based on the BL-
tag system by introducing a bulky photoprotecting group on
the carboxy group (Figure 1a). A similar caged b-lactam
antibiotic was developed to optically control bacterial growth
inhibition.[30] It was assumed that the protected ligand would
not be labeled to the BL-tag protein until light-induced
deprotection was conducted. To validate this concept, we
synthesized two caged derivatives of a fluorescent BL-tag
ligand, FPA-DEACM and FPA-DMNPE, having different
caging moieties, [7-(diethylamino)coumarin-4-yl)methyl
group is a promising photoprotecting group that can be used
in the construction of caged BL-tag ligands.
We next designed the caged BL-tag-HaloTag dimerizer
(CBHD) (Figure 2a), a photoactivatable chemical dimerizer,
for constructing a covalent photo-CID system to link BL-tag
with HaloTag, a mutant bacterial haloalkane dehalogenase
that specifically forms a covalent bond with a chloroalkane
moiety.[31] We assumed that CBHD would initially form
a covalent bond with a HaloTag-fused protein, and then form
a covalent bond to induce heterodimerization with a BL-tag-
fused protein upon UV illumination. CBHD was synthesized
according to the process outlined in Scheme S2. HPLC
analysis did not reveal hydrolytic conversion from CBHD to
BHD in dark for several hours in neutral HEPES buffer or in
10% FBS-supplemented medium (Figure S3). The findings
indicate that CBHD is suitable for long-term cellular
applications. The photo-conversion of CBHD was investigat-
ed under illumination with a Xe lamp at 365 Æ 5 nm (Fig-
ure 2b). Under UV irradiation, the HPLC peak intensity at
12 min corresponding to CBHD decreased, whereas that at
9.5 min, corresponding to the uncaged dimerizer BHD,
increased, in an irradiation time-dependent manner. This
efficient photo-uncaging of CBHD was also expected owing
to the sufficient absorption at approximately 365 nm (Fig-
ure S4). Based on the mass spectrometry (MS) results, the
peak formed at 9 min was suggested to result from a derivative
of the DMNPE group, and a peak formed at the same
retention time was also observed in the uncaging reaction of
FPA-DMNPE.
(DEACM)
and
1-(4,5-dimethoxy-2-nitrophenyl)ethyl
(DMNPE) groups, respectively (Figure 1a). FPA-DEACM
was labeled with BL-tag at moderate levels even in dark
(unpublished data). Conversely, the labeling of FPA-DMNPE
was largely suppressed in the dark, and the specific labeling
property was recovered upon UV irradiation in both in vitro
and live-cell experiments (Figures 1b–d and S1). The weak
fluorescent band in the gel observed under dark conditions
was considered to be formed due to slow labeling by FPA-
DMNPE, as FPA-DMNPE was not hydrolyzed to FPA in
neutral HEPES buffer (Figure S2a). Conversely, FPA was
generated at trace levels in 10% FBS-supplemented medium
after 2 h of incubation (Figure S2b). Therefore, the faint
fluorescence signals obtained in the live-cell imaging with
FPA-DMNPE in the absence of UV irradiation could be
attributed to the low levels of FPA labeling owing to the
hydrolysis of FPA-DMNPE. However, the signals were
Next, we confirmed the light-induced dimer formation of
BL-tag and HaloTag using CBHD in vitro. CBHD (5 mm) was
incubated with an N-terminal His-tagged HaloTag (His-Halo,
10 mm) for 30 min for covalent labeling. A maltose-binding
protein fused at the C-terminal with BL-tag (MBP-BL,
10 mm) was added to the sample, and the sample was
Figure 1. a) Chemical structures of the synthesized caged BL-tag ligands (top), and schematic illustration of light-induced fluorescence labeling of
the BL-tag-fused protein (bottom). b) SDS-PAGE analysis of the light-induced labeling reaction of BL-tag with FPA-DMNPE. Coomassie Brilliant
Blue (CBB)-stained and fluorescence gel images. [FPA-DMNPE]=15 mm, [BL-tag]=10 mm in 100 mm HEPES buffer (pH 7.4). Uncaging light:
365Æ5 nm, 9.3 mWcmÀ2, for 5 min at rt. Excitation: 470 nm. Detection: 590Æ40 nm. c) Fluorescence intensities of the bands corresponding to
the labeled BL-tags in the SDS-PAGE analysis shown in (b). The error bars represent s.d. (n=4). d) Confocal laser scanning microscopy images of
BL-tag-fused EGFR (BL-EGFR) labeled with 100 nm FPA-DMNPE. For the condition of UV (+), FPA-DMNPE was uncaged by UV irradiation before
probe addition to the live cells. Uncaging light: 365Æ5 nm, 9.3 mWcmÀ2, for 5 min at rt. Scale bar: 40 mm.
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Figure 2. a) Chemical structure of the caged dimerizer, CBHD (top),
and schematic illustration of the covalent photo-CID system based on
BL-tag and HaloTag (bottom). b) HPLC analysis of the photo-uncaging
reaction of CBHD. c) SDS-PAGE analysis of protein dimerization
induced by CBHD and UV irradiation. The polyacrylamide gel was
stained using CBB. [CBHD]=5 mm, [His-Halo]=10 mm, [MBP-
BL]=10 mm in 100 mm HEPES buffer (pH 7.4). Uncaging light:
365Æ5 nm, 9.6 mWcmÀ2, for 5 min at rt. Gel: 5%–15% polyacryl-
amide.
irradiated with UV light for 5 min. After incubation for
60 min, the sample was analyzed using SDS-PAGE (Fig-
ure 2c). In the lane loaded with the UV-irradiated sample
with CBHD, a new band was observed at approximately
110 kDa corresponding to the dimer of His-Halo (36 kDa)
and MBP-BL (73 kDa). Conversely, the lane loaded with the
non-UV-irradiated sample did not show a dimer band;
however, a new band appeared slightly above the position
corresponding to His-Halo, which supposedly corresponded
to CBHD-labeled His-Halo (37 kDa). These results demon-
strated that the formation of a covalent heterodimer com-
prising a BL-tag-fused protein and a Halo-Tag-fused protein
was induced by UV light irradiation in the presence of CBHD.
We next evaluated intracellular protein translocation as
an application of the covalent photo-CID system in live-cell
studies (Figure 3a). HEK293T cells were transfected to
express both BL-tag-fused EGFP (BL-EGFP) and HaloTag-
fused mCherry with a CAAX sequence at the C-terminus
(Halo-mCherry-CAAX) targeting the cytosolic interface of
the plasma membrane.[32] After the cells were incubated with
10 mm CBHD for 30 min, followed by 30 min incubation in
fresh medium for washing out unreacted CBHD, images were
acquired using confocal fluorescence microscopy. CBHD
formed a covalent bond with membrane-anchored Halo-
Figure 3. a) Schematic illustration of the photo-CID system using
CBHD. b,c) Time-lapse imaging of BL-EGFP translocation to the
cytosolic interface of the plasma membrane using CBHD in the
absence (b) or presence (c) of UV irradiation. Scale bars: 15 mm.
d) Time-course of normalized EGFP fluorescence intensity in the
cytosol. The error bars represent the standard error of means (n=11
(for UV (À)) or 12 cells (for UV (+)) from two independent experi-
ments).
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mCherry-CAAX before UV irradiation. Therefore, the green
fluorescence signals of BL-EGFP and red fluorescence signals
of Halo-mCherry-CAAX were observed from the entire cell
and the plasma membrane, respectively, without significant
overlap, and no changes were observed in the localization of
BL-EGFP for 70 min (Figure 3b). Conversely, UV irradiation
for 10 s through the objective lens using an Hg lamp rapidly
initiated the translocation of BL-EGFP to the plasma
membrane (Figure 3c), which was almost completed approx-
imately within 30 min (Figure 3d). The Pearson correlation
coefficient (PCC) changed from 0.39 Æ 0.04 to 0.84 Æ 0.04
(mean Æ s.e.m., n = 8 cells) upon UV irradiation, indicating
that most of the BL-EGFP were translocated to the plasma
membrane.
To demonstrate the versatility of the covalent photo-CID
system, light-induced protein translocation to different sub-
cellular regions was also examined. Halo-mCherry-NLS and
Tom20-mCherry-Halo were used instead of Halo-mCherry-
CAAX for translocation to the nucleus and mitochondrial
outer membrane, respectively, where NLS is a nuclear local-
ization signal from the SV40 large T antigen,[33] and Tom20 is
the N-terminal 33 amino acids of the mitochondrial import
receptor Tom20.[34] In both cases, light-induced protein
translocation was implemented successfully (Figures 4a,b,
S5, and S6), as was translocation to the plasma membrane. In
addition, the nuclear translocation of BL-EGFP required
50 min (Figure S5), which was longer than the duration
required for complete translocation to the plasma membrane
(30 min) or the mitochondrial outer membrane (20 min,
Figure S6). Possibly, the presence of protein diffusion barriers,
such as nuclear pores, delayed the protein translocation
process. Colocalization of the two fluorescent proteins was
confirmed by calculating the PCCs, 0.42 Æ 0.05 (dark) and
0.87 Æ 0.02 (UV irradiation) for the nucleus (mean Æ s.e.m.,
n = 8 cells), and 0.53 Æ 0.05 (dark) and 0.83 Æ 0.02 (UV
irradiation) for the mitochondria (mean Æ s.e.m., n = 8 cells).
When the cells expressing BL-EGFP and Halo-mCherry-NLS
were subjected to western blotting analysis, it was confirmed
that covalent protein dimerization could be induced by
CBHD under whole-sample UV irradiation with a Xe lamp
(Figure S7). This application is one of the advantageous
properties of the covalent photo-CID system.
Contrary to our expectation, light-triggered protein
translocation required several tens of minutes for completion.
Although a ten-fold reduction in the concentration of the BL-
EGFP-encoding plasmid did not lead to any significant
difference in the translocation rates (Figure S8), we believe
that the expression levels of both proteins affected the
translocation rate; further investigation will be required to
confirm this. We also constructed the reverse fusion pair, with
BL-EGFP-CAAX expressed in the plasma membrane and
Halo-mCherry in the cytosol (Figure S9a). When the cells
were transfected with plasmids for BL-EGFP-CAAX and
Halo-mCherry in a 1:1 ratio (1250 ng/1250 ng), photo-CID-
based translocation was barely observed. However, when the
ratio of BL-EGFP-CAAX- and Halo-mCherry-encoding
plasmids was altered to 10:1 (909 ng/91 ng), the light-induced
translocation of Halo-mCherry from the cytosol to the plasma
membrane was clearly observed (Figures S9b,c). In the
Figure 4. a,b) Light-induced protein (BL-EGFP) translocation to the
nucleus (Halo-mCherry-NLS) (a) and mitochondrial outer-membrane
(Tom20-mCherry-Halo) (b). Scale bars: 15 mm. c) Laser-induced quick
protein (BL-EGFP) translocation to the irradiated microregion (square)
in the mitochondrial outer membrane. Scale bar: 10 mm.
experiments performed using this construct pair, the protein
translocation was completed within 15 min, although the total
concentration of the translocated protein was less than that of
the BL-EGFP/Halo-mCherry-CAAX pair (Figure S9d).
These results indicate that the kinetics and efficiency of
photo-CID-based protein translocation are affected by the
anchoring and diffusible proteins selected.
Lastly, to verify the potential of the developed photo-CID
system, we attempted subcellular-level light-induced protein
translocation. BL-EGFP and Tom20-mCherry-Halo were
expressed in the whole-cell area and mitochondrial outer
membrane, respectively, and CBHD was labeled to Tom20-
mCherry-Halo. After a microregion in a single HEK293T cell
was illuminated using a 405 nm laser, EGFP fluorescence
signals were rapidly increased in the illuminated region
(Figure 4c). This result indicated that photoinduced protein
labeling and translocation occur considerably rapidly in the
subcellular microregion. The EGFP fluorescence signals in
the microregion were restored to levels similar to those in the
surrounding regions within 19 min (Figure S10). This could be
attributed to the diffusion of the mitochondria in the cell.
However, the protein dimer could be retained for a prolonged
period in the microregion owing to the fact that the covalent
bonds between CBHD and the tag proteins were maintained
even at low concentrations. Single-cell-specific light-induced
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translocation of Halo-mCherry-NLS was also achieved (Fig-
ure S11). Overall, our covalent photo-CID system with
CBHD can be applied to subcellular protein dimerization
and translocation, and has the potential for local activation of
signal transduction pathways at the organelle level.
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Conclusion
We developed a photoactivatable covalent protein-label-
ing system based on our original BL-tag technology by
incorporating a photoprotecting DMNPE group into the BL-
tag ligand structure. We also used this system to implement
photoactivatable protein dimerization using the caged chem-
ical dimerizer CBHD, which covalently links BL-tag and
HaloTag. Using this covalent photo-CID system, we achieved
the light-induced translocation of cytosolic proteins to various
subcellular regions such as the plasma membrane, nucleus,
and mitochondrial outer membrane. To our knowledge, this is
the first study to achieve light-induced covalent protein dimer
formation in living cells. Once a stable covalent bond is
formed between the ligand and tag-protein, the resulting
protein dimer is maintained stably for a long duration
regardless of the ligand–protein complex concentration. This
advantageous property of covalent bond formation was also
confirmed by the detection of the stable protein dimer in the
gel electrophoresis assay. Light-induced subcellular-level
protein dimerization has various potential biological applica-
tions, such as the sustained activation of ERK in the MAPK
signaling pathway[35] in a specific single cell.
Acknowledgements
This work was supported by MEXT KAKENHI Grant
Numbers JP18H04605 and JP19H05284, by JSPS KAKENHI
Grant Numbers JP17K05921, JP18H02102, JP18F18340,
JP19K22241, and JP20K05702, and by the Uehara Memorial
Foundation, the Takeda Science Foundation, the Astellas
Foundation for Research on Metabolic Disorders, the Tokyo
Biochemical Research Foundation, the Kowa Life Science
Foundation, the Nakatani Foundation, and the “Dynamic
Alliance for Open Innovation Bridging Human, Environment
and Materials” Research Program in the “Network Joint
Research Center for Materials and Devices”. We appreciate
the experimental assistance of Hiroto Takahashi, Naoki
Honzawa, and Kashfia Ahamed. We thank Tagen Central
Analytical Facility for providing the NMR and MS instru-
ments.
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Conflict of interest
The authors declare no conflict of interest.
Keywords: caged compound ·dimerization ·photochemistry ·
protein–protein interactions ·proteins
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Manuscript received: December 16, 2020
Revised manuscript received: February 15, 2021
Accepted manuscript online: February 28, 2021
Version of record online: April 6, 2021
Angew. Chem. Int. Ed. 2021, 60, 11378 –11383
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