Cell-permeant and photocleavable chemical inducer of dimerization

Article abstract of DOI:10.1002/anie.201310969

Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivi

Full text of DOI:10.1002/anie.201310969

Angewandte
Chemie
DOI: 10.1002/anie.201310969
Protein–Protein Interactions
Cell-Permeant and Photocleavable Chemical Inducer of
Dimerization**
Mirjam Zimmermann, Ruben Cal, Elia Janett, Viktor Hoffmann, Christian G. Bochet,
Edwin Constable, Florent Beaufils,* and Matthias P. Wymann*
Abstract: Chemical inducers of dimerization (CIDs) have
been developed to orchestrate protein dimerization and
translocation. Here we present a novel photocleavable Halo-
Tag- and SNAP-tag-reactive CID (MeNV-HaXS) with excel-
lent selectivity and intracellular reactivity. Excitation at 360 nm
cleaves the methyl-6-nitroveratryl core of MeNV-HaXS.
MeNV-HaXS covalently links HaloTag- and SNAP-tag
fusion proteins, and enables targeting of selected membranes
and intracellular organelles. MeNV-HaXS-mediated translo-
cation has been validated for plasma membrane, late endo-
somes, lysosomes, Golgi, mitochondria, and the actin cytoske-
leton. Photocleavage of MeNV-HaXS liberates target proteins
and provides access to optical manipulation of protein
relocation with high spatiotemporal and subcellular precision.
MeNV-HaXS supports kinetic studies of protein dynamics and
the manipulation of subcellular enzyme activities, which is
exemplified for Golgi-targeted cargo and the assessment of
nuclear import kinetics.
precision. Caged small molecules and enzyme substrates have
been developed for a number of applications.[2]
Naturally occurring light-sensitive protein domains have
been used to design genetically encoded light-controlled
protein–protein interaction modules. These so-called optoge-
netic systems contain a photoisomerizable chromophore,
which undergoes a conformational change upon illumination
at a defined wavelength. Optogenetic systems have been used
to control the activation of single signaling proteins by protein
caging (light-inducible GTPase Rac),[3] or in a more modular
approach to indirectly manipulate cellular signaling, through
the light-dependent dimerization of two protein modules.[4]
Optogenetic light-activated dimerization systems are versa-
tile tools, but suffer from several drawbacks such as large
photosensory protein tags,[4,5] the requirement of exogenous
cofactors,[4] slow kinetics,[5] formation of unwanted homo-
dimers,[6] and sensitivity to environmental light, and/or over-
lap with excitation wavelength of popular fluorescent
reporter proteins.[6,7] Another approach to control protein
localization and enzyme activity are chemical inducers of
dimerization (CIDs)[8] and self-localizing ligands,[9] which
have been successfully used to manipulate signaling pathways
including phosphoinositide turnover,[10] and small GTPases.[11]
Presently, cell-permeable CIDs that can be efficiently
manipulated intracellularly have not been reported.[8] Some
spatial selectivity has been achieved with photocleavable,
biotinylated a-methylnitrobenzylrapamycin, which has been
used to control small GTPase activity.[12] This caged rapamy-
cin was targeted to an extracellular location by means of its
biotin moiety, required, however, extracellular photolytic
removal of the caging group before rapamycin was released to
diffuse across the cell membrane.[12] Another photocaged
rapamycin derivative is pRap.[13] Both of these noncovalent,
photocleavable CIDs provide a source of highly diffusible
dimerizer, limiting local target manipulation.
L
ocalization of signaling enzymes is key to controlling
protein and lipid kinase cascades in physiology and disease.[1]
Control of protein localization and enzyme activity by
illumination provides unique access to the manipulation of
biological processes in living cells with high spatiotemporal
[*] M. Zimmermann,[+] R. Cal,[+] V. Hoffmann, Dr. F. Beaufils,
Prof. Dr. M. P. Wymann
University of Basel, Department of Biomedicine
Mattenstrasse 28, Basel (Switzerland)
E-mail: Florent.Beaufils@UniBas.CH
Matthias.Wymann@UniBas.CH
E. Janett, Prof. Dr. C. G. Bochet
University of Fribourg, Department of Chemistry
Chemin du Musꢀe 9, Fribourg (Switzerland)
Prof. Dr. E. Constable
University of Basel, Department of Chemistry
Spitalstrasse 51, Basel (Switzerland)
Here we present a novel photocleavable CID, which forms
a covalent link between HaloTag-[14] and SNAP-tag[15]-fused
proteins. The photocleavable methyl-6-nitroveratryl (MeNV)
group was introduced into the core module linking the
HaloTag-reactive chloroalkane ligand and the SNAP-tag-
reactive O6-benzylguanine, and the cell permeability of the
resulting CID molecule is retained (dubbed MeNV-HaXS;
Figure 1). The combination of chemical-induced dimerization
and the possibility of a subsequent light-induced reversal of
the protein–protein interaction combines the advantage of
a modular approach of genetically encodable tags with
a highly specific spatiotemporal control by light.
[+] These authors contributed equally to this work.
[**] We thank Takanari Inoue for FRB-YFP-Giantin and Stephan Hꢁbner
for NLS-CFP-FKBP expression constructs. This work was supported
by Swiss National Science Foundation (205320-138302, 205320-
143699), the ESF EuroMEMBRANE Programme (31EM30-126143),
and the Novartis (Jubilꢀe) Foundation.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310969.
ꢂ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
As depicted schematically in Figure 1, MeNV-HaXS
penetrates cells and induces the dimerization of HaloTag
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Figure 2. MeNV-HaXS induces the formation of intracellular dimers of
HaloTag and SNAP-tag fusion proteins, which are cleaved upon UV
illumination. a) HeLa cells transfected with expression constructs for
SNAP-tag-GFP (SNAP-GFP) and HaloTag-GFP (Halo-GFP) fusion pro-
teins were exposed to 5 mm MeNV-HaXS or 5 mm light-insensitive
HaXS8 for the indicated times in cell culture medium at 378C.
Subsequently, cells were lysed and proteins were subjected to SDS-
PAGE and immunoblotting. Tagged proteins were detected using anti-
GFP (primary) and horseradish peroxidase labeled (secondary) anti-
bodies, and chemiluminescence (meansÆSEM, n=3). b) HeLa cells
expressing SNAP-GFP and Halo-GFP as in (a) were incubated with
5 mm MeNV-HaXS or HaXS8 for 15 min. Cells were then washed and
submerged in phosphate-buffered saline (PBS) to remove unreacted
compounds, and illuminated with a high-intensity UV lamp (100 W,
5 cm distance) for 10 min. Analysis of dimerization products was
performed as in (a); values represent meansÆSEM, n=3; * indicates
p<0.05.
Figure 1. A photocleavable, cell-permeable HaloTag- and SNAP-tag-
reactive CID with a methyl-6-nitroveratryl (MeNV) core was generated
(MeNV-HaXS). After cell entry, MeNV-HaXS dimerizes HaloTag- and
SNAP-tag-fused proteins of interest (POI). Illumination of MeNV-HaXS
(360 nm; e=4058mÀ1 cmÀ1; quantum yield=0.075) cleaves the link
between the POIs, and releases them from the covalent complex. For
the synthesis of MeNV-HaXS see the Supporting Information.
exposure to UV light revealed that MeNV-HaXS-induced
dimers were quantitatively cleaved after 10 min of illumina-
tion, whereas HaXS8-containing dimers remained intact
(Figure 2b).
and SNAP-Tag fusion proteins to form a covalently stabilized
complex. Upon illumination (360 nm), the MeNV group
undergoes photolysis, which triggers the cleavage of the
protein dimer and the release of cargo proteins.
MeNV-HaXS thus offers the possibility to trigger covalent
dimerization, and to subsequently release associated proteins
in a controlled way. This allows the manipulation of protein
localization, which can be exploited to mimic cellular signal-
ing events in a timed and localized fashion. Many signaling
events take place at defined intracellular locations. The
combination of a tagged anchor protein and MeNV-HaXS
exposure permits the depletion of tagged signaling enzymes
from their productive sites. Presently, MeNV-HaXS has been
validated for the targeting of tagged proteins to intracellular
organelles such as Golgi (Figure 3a), plasma membrane,
lysosomes, mitochondria, and the actin skeleton (Figure S2).
Targeted irradiation of MeNV-HaXS-anchored protein
complexes using a microscope equipped with an XY scanning
excitation laser for FRAP (fluorescence recovery after
photobleaching; 355 nm) indeed released tagged proteins
from the illuminated spots. HeLa cells were cotransfected
with cytosolic teal fluorescent (cyan) SNAP-tag fusion
protein (SNAP-mTFP1) and the Golgi anchor Halo-RFP-
Giantin, which was constructed by the fusion of a red
fluorescent protein (monomeric RFP; TagRFP) to a HaloTag
and a C-terminal Golgi-targeting motif derived from gian-
tin.[17]
MeNV-HaXS was optimized to match the cell perme-
ability of the noncleavable dimerizer of HaloTag and SNAP-
tag fusion proteins called HaXS8.[16] Time-dependent dime-
rization of HaloTag-GFP and SNAP-tag-GFP fusion proteins
expressed in HeLa cells was studied in response to the
addition of MeNV-HaXS and HaXS8, and we found that
MeNV-HaXS and HaXS8 produced dimers at a similar rates.
Slight differences were measurable at earlier time points (t <
10 min, Figure 2a), which became insignificant after 15 min of
treatment. These slight differences may be explained by the
increased molecular weight and higher polarity of MeNV-
HaXS, resulting from the incorporation of the PEG6 element.
MeNV-HaXS-induced HaloTag-SNAP-tag dimers were
stable for > 5 h, and exposure to ambient light did not
affect the stability of dimers.
Due to the matched dimerization properties of MeNV-
HaXS and HaXS8, the noncleavable HaXS8 is a valuable
control compound to monitor the efficiency of photocleavage,
and to detect potential side effects exerted by UV irradiation.
The efficiency of the intracellular photocleavage of MeNV-
HaXS was tested in HeLa cells expressing HaloTag-GFP and
SNAP-tag-GFP fusion proteins. A HaloTag-SNAP-tag com-
plex preformed with MeNV-HaXS could be cleaved in bulk
by illumination with a 360 nm lamp (Blak-Ray, B-100A,
UVP). Quantification of protein dimers before and after
Incubation with 5 mm of MeNV-HaXS or 5 mm of HaXS8
efficiently translocated cytosolic SNAP-mTFP1 to the cyto-
solic surface of the Golgi membrane (Figure 3a). After an
illumination pulse (8 ꢀ 5 ms at 355 nm) of a subset of Golgi-
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derived vesicles, SNAP-mTFP1 was efficiently and selectively
released from illuminated, but not from the non-illuminated
vesicular compartments (Figure 3a,c). No significant loss of
fluorescence intensity was observed when the light-insensitive
HaXS8 was used to anchor SNAP-mTFP1 at Golgi mem-
branes (Figure 3b,c), confirming that the observed light-
triggered decrease in fluorescence of SNAP-mTFP1 tethered
with MeNV-HaXS results from the release, and not from the
photobleaching of SNAP-mTFP1 fluorescence. Moreover,
membrane-anchored SNAP-mTFP1 detaches rapidly from
membranes after a laser pulse (t < 1 s). This demonstrates that
MeNV-HaXS can be utilized to manipulate protein local-
ization with excellent subcellular precision on a timescale of
seconds.
As a scanning FRAP laser is not part of standard
fluorescence microscope equipment, we explored the possi-
bility of using global field of view illumination with standard
DAPI excitation filters (377 Æ 25 nm; standard mercury
halide lamp) to induce the photocleavage of MeNV-HaXS-
induced protein complexes. We found that an illumination
time of < 20 s was sufficient to completely liberate SNAP-
mTFP1 from Golgi membranes (Figure 3d,e). A correspond-
ing increase of mTFP1 fluorescence intensity in the cytoplasm
demonstrates that the light-induced fluorescence decrease of
SNAP-mTFP1 at vesicles results from the release and not
from global photobleaching of SNAP-mTFP1 fluorescence.
Although slower due to the limited excitation energy,
photocleavage using DAPI excitation filters on a conventional
fluorescence microscope greatly expands the range of appli-
cations offered by the MeNV-HaXS system.
In contrast to previously reported CIDs, MeNV-HaXS
can instantaneously release its cargo when illuminated. To
further evaluate this concept, a nuclear probe was forced to
dock on a Golgi anchor. Expressed alone in HeLa cells, the
nuclear localization sequence (NLS)-containing cyan fluores-
cent probe (NLS-CFP-SNAP) accumulated in the nucleus. In
the presence of co-expressed Golgi anchor (Giantin-RFP-
Halo) and added MeNV-HaXS, NLS-CFP-SNAP was trap-
ped at perinuclear sites on the Golgi (Figure 4a). Subsequent
irradiation of cells triggered the release of NLS-CFP-SNAP
from the Golgi to the cytosol within seconds (Figure 4b).
Nuclear import of liberated NLS-CFP-SNAP was delayed
(Figure 4b), reflecting nuclear import processes.[18] This
illustrates that photocleavage of MeNV-HaXS is rapidly
achieved, and permits the study of nuclear import kinetics in
real time in a simple experimental setup.
In summary, MeNV-HaXS is the first cell-permeant CID
giving rise to the formation of covalently linked and photo-
Figure 3. Translocation of cytosolic SNAP-mTFP1 proteins to the Golgi
followed by their release upon UV illumination. a,b) HeLa cells
expressing SNAP-mTFP1 and Halo-RFP-Giantin were exposed to
a) 5 mm MeNV-HaXS or b) 5 mm HaXS8 in cell culture medium for
15 min at 378C, both of which induced translocation of cytosolic
SNAP-mTFP1 to the Golgi. SNAP-mTFP1 intensity was monitored in
the indicated circular regions of interest by live-cell microscopy, before
and after illumination of a subcellular region within the cell (white,
dotted square) with a scanning FRAP laser (8 areas ꢃ 5 ms at
355 nm). c) Quantification of mTFP1 fluorescence intensity in selected
regions of interest (labeled circles) for SNAP-mTFP1 after addition of
MeNV-HaXS (circles) or HaXS8 (squares). Quantifications of mTFP1
intensity in illuminated areas (green curves) and non-illuminated Golgi
vesicles (black curve); values are means ÆSEM, n=10, error bars not
shown when smaller than symbols. d) HeLa cells as in (a) were
exposed to 5 mm MeNV-HaXS and illuminated for 20 s using a standard
DAPI filter set on a conventional fluorescence microscope (t=20 s,
377Æ25 nm). e) Quantification of mTFP1 fluorescence intensity in
selected regions of interest (circles) at Golgi-derived vesicles (v), and
in the cytoplasm (c) before and after illumination as described in (d);
values are means ÆSEM, n=10, error omitted when smaller than
symbols; for more controls see Figure S3.
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Figure 4. Control of nuclear export and import by MeNV-HaXS. Translocation of the NLS-CFP-SNAP
probe from the nucleus to the Golgi was controlled by MeNV-HaXS and reversed by subsequent UV
illumination. a) HeLa cells expressing Halo-RFP-Giantin and NLS-CFP-SNAP were exposed to 5 mm
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cleavable protein complexes. Like other CID approaches, the
design of fusion proteins bearing interacting functional
protein domains must be carefully considered. The covalent
link formed by MeNV-HaXS simplifies monitoring and
validation of protein complexes greatly. MeNV-HaXS is
therefore a novel valuable tool that combines intracellular
protein dimerization with photocleavage triggered by a wave-
length compatible with widely used fluorescent reporter
proteins. The possibility to control protein localization by two
independent events can be exploited to sequester any protein
of interest away from its functional compartment. CID-
dependent trapping of enzymes to nonfunctional sites has
been established as an elegant approach to interrupt signaling
pathways.[19,20] Optically guided cleavage of MeNV-HaXS can
release anchored proteins and restore their function. Many
more scenarios are possible, for example the deployment of
on-off-on and off-on-off protocols and the orthogonal use of
MeNV-HaXS with other CIDs. The choice between global
and local illumination triggering dissociation of the CID-
mediated complex opens a wide range of applications, such as
investigation of cell-compartment-associated signaling, and
the simulation of cell-wide physiological and pathological
signaling dynamics.
Received: December 18, 2013
Published online: March 26, 2014
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Keywords: chemical inducers of dimerization · fusion proteins ·
.
photocleavable linkers · photolysis · protein–protein interactions
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