Light-activated regulation of cofilin dynamics using a photocaged hydrogen peroxide generator

Article abstract of DOI:10.1021/ja107783j

Hydrogen peroxide (H₂O₂) can exert diverse signaling and stress responses within living systems depending on its spatial and temporal dynamics. Here we report a new small-molecule probe for producing H ₂O₂ on demand upon photoactivation and its application for optical regulation of cofilin-actin rod formation in living cells. This chemical method offers many potential opportunities for dissecting biological roles for H₂O₂ as well as remote control of cell behavior via H₂O₂-mediated pathways.

Full text of DOI:10.1021/ja107783j

Published on Web 11/15/2010
Light-Activated Regulation of Cofilin Dynamics Using a Photocaged
Hydrogen Peroxide Generator
Evan W. Miller,† Nicolas Taulet,§ Carl S. Onak,† Elizabeth J. New,† Julie K. Lanselle,†
Gillian S. Smelick,† and Christopher J. Chang*,†,‡
Department of Chemistry and the Howard Hughes Medical Institute, UniVersity of California, Berkeley,
California 94720, United States and Department of Immunology and Microbial Science and Department of Cell
Biology, The Scripps Research Institute, La Jolla, California 92037, United States
Received August 29, 2010; E-mail: chrischang@berkeley.edu
Scheme 1. Photolysis of CPG1 Releases 1,2,4-Trihydroxybenzene
Abstract: Hydrogen peroxide (H2O2) can exert diverse signaling
and stress responses within living systems depending on its
spatial and temporal dynamics. Here we report a new small-
molecule probe for producing H2O2 on demand upon photoacti-
vation and its application for optical regulation of cofilin-actin rod
formation in living cells. This chemical method offers many
potential opportunities for dissecting biological roles for H2O2 as
well as remote control of cell behavior via H2O2-mediated
pathways.
(4), which Sequentially Reduces Molecular Oxygen to Give H2O2
via a Superoxide Intermediate
H2O2 in cell signaling and stress cascades as well as real-time, remote
control of cell behavior via H2O2-mediated pathways.
The design and action of CPG1 is shown in Scheme 1. We
reasoned that electron-rich ortho- or para-hydroquinones, which
are known to produce H2O2 by reduction of molecular oxygen with
concomitant oxidation to the corresponding quinone species via
semiquinone radical and superoxide (O2-) intermediates,35 would
provide a platform amenable to delivery of caged H2O2 in a fashion
similar to that for endogenous H2O2-producing NAD(P)H oxidases.
Hydrogen peroxide (H2O2) is a potent small-molecule oxidant
that can influence the growth, development, and fitness of living
organisms in a wide variety of ways.1 Aberrant production of H2O2
leads to oxidative stress and damage cascades connected to aging2
and diseases ranging from cancer2 to neurodegeneration,3 whereas
regulated H2O2 fluxes are used in phagocytic killing of invading
pathogens.4 In addition, newer studies link H2O2 signaling5-9 to
cell growth, proliferation, and migration events that form the basis
for beneficial processes like wound healing10 and neurotransmis-
sion.11
-
Whereas in both systems O2 is the initial ROS generated upon
reduction of O2, the relative cellular stability of H2O2 compared to
O2 support the latter as the primary signaling agent.1 In initial
-
experiments we screened several hydroquinones for their ability to
produce H2O2 in water as measured using the H2O2-specific
fluorescent probe Peroxyfluor 1 (PF1).12 The electron-rich polyphe-
nol 1,2,4-trihydroxybenzene 4 produces more H2O2 than its
structural isomer pyrogallol (Figures S1 and S2). H2O2 production
by 4 is completely abrogated by the addition of 25 U/mL catalase,
whereas addition of 50 U/mL superoxide dismutase (SOD) reduces
H2O2 levels by 50%, establishing the role of superoxide as an
intermediate in the CPG1-mediated production of H2O2 and
suggesting that oxidation of 4 or its semiquinone radical competes
with the spontaneous dismutation of superoxide in the absence of
enzymes (Figure S3). When loaded into HEK 293 cells, the
peracetylated form of 4 is membrane-permeable and, after in situ
ester hydrolysis by intracellular esterases, generates levels of H2O2
that are detectable by PF1 (Figure S4).
Based on these results, we synthesized CPG1 as shown in
Scheme S1. Installation of a photolabile ortho-nitrobenzyl ether at
the 1-hydroxy position effectively blocks quinone formation by
imposing a meta arrangement between the two remaining hydroxyl
groups, rendering CPG1 oxidatively inert. In turn, removal of this
protecting group by photolysis furnishes the reactive hydroquinone
H2O2 generator. Alkylation of bis-MEM protected phenol 1 with
2-nitrobenzyl bromide in acetone affords nitrobenzyl ether 2 in 86%
yield. Removal of the MEM groups using para-toluenesulfonic acid
monohydrate in ethanol delivers nitrobenzyl-caged CPG1 3 in 74%
yield. In DPBS at pH 7, CPG1 features a moderate-intensity UV
absorbance band centered at 280 nm (ꢀ ) 6400 M-1 cm-1).
Because the localized dynamics of H2O2 are intimately linked to
disparate physiological and/or pathological consequences, a major
challenge in dissecting roles for H2O2 and its downstream effects is a
dearth of methods for directly probing this reactive oxygen metabolite
in complex biological settings. In this context, the vast majority of
chemical tools developed to study H2O2 in living environments are
fluorescent probes,12-21 but another potentially powerful chemical
approach for interrogating H2O2 biology is through the use of
photocaged compounds,22-24 which have been employed to release
bioactive molecules such as ATP,25 neurotransmitters,26-28 metal
cations,29-31 peptides,32 and proteins33,34 in living cells with spatial
and temporal fidelity by unmasking a photolabile protecting group.
Such synthetic systems offer the opportunity to turn on a specific type
of chemical reactivity within live biological specimens with precise
spatial and temporal control. Here we report the synthesis and
properties of Caged Peroxide Generator 1 (CPG1), a new type of
synthetic small-molecule probe that can produce H2O2 upon photo-
activation. Molecular imaging with H2O2-sensitive fluorescent reporters
establishes that CPG1 can deliver H2O2 on demand to living cells by
photochemical manipulation. Moreover, we have applied the photo-
caged H2O2 reactivity of CPG1 for optical control of cytoskeleton
dynamics through redox-regulated cofilin-actin rod formation, presag-
ing the utility of this new chemical method for elucidating roles for
† Department of Chemistry, University of California, Berkeley.
‡ Howard Hughes Medical Institute, University of California, Berkeley.
§ The Scripps Research Institute.
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Figure 2. Live-cell imaging of H2O2-induced cofilin-actin rod formation
in eGFP-cofilin expressing HeLa cells by exogenous H2O2 addition or light-
induced generation of H2O2 with CPG1. (a) eGFP-cofilin HeLa cells treated
with 10 µM H2O2 show no rod formation. (b) Cells stimulated with 50 µM
H2O2 produce a regulated pattern of rod formation that localizes to the
periphery of the cell membrane. (c) Samples treated with 100 µM H2O2
form numerous intracellular cofilin-actin rods. (d) Samples treated with 500
µM H2O2 also form numerous intracellular cofilin-actin rods. (e) Control
eGFP-cofilin HeLa cells without CPG1 or UV irradiation show no rod
formation. (f) Control eGFP-cofilin HeLa cells loaded with 200 µM Ac-
CPG1 without UV irradiation do not show rod formation. (g) Control cells
without CPG1 that were treated with an identical pulse of UV excitation
show negligible rod formation, establishing that cofilin activation requires
light-initiated decaging of CPG1 to elicit H2O2 production. (h) CPG1-loaded
HeLa cells activated with UV light exhibit controlled rod formation in a
global pattern similar to that observed upon stimulation with 50 µM H2O2.
(i) Control cells from the same imaging dish as (h), which were not exposed
to UV light and do not show rod formation. All images displayed were
taken 26 min after treatment with either H2O2 or UV light, except for (i),
which was taken immediately after (h). Cells treated with Ac-CPG1 were
loaded for 10 min at 37 °C, washed, and then imaged. UV light was provided
in 1 s pulses for the first 9 min of the experiment. Scale bar ) 30 µm.
Figure 1. Controlled generation of intracellular H2O2 by photolysis of
CPG1. (a) Live-cell imaging of HyPer-expressing HEK 293 cells loaded
with 100 µM Ac-CPG1. (b) Live-cell imaging of the same cells after
photodecaging of CPG1 with UV light to initiate H2O2 production from
1,2,4-trihydroxybenzene. (c) Real-time fluorescence intensity readouts of
HyPer-expressing HEK cells upon UV irradiation (40 s) in the presence or
absence of CPG1, showing cells loaded with Ac-CPG1 and activated by
UV light (black line), cells loaded with Ac-CPG1 but not exposed to UV
irradiation (blue line), cells without CPG1 under UV irradiation (red line),
and cells without CPG1 that are also not exposed to UV activation (green
line). HyPer fluorescence is calculated from 10-15 cells per experiment.
Error bars are (SEM for at least three separate experiments. * ) p < 0.05.
*** ) p < 0.01 (Student’s t test). (d) Temporal control of phototriggered
release of H2O2 in living HEK cells by CPG1, as monitored by HyPer
fluorescence. HyPer-expressing HEK cells were loaded with Ac-CPG1 as
in panel (a) and then washed and imaged on stage. UV light (40 s) was
delivered at varying times, and plots show data where UV excitation to
elicit CPG1-mediated generation of H2O2 occurs after 5 min (black line),
11 min (red line), 17 min (blue line), or 23 min (green line) in separate
experiments.
Photolysis of a 50 µM CPG1 solution with a hand-held UV lamp
(304 nm, 8 W) results in almost complete cleavage of the
nitrobenzyl cage after 30 min and the liberation of 1,2,4-trihy-
droxybenzene 4 as confirmed by LC-MS (Figure S5). The quantum
yield for CPG1 photolysis was determined to be Φ ) 0.19 using
caged Pi as a standard (Supporting Information). H2O2 production
by photodeprotection of CPG1 can also be monitored using PF1.
Photolysis of a 200 µM CPG1 solution for 30 min (304 nm, 8 W)
produces ca. 20-30 µM H2O2 by fluorometric analysis with PF1,
a level suitable for triggering cellular responses (Figure S6).
We next tested the ability of CPG1 to deliver H2O2 to living
cells with spatial and temporal control using light as a trigger. To
avoid potential issues of small-molecule fluorescent dye uptake and
retention, we monitored the photoinitiated production of intracellular
H2O2 by CPG1 using the genetically encodable protein sensor
HyPer. HyPer-expressing HEK 293 cells loaded with 100 µM of
the acetylated, cell-permeable form of CPG1 (Ac-CPG1) for 30
min exhibit basal levels of intracellular fluorescence as determined
by epifluorescence microscopy (Figure 1a). Upon CPG1 photolysis,
cells show a prompt 20% rise in H2O2-induced HyPer fluorescence
within 2 min of UV irradiation as seen in Figure 1b-1c.
Importantly, control experiments displayed in Figure 1c establish
that no significant increases in HyPer fluorescence are observed
under conditions where CPG1-loaded cells are not irradiated with
UV light, cells without CPG1 are irradiated with UV light, or cells
without CPG1 are not exposed to UV light, verifying that observed
increases in intracellular H2O2 detected by HyPer depend upon both
the presence of CPG1 and light activation. Moreover, CPG1 also
allows for temporal control of H2O2 delivery to living cells by
varying the time at which UV light is delivered to the sample as
shown in Figure 1d.
Finally, we sought to employ this new caged H2O2 system to
simultaneously control and observe downstream effects elicited by
cellular H2O2 reactivity by releasing this molecular signal in a light-
dependent manner. In this regard, recent studies establish that H2O2
produced by membrane-bound NAD(P)H oxidase (Nox) enzymes
can direct and facilitate the leading edge progression of cell
populations and their membrane dynamics.36 A primary molecular
target of H2O2 in these cascades is the actin depolymerization factor
(ADF) cofilin, which results in the regulated assembly of cofilin-
actin rods upon oxidative activation. To probe cofilin-actin rod
formation mediated by H2O2 signaling, we utilized a HeLa cell line
stably expressing eGFP-cofilin. In the absence of a redox signal,
eGFP-cofilin exhibits a diffuse cytosolic staining pattern in these
modified HeLa cells (Figure 2e and 2f). Treatment of these live
specimens with H2O2 in a dose-dependent manner initiates forma-
tion of bright bands of rods to various extents as eGFP-cofilin is
incorporated into cofilin-actin rod structures. Although a variety
of stimuli,37 including excessive glutamate or AMPA stimulation
in cultured neurons or ATP depletion,38 induce cofilin rod forma-
tion, treatment with H2O2 alone provides sufficient impetus for the
formation of cofilin-actin rods.36 Stimulation of the eGFP-cofilin
HeLa cells with 50 µM H2O2 produces a regulated pattern of rod
formation that localizes to the periphery of the cell (Figure 2b),
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whereas addition of 10 µM H2O2 fails to generate a rod-forming
response after extended treatment (Figure 2a). Higher doses of H2O2
(100 or 500 µM) result in intense intracellular rod formation (Figure
2c and 2d). To our delight, loading the eGFP-cofilin cells with 200
µM Ac-CPG1 followed by irradiation with UV light triggers a rod
formation pattern that is strikingly similar in appearance to treatment
with the 50 µM H2O2 dose (Figure 2h), suggesting that CPG1-
derived H2O2 production in this context is on the order of tens of
micromolar. More importantly, control cells in the same imaging
dish that were not exposed to UV irradiation did not show H2O2-
induced coflin-actin rod patterns, highlighting the utility of CPG1
for delivering H2O2 within precise, spatially defined regions in the
same experiment (Figure 2i). In addition, control cells that are either
loaded with CPG1 and not activated with UV light or not loaded
with CPG1 and treated with UV excitation do not form rods (Figure
2f and 2g, respectively), establishing that the downstream phenotype
is due to controlled photoactivation of CPG1 to release the
molecular signal H2O2. As further evidence that photogeneration
of H2O2 from CPG1 and not a miscellaneous factor induces rod
formation, we treated cells loaded with a nitrobenzyl-protected
resorcinol compound. UV-irradiation releases the nonredox active
resorcinol along with the nitrosobenzaldehyde side product resulting
from photodeprotection. No rod formation was observed under
conditions identical to cells in Figure 2, confirming byproducts of
photodecaging do not contribute to rod formation (Figure S7).
Finally, analysis of the cellular morphology and corroboration by
MTT viability assay following experimental treatments confirm that
CPG1 is nontoxic to HeLa cells at concentrations up to 300 µM
(Figure S8).
for the eGFP-cofilin HeLa cell line. We dedicate this manuscript
to the memory of Dr. Gary Bokoch.
Supporting Information Available: Synthetic and experimental
details. This material is available free of charge via the Internet at http://
pubs.acs.org.
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Acknowledgment. We thank the Packard and Sloan Founda-
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