Light-Controlled Cell-Cycle Arrest and Apoptosis

Article abstract of DOI:10.1002/anie.202008267

Cell-cycle interference by small molecules has widely been used to study fundamental biological mechanisms and to treat a great variety of diseases, most notably cancer. However, at present only limited possibilities exist for spatio-temporal control of the cell cycle. Here we report on a photocaging strategy to reversibly arrest the cell cycle at metaphase or induce apoptosis using blue-light irradiation. The versatile proteasome inhibitor MG132 is photocaged directly at the reactive aldehyde function effectively masking its biological activity. Upon irradiation reversible cell-cycle arrest in the metaphase is demonstrated to take place in vivo. Similarly, apoptosis can efficiently be induced by irradiation of human cancer cells. With the developed photopharmacological approach spatio-temporal control of the cell cycle is thus enabled with very high modulation, as caged MG132 shows no effect on proliferation in the dark. In addition, full compatibility of photo-controlled uncaging with dynamic microscopy techniques in vivo is demonstrated. This visible-light responsive tool should be of great value for biological as well as medicinal approaches in need of high-precision targeting of the proteasome and thereby the cell cycle and apoptosis.

Full text of DOI:10.1002/anie.202008267

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Accepted Article
Title: Light Controlled Cell-Cycle Arrest and Apoptosis
Authors: Edgar Uhl, Friederike Wolff, Sriyash Mangal, Henry Dube,
and Esther Zanin
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10.1002/anie.202008267
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Light Controlled Cell-Cycle Arrest and Apoptosis
a
b
b
b
†
Edgar Uhl, † Friederike Wolff, Sriyash Mangal, Henry Dube,a,c Esther Zanin *
a Ludwig-Maximilians-Universität München, Department of Chemistry and Center for Integrated
Protein Science CIPSM, Butenandtstr. 5-13, 81377 München, Germany
b Ludwig-Maximilians-Universität München, Center for Integrated Protein Science CIPSM,
Department Biology II, Planegg-Martinsried, 82152 München, Germany
c Current address: Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Chemistry and
Pharmacy, Nikolaus-Fiebiger-Str. 10, 91058 Erlangen, Germany
* E-mail: zanin@biologie.uni-muenchen.de
† both authors contributed equally to the work
In memoriam of François Diederich.
Abstract
Cell-cycle interference by small molecules has widely been used to study fundamental
biological mechanisms and to treat a great variety of diseases, most notably cancer.
However, at present only limited possibilities exist for spatio-temporal control of the cell
cycle. Here we report on a photocaging strategy to reversibly arrest the cell cycle at
metaphase or induce apoptosis using blue light irradiation. The versatile proteasome
inhibitor MG132 is photocaged directly at the reactive aldehyde function effectively
masking its biological activity. Upon irradiation reversible cell cycle arrest in metaphase
is demonstrated to take place in vivo. Similarly, apoptosis can efficiently be induced by
irradiation of human cancer cells. With the developed photopharmacological approach
spatio-temporal control of the cell cycle is thus enabled with very high modulation, as
caged MG132 shows no effect on proliferation in the dark. In addition, full compatibility
of photo-controlled uncaging with dynamic microcopy techniques in vivo is
demonstrated. This visible-light responsive tool should be of great value for biological as
well as medicinal approaches in need of high-precision targeting of the proteasome and
thereby the cell cycle and apoptosis.
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Introduction
The development of versatile molecular tools enabling elucidation of fundamental bio-
chemical processes, detailed mechanistic understanding of diseases, sensitive and selective
diagnostics, or efficient treatment of medical conditions is a central goal of chemical biology
today. Towards this goal, significant progress has been made with the introduction of photo-
pharmacological concepts introducing spatio-temporal control to the bioactivity of molecular
agents.[1] Such an approach is not only valuable in the context of disease-treatment to reduce
side-effects and dosage but also shows high potential in enhancing diagnostics and in
fundamental research in biology, medicine, or pharmaceutical sciences. A great variety of
bioactive components are currently altered into light-responsive versions either by introducing
photoswitch motives[1d, 2] or by photolabile protecting groups[1f, 3] that cage the crucial
bioactive function. In general, photoswitchable variants offer intrinsically greater spatial
resolution due to the possibility of turning off their activity at places where it is not wanted.
Photocaging approaches however, possess the advantage of very high ON/OFF modulation of
activity as residual activity of the caged forms is typically extremely small. In biological
applications it is further highly desirable to use nondamaging visible or even near infrared
light for photocontrol. Consequentially fundamental research developing such photoswitches
and cages is currently a very active area in itself (for recent developments see selected ref. [4]
for photoswitches and ref. [5] for cages).
Using photoresponsive tools for the photoinduction of cell death in a selective fashion is of
particular interest not least for drug development. Different strategies are followed that
include photoswitching and photocaging,[1b, 2a, 2b, 3d, 6] or optogenetics,[7] as well as classic
photodynamic therapy approaches (for selected references see [8]). Photoinduction of
apoptosis has been achieved either by controlling cellular uptake of proapototic peptides[8a, 9]
or caspase 3,[10] optogenetically modified caspases,[7, 11] or photoswitchable BH3 peptides.[12]
Light control of the cell cycle has been achieved by the development of photoswitchable
microtubule inhibitors[13] or by optogenetic methods.[14] Despite these efforts, examples for
precise control of the cell cycle are very scarce at the moment stressing the need for effective
photopharmacological tools in this area. Light-controlled targeting of specifically the
proteasome - a key player in cell cycle regulation - is currently only possible with
photoswitchable Bortezomib© variants[15] enabling up to 5-fold modulation of activity.[15a]
MG132 is a well-established proteasome inhibitor that is used as a versatile biochemical tool
to study various cellular processes including the cell-cycle, apoptosis,[16] proteostasis, or virus
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life cycle[17] (Figure 1). It consists of a simple tri-peptide structure that mainly targets the
chymotrypsin-type catalytic centers at the beta-subunits of the proteasome – a multi-
component enzyme, which is responsible for protein degradation. At higher concentrations
MG132 also inhibits the protease calpain.[18] MG132 forms a covalent hemi-acetal adduct
with the catalytically active threonine in the active site of the proteasome efficiently inhibiting
its proteolytic activity.[19] Proteasome inhibition severely alters protein turnover of the cell. Of
special importance in this regard is inhibition of the proteasomes role in cell-cycle control
leading to cell-cycle arrest in metaphase.[20] Prolonged inhibition of the proteasome ultimately
induces apoptosis, preferentially so in cancer cells as compared to healthy ones.[21] Therefore,
MG132 also represents an interesting candidate for cancer research and antidotes although its
untamed reactivity and rather fast metabolism currently hampers development into a medical
drug.[22] Taken together MG132 can be regarded as a multi-faceted, versatile, and easily
applicable biochemical agent, however control of MG132 activity in time or place is currently
not possible. We envisioned that especially a light-responsive MG132 version would thus
represent a highly valuable tool for a plethora of different applications. In general, such tool
would enable studying fundamental and highly dynamic biological processes directly linked
to proteasome function with precise spatial and temporal resolution. For example dynamic
modulation of the proteasome-dependent protein turnover would become possible during cell
cycle progression, cellular signaling, or immune responses. The dynamic nature and
resolution advantage would be even more critical in the context of tissues or organisms to
instill local and timed effects – e.g. in applications for local cancer treatment, tissue targeting,
or apoptosis studies in developing animals, to name a few exciting possibilities. Furthermore
caging could prevent fast metabolism of MG132 during its distribution in an organism and
thus would potentially alleviate some of the challenges encountered in its drug development.
In this work we present a photocaging strategy allowing us to convert MG132 into a
noneffective agent in its protected MG132-Cage (1) form but trigger full functionality upon
irradiation with blue light (Figure 1). For this purpose the covalent binding moiety i.e. the
aldehyde function of MG132 is protected as mixed acetal of the photolabile 4,5-dimethoxy-2-
nitrobenzyl (DMNB) cage,[23] which fully blocks binding of the proteasome. This caging
approach therefore is highly effective as it targets the main reactivity of MG132 directly while
also removing potential toxicity of the aldehyde, i.e. side effects and unselective reactions
with nucleophiles within a cell, before uncaging. Blue light irradiation at 405 nm enables full
recovery of the MG132 structure at a given time and place and thus allows to instill spatio-
temporal control of the inhibitor function in living cells. The high potential of this new light-
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responsive proteasome inhibitor for biological as well as pharmacological research is
showcased by light-induced cell-cycle arrest as well as apoptosis induction in living human
tissue culture cells.
Figure 1
Transformation of the versatile covalent proteasome inhibitor MG132 into a blue
light-controlled biomolecular tool MG132-Cage (1) enabling spatio-temporal control
of various fundamental biological phenomena. The reactive aldehyde anchorpoint of
MG132 is caged by a photolabile protecting group in the form of an acetal rendering
the resulting compound MG132-Cage (1) fully inactive. Upon blue light irradiation
the aldehyde function is restored and covalent proteasome binding and inhibition is
triggered.
Results and Discussion
Synthesis and Photochemical Assessment
To be able to effectively mask the biological activity of MG132, the main functionality
responsible for covalent binding to the proteasome, i.e. the aldehyde function, was targeted
for protection with a photolabile group. To regain the aldehyde upon irradiation, the oxidation
state of its carbon atom should not be altered during uncaging and thus protection as acetal
was deemed the most straight-forward approach. To the best of our knowledge such an
aldehyde caging/uncaging method - albeit recently introduced for simple organic aldehydes[24]
- has not been used for bioactive compounds or in a biological setting so far. The DMNB
photolabile group was chosen for its well established photocaging properties and particularly
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because of its blue light responsiveness, which is favorable for biological applications.[23]
MG132-Cage (1) was synthesized in a convergent and iterative peptide-coupling sequence as
shown in Scheme 1 (for full details see Supporting Information, Scheme S1-S4, and Figure
S1-S8). Carboxybenzyl (Z)-protected leucine 2 was first coupled with leucine methyl ester
and after subsequent ester hydrolysis dipeptide 3 was obtained in 57% yield. The DMNB
photolabile protection group was introduced to another tert-butyloxycarbonyl (Boc)-protected
leucine (4) via an ester linkage to give the Boc-protected amine 5 in 69% yield. After Boc-
deprotection of 5 using trifluoroacetic acid (TFA) the corresponding free amine 7 was joined
with 3 to the tripeptide 6 using standard peptide coupling conditions in 77% over the two
steps. A final one-pot ester reduction and mixed acetal formation sequence gave the desired
MG132-Cage (1) in acceptable 17%. Additionally the DMNB cage was introduced to
propionic acid to obtain the control compound Propionic Acid-Cage (8, for details see the
Supporting Information).
Scheme 1
Synthesis of photocaged MG132-Cage (1). The synthetic precursor MG132-Ester-
Cage (6) is used as control compound in the biological experiments. The caging group
DMNB is introduced via an ester link early in the synthesis. After successive peptide
couplings a final reductive step yields MG132-Cage with the aldehyde protected as
acetal. Conditions: i) Leucine methyl ester x HCl, PyBOP, DIPEA; ii) NaOH 57%
yield over two steps; iii) DMNB-alcohol, DCC, DMAP 69% yield; iv) 5, TFA; v) 3,
DIPEA, DCC, HOBt, 77% yield over two steps; vi) DIBAL-H, acetic anhydride, 17%
yield.
1
Photodeprotection efficiency of 1 was tested using UV/Vis and especially H NMR
spectroscopy as quantitative tool (Figure 2a and Figure S13 and S15). Irradiation with various
wavelengths was conducted showing 405 nm to be the longest wavelength at which full
photodeprotection proceeded in reasonable time scales, i.e. within several minutes at the high
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NMR concentrations (approximately 2-5 mM in DMSO solution). Concomitant with the
decreasing signals of 1 the known signals of MG132 with the indicative aldehyde signal at 9.5
ppm increased. The responsiveness of MG132-Cage towards 405 nm is owed to its absorption
profile, which tails out but does not yet reach zero at this wavelength. Irradiation at UV/Vis
concentrations (typically 10-5 M in DMSO solution) with 405 nm light shows the typical
spectral changes expected for deprotection of a DMNB group (Figure 2b). Likewise,
photodeprotection of the two control compounds MG132-Ester-Cage (6) and Propionic Acid-
Cage (8) in DMSO solution leads to the analogous absorption spectral changes (see Figure
S11 and S12, respectively) proving effective uncaging also in these cases. Quantum yield
measurements were conducted for MG132-Cage in DMSO solution due to solubility issues in
buffer media. Consistently with a previous literature report for the same DMNB photocaging
group and photorelease of alcohols,[25] we measured a quantum yield of 1.0% for 405 nm
irradiation (for details see Supporting Information and Figure S14-S16). This quantum yield
is high enough to warrant effective photodeprotection within minutes under the biological
experimental conditions. In a typical biological setup (see below) 1 mL of 5 or 10 µM
solutions of caged compound are used (5 or 10 nmol), which are irradiated with a 105 mW
405 nm LED or a 375 mW 405 nm LED positioned in ca. 2 cm distance from the sample
solutions.
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Figure 2
Blue light irradiation of MG132-Cage (1) leads to release of MG132 and restoration
1
of its covalently binding aldehyde function. a) H NMR monitoring of the uncaging
process shows almost complete photodeprotection while at the same time the signal of
the aldehyde proton of MG132 increases. The spectrum of pure MG132 is shown at
the top, and the spectrum of pure MG132-Cage directly below. Spectra recorded in 1
min intervals during irradiation of a DMSO-d6 solution of MG132-Cage with a 405
nm LED are shown starting with the third spectrum from the top. b) Changes of the
UV/Vis absorption recorded in intervals during 405 nm irradiation of MG132-Cage in
DMSO (blue starting point to red final spectrum).
Light-induced metaphase arrest in HeLa cells
After demonstrating proper photochemical response of MG132-Cage we moved to in vivo
studies to showcase its functionality in biological context. For this purpose, light-induced cell
cycle control was evidenced using a quantitative imaging assay in HeLa cells (Figure 3a, b
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and Figure S17). The cell cycle consists of interphase, during which the cell content including
the DNA is duplicated, and the mitotic phase during which the content of the mother cell is
distributed to the two daughter cells. The mitotic phase starts with the prometaphase, followed
by metaphase, anaphase and telophase. During prometaphase the DNA condenses and at
metaphase the chromosomes align at the metaphase plate. After chromosome alignment on
the metaphase plate the cell proceeds into anaphase and distributes the chromosomes to the
two daughter cells. Finally, the mother cell is split into two daughter cells during telophase.
During cell division the cell needs to ensure that it only proceeds into anaphase after all
chromosomes are correctly attached to the mitotic spindle and aligned at the metaphase plate.
Chromosome alignment is monitored by the spindle assembly checkpoint (SAC).[26] In case
not all chromosomes are aligned properly at the metaphase plate the SAC keeps the cell in
metaphase. To allow metaphase to anaphase transition the proteasome needs to be active to
degrade key cell-cycle regulators for example cyclin B[20] and securin.[27] Our first aim was to
arrest cells with light in metaphase by using MG132-Cage. HeLa cells were synchronized to
enrich for mitotic cells. Cells were then incubated with 5 or 10 µM DMSO solutions of
MG132-Cage and either exposed to low power 405 nm LED light for 5 or 10 min or
maintained in the dark. After light exposure cells were incubated for 2 h after which they were
fixed and stained for α-tubulin, filamentous actin (F-actin), and DNA to determine the cell-
cycle stage by confocal microscopy (Figure 3b). Over >95% of the mitotic cells were arrested
in metaphase when cells were exposed to 10 min light irradiation (Figure 3c, d, and Figure
S17). Similar metaphase arrest was observed when cells were treated directly with MG132 in
5 µM and 10 µM concentrations with and without 10 min LED light irradiation (Figure 3b and
Figure S17b). As a control MG132-Cage-treated cells were maintained in the dark for the
same time period. We observed that 58% of those cells were in ana-/telophase, which was
indistinguishable from untreated cells or cells treated with DMSO (Figure 3d and Figure
S17b). Since mitotic cells spend the same amount of time in metaphase as in ana- and
telophase combined (Figure 3b, top) a 1:1 ration of metaphase to ana-/telophase is expected
for non-disturbing conditions. The control experiments thus confirm that uncaging of MG132-
Cage in the absence of light is neglectable in vivo. To exclude that 405 nm light treatment
used for uncaging results in an unspecific metaphase arrest, we also exposed DMSO-treated
and untreated cells to irradiation. We found that DMSO-treated and untreated cells that were
exposed to 405 nm light for 10 min behaved indistinguishably from control cells kept in the
dark. In both cases the cells proceeded readily to ana-/telophase (Figure 3d and Figure S17b).
This demonstrated that 405 nm irradiation does not induce cell-cycle arrest at metaphase. To
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test whether the light-induced arrest in metaphase was reversible, an inhibitor-washout
protocol was applied 2 h after light exposure of MG132-Cage treated cells (Figure 3b). After
another 2 h waiting time the typical 51% of the mitotic cells were in ana-/telophase
evidencing light-induced metaphase arrest can be fully reversed and cells proceed normally
into anaphase.
During photodeprotection MG132-Cage is split into MG132 and a nitrosobenzaldehyde. To
exclude that the nitrosobenzaldehyde induces metaphase arrest we used the synthetic
precursor MG132-Ester-Cage (compound 6) as well as propionic acid, which was caged with
the same photolabile DMNB cage. Upon photoirradiation MG132-Ester-Cage as well as
Propionic-Acid-Cage release the corresponding carboxylic acids (the simple tripeptide Z-Leu-
Leu-Leu and propionic acid), which are nontoxic to cells and do not inhibit the proteasome.
Both caged control compounds also release the very same nitrosobenzaldehyde. Cells that
were incubated with 10 µM MG132-Ester-Cage or Propionic-Acid-Cage and irradiated for 10
min did not arrest in metaphase (Figure 3d and Figure S17b). Therefore, light exposed
MG132-Cage-treated cells arrest in metaphase due to the release of MG132 and not due to the
presence of released nitrosobenzaldehyde. Finally, we addressed whether uncaging of
MG132-Cage changes its diffusion across the plasma membrane. To test if uncaging of
MG132-Cage promotes its cellular uptake we incubated cells for 1 h with MG132-Cage in the
dark and subsequently exchanged the MG132-Cage containing medium with fresh medium
without MG132-Cage just before irradiation with 405 nm light. We found 100% of the cells
were arrested in metaphase after another 2 h incubation in the dark (Figure 3d, prewash
condition). This result was similar to the one from the analogous experiment lacking a
prewash step. It shows that MG132-Cage is taken up by the cells and is present in the
cytoplasm prior photoactivation. Light-induced metaphase arrest is thus due to the release of
the active compound MG132 and not due to allowing cellular uptake of released MG132.
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Figure 3
Cell-cycle control with blue light. a) General mechanism of cell cycle arrest induced
by photoactivated MG132-Cage. Blue light irradiation releases MG132, which
inhibits the proteasome leading to arrest in metaphase. For depiction of the human
[28]
proteasome crystallographic data (PDB code 5GJR) from Ref.
were used. b) Key
experimental setups to quantify blue light induced metaphase arrest. c) Confocal
microscopy images of HeLa cells incubated with MG132-Cage and either maintained
in the dark (left) or exposed for 10 min to 405 nm light (right). Cells were stained for
DNA (blue), F-actin (green), and α-tubulin (orange). Metaphase cells are marked with
an asterisk and ana-/telophase cells with a green bar. Representative confocal images
of a metaphase, anaphase, and telophase cell stained for DNA, F-actin, and α-tubulin
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are shown at the bottom. d) Mean percentages of mitotic cells in metaphase and ana-
/telophase for the indicated conditions. The mean of 3-7 independent experiments is
shown and error bars represent standard deviations. For the different conditions a
minimum of 110 and maximum of 476 cells were analyzed individually.
Light-induced apoptosis in HeLa cells
Proteasome inhibitors induce apoptosis in many cell types including cancer cells. Inhibition of
the proteasome results in an increase of proapoptotic factors such as p53 and caspases.[29] For
this reason proteasome inhibitors are promising candidates in cancer therapies, bortezomib©
for example is used to treat multiple myeloma.[30] Our next goal was to induce apoptosis with
blue light in HeLa cells using MG132-Cage (Figure 4a). HeLa cells were incubated with
DMSO or MG132-Cage and either exposed to 405 nm light for 10 min or kept in the dark. To
monitor cell viability over time the number of healthy cells was counted using fixed samples 0
h, 12 h, 18 h, and 24 h after light exposure (Figure 4b). In the negative control experiments
the number of cells doubled within 24 h (200%). However, for cells incubated with MG132-
Cage and exposed to light for 10 min the cell number was reduced to 41% after 18 h and 2%
after 24 h (Figure 4c, d). This decrease in cell number over time was comparable to cells
treated with 10 µM MG132 (Figure S18a) suggesting that MG132-Cage is very effectively
uncaged in vivo. Cells incubated with MG132-Cage but kept in the dark continued to
proliferate similar to DMSO-treated cells (Figure 4d). This suggests that MG132-Cage is not
uncaged in the absence of light even over prolonged incubation time of several hours. After
irradiating control cells with 405 nm light for 10 min we found that light exposure had no
effect on cell number suggesting that the amount of light used to uncage MG132-Cage is not
toxic. We also performed an inhibitor-washout experiment to determine whether the effects of
2 h exposure to uncaged MG132-Cage can be reversed. Cells treated with MG132-Cage,
DMSO, or MG132 were exposed to 405 nm light for 10 min and incubated for 2 h followed
by inhibitor washout. When we counted cell numbers 24 h later we found that MG132-Cage,
DMSO, or MG132 treated cells that had undergone washout displayed similar cell numbers
like DMSO-treated control cells without applied washout (Figure S18b). To exclude that the
co-released nitrosobenzaldehyde reduces cell viability we incubated cells with 10 µM MG132-
Ester-Cage as well as with 10 µM Propionic Acid-Cage and exposed them to 405 nm light for
10 min. After 24 h the healthy cell number in these two control experiments were comparable
to the ones observed for DMSO-treated cells indicating that the co-released
nitrosobenzaldehyde does not negatively affect cellular growth (Figure S18b). To corroborate
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these findings we quantified cell viability using a resazurin-based assay. In living cells
resazurin is reduced to strongly fluorescent resorufin.[31] When we plotted fluorescence
intensity for MG132-Cage (with and without 405 nm light), MG132-Ester-Cage (with 405 nm
light), Propionic Acid-Cage (with 405 nm light), MG132 (dark) treated cells against DMSO-
treated control cells similar results were obtained as for the single-cell fixed analysis (Figure
S19).
To evaluate whether uncaging of MG132-Cage kills cells with the same efficiency as MG132
we performed dose-response experiments. Cells were incubated with different concentrations
of MG132 (dark) or MG132-Cage (irradiated with 405 nm light). After 24 h cells were fixed
and stained and the number of healthy cells was counted and plotted relative to DMSO-treated
cells (Figure S20). For both conditions the cell number started to increase at ~1,5 µM and
reached control levels at ~0,1 µM concentrations. Our dose-response experiments are in very
good agreement with a previous literature report of the dose response of MG132 induced cell
death.[32] These experiments confirm that blue light irradiation of MG132-Cage kills cells in a
dose-dependent manner.
To determine whether cells are dying indeed via the apoptosis pathway we also analyzed the
cleavage of poly (ADP-ribose) polymerase-1 (PARP). In healthy cells PARP mediates DNA
damage repair and with the induction of apoptosis PARP is cleaved by caspases. PARP is a
114 kDa protein and upon caspase activation it is cleaved into a 89 and a 24 kDa fragment.[33]
PARP cleavage was analyzed by immunoblots and cells that were incubated with MG132-
Cage and exposed to 405 nm light for 10 min showed a reduction in the 114 kDa band and an
increase in the 89 kDa fragment, which was similar to cells treated with MG132 (Figure S21).
DMSO-treated cells and MG132-Cage treated cells that were maintained in the dark showed a
prominent band at 114 kDa and were indistinguishable from each other.
During apoptosis phosphatidylserine translocates from the inner to the outer leaflet of the
plasma membrane to generate an ‘eat me’ signal for macrophages.[34] To further confirm that
MG132-Cage treated and irradiated cells die by apoptosis, cells were stained with annexin V,
which strongly binds to phosphatidylserine.[35] The cells were additionally incubated with
propidium iodide (PI), which is a membrane impermeable dye that only labels dead cells.
More than 99% of the DMSO-treated cells that were either incubated in the dark or irradiated
with 405 nm light for 10 min were negative for annexin V and PI (Figure 4e, f). Treatment of
cells with MG132-Cage and subsequent exposure to 405 nm light for 10 min resulted in 53%
annexin V positive and PI negative cells indicating that these cells were still alive but initiated
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apoptosis. In addition, 7% of the cells were positive for both annexin V and PI showing that
these cells were dead. Importantly cells incubated with MG132-Cage but maintained in the
dark were indistinguishable from DMSO-treated control cells. Together with the dose-
response results these experiments confirm that release of MG132 upon blue light irradiation
induces apoptosis in a dose-dependent manner.
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Figure 4
Induction of apoptosis in HeLa cells with blue light. a) Simplified mechanism of
apoptosis induction by photoactivated MG132-Cage. Blue light irradiation triggers
MG132 release, which inhibits the proteasome leading to apoptosis of the respective
cell after prolonged (t) exposure. b) Experimental setups to quantify blue light induced
cell death over time. c) Confocal microscopy images of HeLa cells incubated with 10
µM MG132-Cage and either maintained in the dark for 24 h (left) or exposed to 405
nm light for 10 min and then cultured for 24 h (right, the few remaining cells display
hallmarks of apoptosis such as cell shrinkage and DNA condensation). d) Mean
percentages of healthy cells at different time points under the indicated conditions.
Photodeprotection of 10 µM MG132-Cage results in complete cell death 24 hours after
irradiation. Mean of 2 independent experiments is shown and error bars represent
standard deviation. e) Maximum z-projections of five images of HeLa cells stained
with Hoechst dye to label all nuclei (DNA, blue), annexin V (green), and PI (red).
HeLa cells were treated with 0.1% DMSO or 10 µM MG132 and maintained in the
dark, or treated with 10 µM MG132-Cage and either exposed to 405 nm light for 10
min or kept in the dark. The 20 h time point was chosen to monitor annexin and PI
labeling while cells are dying. f) Percentages of cells that were labeled by annexin V
and/or PI for the indicated conditions. The mean of 3 independent experiments is
shown and error bars represent standard deviation. For the different conditions a
minimum of 123 and maximum of 409 cells were analyzed individually.
To dynamically follow the light-induced cell cycle arrest as well as execution of apoptosis
upon uncaging of MG132-Cage a live-cell imaging approach was chosen. Transmission and
confocal images of mitotic HeLa cells expressing fluorescently labeled α-tubulin (mKate-α-
tubulin) were acquired. For transmission images a 470 nm cut-off filter was placed in the light
path to exclude the blue parts of the imaging light and thus to prevent uncaging by image
acquisition. Cells were treated with 10 µM MG132-Cage and either exposed to 405 nm LED
light for 5 min or maintained in the dark. After light exposure imaging was resumed and
continued for 20 h. As expected from our analysis of fixed cells, we found that 405 nm light
exposure resulted in efficient cell cycle arrest at metaphase for several hours (Figure 5a,
Suppl. Movie 1). Following 405 nm light exposed cells for longer time revealed that after ~12
h cells started to shrink and display strong plasma membrane blebbing, which are both
hallmarks of apoptosis (Figure 5b, Suppl. Movie 2). 20 h post light exposure the majority of
cells were undergoing apoptosis. In contrast, cells treated with MG132-Cage but not exposed
to 405 nm LED light continued to proliferate and did not show any signs of apoptosis. To
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10.1002/anie.202008267
Angewandte Chemie International Edition
dynamically follow reversibility of blue light induced metaphase arrest an additional washout
experiment was performed using live-cell imaging. MG132-Cage treated cells were exposed
to 405 nm LED light which resulted in metaphase arrest (Figure 5c, Figure S22, and Suppl.
Movie 3). After 2 h cells were washed and imaging was continued. We observed that
metaphase cells entered anaphase after the washout and completed cell division. Furthermore,
long-term imaging revealed that the cells continued to proliferate and did not display signs of
apoptosis. These results demonstrate that uncaging of MG132-Cage can easily be combined
with live-cell imaging techniques allowing the investigation of dynamic proteasome-
dependent processes in living cells or organisms at high spatial and temporal resolution.
Figure 5
Live-cell imaging of blue light induced metaphase arrest and apoptosis in HeLa cells.
Cells treated with 10 µM MG132-Cage were either exposed to 405 nm LED light for 5
min (top row) or maintained in the dark (bottom row). Merged transmission and
confocal mKate-α-tubulin (maximum z-projection shown in green) images for
indicated time points are shown. Scale bars represent 20 µm. a) Prometaphase cells
exposed to 405 nm light arrested in metaphase (top row). Control cells in the dark
entered anaphase and completed cell division (bottom row). Selected time frames are
taken from Suppl. Movie 1. b) First apoptotic cells were observed ~12 h after 405 nm
15
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10.1002/anie.202008267
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light exposure (top row). Cells maintained in the dark continued proliferation without
undergoing apoptosis (bottom row). Selected time frames are taken from Suppl. Movie
2. c) Prometaphase cells exposed to 405 nm light arrested in metaphase for 2 h. After
washing the cells proliferation continues establishing reversibility of the arrest. After
20 h no signs of apoptosis are seen. Selected time frames are taken from Suppl. Movie
3.
In summary, we present an efficient approach for establishing spatio-temporal control over
the cell cycle stage as well as apoptosis of human cancer cells by blue light irradiation. To this
end a photocaging strategy is used, which effectively masks the reactive and covalently
binding aldehyde function of the versatile proteasome inhibitor MG132. In the presence of
MG132-Cage without light, cell viability and proliferation remained unperturbed in vivo.
However, after 5-10 min blue light irradiation prometaphase cells treated with MG132-Cage
are arrested in metaphase for several hours. This metaphase arrest can be alleviated by
applying a washing protocol. If cells are irradiated in the presence of MG132-Cage and
subsequently cultured for prolonged time in the dark almost quantitative apoptosis is induced.
Both metaphase arrest as well as apoptosis were followed dynamically with a fluorescent live-
cell imaging approach proving full compatibility of photo-controlled uncaging with dynamic
microscopy techniques in vivo. With this approach a highly promising photopharmacological
tool for full spatio-temporal control of cell cycle, apoptosis, and cancer treatment via simple
non-toxic blue light irradiation is presented. We believe that MG132-Cage will be of great
interest for a broad variety of biologically and medically oriented research. Our future efforts
are directed at establishing full spatial control over proteasome activity in tissues and
organisms as well as studying highly dynamic processes related to the biochemistry of the
proteasome.
Acknowledgements
E. Zanin thanks the DFG for an Emmy Noether fellowship (ZA619/3). S. Mangal was a
member of the International Max Planck Research School for Molecular Life Sciences. H.
Dube thanks the Deutsche Forschungsgemeinschaft (DFG) for an Emmy Noether fellowship
(DU 1414/1-1). We further thank the Deutsche Forschungsgemeinschaft (SFB 749, A12) and
the Cluster of Excellence ’Center for Integrated Protein Science Munich’ (CIPSM) for
financial support. Microscopy was performed at the center for advanced light microscopy
16
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10.1002/anie.202008267
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(CALM) at the LMU. We also thank Stefan Wiedemann for help with the synthesis as well as
Martin Parniske and Arne Weiberg for access to the plate reader and Constance Tisserant for
technical advice.
Keywords: chemical biology • cell cycle • apoptosis • proteasome • photopharmacology •
MG132
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TOC Figure
Blue light control of the proteasome is achieved using a new photocaging approach.
Photolabile protection of the versatile proteasome inhibitor MG132 at the reactive aldehyde
function abolished proteasome binding. After irradiation MG132 activity is restored resulting
in cell cycle arrest at metaphase or apoptosis. This visible light responsive tool opens up new
avenues for spatiotemporal control of the proteasome in basic and medicinal research.
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