Selective uncoupling of individual mitochondria within a cell using a mitochondria-targeted photoactivated protonophore

Article abstract of DOI:10.1021/ja2077922

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and r

Full text of DOI:10.1021/ja2077922

Communication
pubs.acs.org/JACS
Selective Uncoupling of Individual Mitochondria within a Cell Using
a Mitochondria-Targeted Photoactivated Protonophore
Susan Chalmers,‡ Stuart T. Caldwell,† Caroline Quin,† Tracy A. Prime,§ Andrew M. James,§
Andrew G. Cairns,† Michael P. Murphy,§ John G. McCarron,*,‡ and Richard C. Hartley*,†
†WestCHEM School of Chemistry, University of Glasgow, Glasgow G12 8QQ, U.K.
‡Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, U.K.
§MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, U.K.
S
* Supporting Information
calcium ion uptake by a chosen single mitochondrion or
localized subpopulation of mitochondria within a cell (Figure
1).
ABSTRACT: Depolarization of an individual mitochond-
rion or small clusters of mitochondria within cells has been
achieved using a photoactivatable probe. The probe is
targeted to the matrix of the mitochondrion by an
alkyltriphenylphosphonium lipophilic cation and releases
the protonophore 2,4-dinitrophenol locally in predeter-
mined regions in response to directed irradiation with UV
light via a local photolysis system. This also provides a
proof of principle for the general temporally and spatially
controlled release of bioactive molecules, pharmacophores,
or toxins to mitochondria with tissue, cell, or mitochond-
rion specificity.
MitoPhotoDNP has three constituent parts, a caged
protonophore, a photocleavable linker and a mitochondria-
targeting unit. We chose to target a protonophore to
mitochondria as this is a way of selectively decreasing the
mitochondrial membrane potential, which is at the heart of
mitochondrial function. To produce ATP by oxidative
phosphorylation the mitochondrial electron transport chain
uses the oxidation of substrates by oxygen to pump protons out
of the matrix to generate a proton electrochemical potential
gradient across the mitochondrial inner membrane (MIM),
which is composed mainly of a membrane potential (150−170
mV, negative inside). Protons flow down this gradient through
the ATP synthase to form ATP. Ca2+ uptake into the
mitochondria through the calcium uniporter is also driven by
the membrane potential and provides a mechanism of
modulating the mitochondrial and cytosolic Ca2+ concentration
locally, which is necessary for many cellular processes.13
Oxidation can be uncoupled from ATP synthesis and Ca2+
transport by dissipating the membrane potential, typically by
mildly acidic lipophilic compounds, which act as protono-
phores. These protonophores, or uncouplers, are deprotonated
in the mitochondrial matrix to form lipophilic anions, which
can cross the MIM, pick up a proton, and return, so abolishing
the membrane potential and switching off mitochondrial ATP
production. The classic mitochondrial uncoupler is dinitrophe-
nol (DNP)14−16 (pKa = 4.117) and in MitoPhotoDNP DNP is
caged by linking to an o-nitrobenzyl group, which is a well-tried
photoactivatable linker.14,18,19 The TPP cation is included as a
mitochondria-targeting group because it easily permeates
biological membranes due to its hydrophobicity and large
ionic radius, and it accumulates several hundred fold in the
mitochondrial matrix within cells in vivo due to the large
membrane potential across the MIM, in accordance with the
Nernst equation.1,2 It also has very low toxicity; indeed, a TPP-
containing antioxidant, MitoQ, has been fed long-term to both
animals and humans without toxicity at its therapeutic dose.20
The most scalable and reproducible synthesis of MitoPho-
toDNP involved alkylation of a commercially available phenol 1
itochondria are central to eukaryotic cells. As well as
M
consuming ∼95% of the O2 inspired to generate most of
the cell’s ATP, they control cell death, contain much of central
metabolism, and modulate calcium and redox signaling.
Consequently there is considerable interest in designing
mitochondria-targeted molecules to report on and manipulate
mitochondrial function.1 The use of lipophilic cations such as
the alkyltriphenylphosphonium (TPP) moiety has enabled the
delivery of a wide range of molecules to mitochondria without
the need to alter genes or protein structure.1,2 However, this
procedure is not selective for particular mitochondria within an
organism. The challenge remains of how to use this system to
deliver molecules to mitochondria within individual tissues or
cells or to individual mitochondria in a cell at a specific time.
The latter point is significant as the organization and
localization of mitochondria within cells is important for their
function3−7 and subpopulations of mitochondria contribute
differently to cell processes depending on their location within
the cell.6−11 Furthermore, temporal control is vital to
understanding cellular mechanisms, so that transient changes
can be observed and causal relationships established. To
address this need we have developed a procedure to target
molecules to mitochondria by coupling them to the lipohilic
TPP cation via a photocleaveable linker,12 enabling the release
of the molecule within chosen mitochondria at a specific
moment by selective irradiation of those mitochondria.
As a first example of this class of molecule here we describe
MitoPhotoDNP, which can release a protonophore within
selected mitochondria thereby stopping ATP production in and
Received: August 17, 2011
Published: December 22, 2011
© 2011 American Chemical Society
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Communication
and the caged uncoupler introduced to complete the synthesis
of MitoPhotoDNP. UV irradiation of a solution of
MitoPhotoDNP in CDCl3 led to release of DNP, detected by
1H NMR spectroscopy.
To assess whether MitoPhotoDNP was taken up by
mitochondria in response to the membrane potential across
the inner membrane we constructed an electrode that
responded selectively to the TPP moiety of MitoPhotoDNP.21
When mitochondria were added in the presence of the
respiratory inhibitor rotenone to prevent development of a
membrane potential there was a decrease in the concentration
of MitoPhotoDNP due to the expected adsorption of the
hydrophobic TPP compound to mitochondrial membranes.22
Subsequent addition of the respiratory substrate succinate led
to the rapid and extensive accumulation of MitoPhotoDNP by
energized mitochondria, and this was reversed by addition of
FCCP [carbonylcyanide 4-(trifluoromethoxy)-
phenylhydrazone] to abolish the mitochondrial membrane
potential.14,23 This semiquantitative approach showed that
MitoPhotoDNP is rapidly and extensively accumulated by
energized mitochondria in response to the membrane potential,
as expected for a TPP cation.1,2
Next, we demonstrated that MitoPhotoDNP can be
photoactivated within cells to uncouple selectively an individual
mitochondrion or a small group of mitochondria. Freshly
isolated colonic smooth muscle cells were loaded with the
mitochondrial membrane potential-sensitive dye tetramethylr-
hodamine ethyl ester (TMRE) and showed a punctate
fluorescent staining of the mitochondria, consistent with
energized mitochondria with high membrane potentials. A
short (85 ms) irradiation of site 1 with UV at 355 nm in the
absence of MitoPhotoDNP did not lead to any change in the
pattern of TMRE fluorescence indicating no change in
mitochondrial membrane potential in response to UV alone
(Figure 2, i). There was no change when the cells were exposed
to a low concentration of MitoPhotoDNP (200 nM), indicating
that MitoPhotoDNP itself did not disrupt mitochondrial
function. However, when site 2 was irradiated with UV light
in the presence of MitoPhotoDNP, there was localized loss of
TMRE fluorescence (red region in panel ii). The depolarization
was maintained for the duration of the experiment, which was
typically limited to 30 min, a period during which there was
little TMRE bleaching and cell performance was unaltered.
When all of the cell’s mitochondria were depolarized with
rotenone an irreversible loss of TMRE fluorescence was
observed throughout the cell (panel iii), confirming that the
depolarization caused by MitoPhotoDNP and UV light was
selective. In control experiments, smooth muscle cells treated
with 200 nM MitoPhotoDNP for the same length of time (30
min) showed no alterations in gross cellular morphology, basal
cytosolic [Ca2+] or responsiveness to calcium-generating
agonists, and the compound was not toxic to C2C12 myoblasts
following chronic exposure (overnight) to concentrations
below 5 μM.
Figure 1. Schematic representing MitoPhotoDNP accumulation
within the mitochondrial matrix of all mitochondria and selective
activation within a single mitochondrion to depolarize the inner
membrane and uncouple electron transport from ATP synthesis and
calcium transport.
(Scheme 1), and then reduction of the resulting aldehyde 2 to
give alcohol 3. This was converted into the phosphonium salt 4
Scheme 1. Synthesis of MitoPhotoDNP
The mitochondrial depolarization remained localized and
was not reversed during the period for which observation of an
individual mitochondrion is feasible (up to 1 h). Further
experiments confirmed that the sustained depolarization was
not the result of local depletion of ATP or mitochondrial
metabolites. Cells were patch clamped3 in whole cell
configuration and using the access afforded by the electrode,
ATP (3 mM), phosphocreatine (5 mM), malate (2.5 mM), and
pyruvate (2.5 mM) were introduced into the cell. Irradiation of
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Communication
Figure 2. Localized mitochondrial depolarization following localized photolysis of MitoPhotoDNP. Freshly isolated colonic smooth muscle cells
loaded with TMRE (10 nM) and wortmannin (10 μM, to prevent cell contraction) display a punctate fluorescent staining (i). The cell displayed
underwent (1) a brief, localized exposure to UV laser light (355 nm for 85 ms in region shown in panel i in the absence of MitoPhotoDNP; (2)
MitoPhotoDNP (200 nM) was washed into the cell’s bathing medium, allowed to equilibrate for 15 min and then a second region of the cell was
exposed to UV light (85 ms, in region shown in panel ii); (3) finally the mitochondrial inhibitors rotenone (2 μM) plus oligomycin (3 μM) were
washed into the bathing medium (panel iii). TMRE fluorescence was detected before and after each of the three treatments and any differences
highlighted by overlaying artificially colored images in which “before” is red and “after” is green, such that no change results in yellow regions, loss of
TMRE staining (hence mitochondrial depolarization) results in red regions, and any gain of fluorescence results in green. A bright-field image of the
cell plus 10 μm scale bar is shown in panel iv. This is a typical result of more than 20 similar experiments.
a region of the cell again resulted in depolarization that was
localized and sustained, and a flickering event4 in the unaffected
region further confirmed that if repolarization had occurred, it
would have been observed (see Supporting Information).
The several-hundred-fold accumulation of MitoPhoto-DNP
within the mitochondrial matrix in cells is vital for the selective
depolarization of mitochondria. This is supported by the fact
that addition of 200 nM DNP has no effect on mitochondrial
membrane potential, with 90 μM DNP being required to
decrease the ratio of fluorescence of TMRE to Mitotracker (a
mitochondrial dye whose uptake is not reversed upon
mitochondrial membrane depolarization) to half maximal
within 10 min. The importance of targeting is further supported
by the finding that no UV-induced depolarization of
mitochondria was observed when an untargeted photo-
activatable DNP 5 (Figure 3) was employed at a concentration
irradiation. As well as releasing DNP, irradiation of
MitoPhotoDNP will also generate TPP-conjugated side
products from the caging group. To determine whether these
side products could affect mitochondrial polarization, cells were
incubated with 3 μM TPP-conjugated o-nitrobenzylic alcohol 4
and irradiation of a small cluster of mitochondria as before gave
no depolarization. o-Nitrobenzylic alcohol 4 is expected to
release water upon irradiation, and HPLC confirmed that it
gives a similar distribution of TPP-conjugated side-products to
MitoPhoto-DNP under the irradiation conditions.
In summary, in this proof of concept study, we have shown
that we can locally activate a mitochondria-targeted compound
within mitochondria inside a cell by combining a mitochondria-
targeting group with a photolyzable linker to deliver a cargo.
We showed that this effectively delivered a protonophore to
mitochondria within cells and thereby led to the selective
uncoupling of either individual or a small number of
mitochondria within a cell when used in conjunction with
fluorescence imaging. This demonstrated exquisite spatial and
temporal control of mitochondrial function. The probe itself
should also allow the investigation of the role of subpopulations
of mitochondria in controlling local ATP supply and calcium
ion regulation in specific parts of cells, particularly in highly
differentiated cells such as neurons and smooth muscle cells. In
neurons, for example, mitochondria cluster at locations with
high energy demand and control subplasma membrane [Ca2+]
to manage exocytosis in nerve terminals25 and adrenal
chromaffin cells,26 but current techniques that involve
inhibiting mitochondrial activity throughout the entire cell
cannot elucidate the effect of the localized activity of the
organelle. Thus, we expect MitoPhotoDNP to be an important
investigational tool for relating intracellular organization of
mitochondria to complex functions. More generally, our
approach could be extended to deliver selectively a wide
range of functional molecules including specific inhibitors or
activators of mitochondrial processes to study intracellular
processes and also provide a means of introducing tissue or cell
specificity to mitochondria-targeted pharmacophores not
previously achievable.
Figure 3. Control compounds.
of 200 nM or even when used at a concentration of 30 μM (an
observation which is consistent with the limited effectiveness of
an earlier untargeted photoactivatable uncoupler24).
While UV alone does not cause depolarization of
mitochondria, it could be suggested that the TPP targeting
group or the nitroarene trigger unit of MitoPhotoDNP, in
conjunction with UV, sensitized mitochondria to photodamage
and thus led to uncoupling. This possibility was eliminated by
incubating cells with 3 μM of the TPP-targeted nitro
compound 6 (Figure 3) that cannot release an uncoupler and
showing that the mitochondria were not depolarized upon
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ASSOCIATED CONTENT
* Supporting Information
Complete refs 2a and 20b; experimental details for the
preparation of 4−6 and MitoPhotoDNP; UV−vis spectra;
NMR spectra showing release of DNP from MitoPhotoDNP in
response to UV irradiation; electrode responses for uptake of
MitoPhoto-DNP into isolated mitochondria; details of experi-
ments with intact cells (including pictures and a movie); HPLC
■
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of irradiated MitoPhotoDNP and 4; toxicity tests; and H and
13C NMR spectra of all compounds synthesized. This material
is available free of charge via the Internet at http://pubs.acs.
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AUTHOR INFORMATION
Corresponding Author
Richard.Hartley@glasgow.ac.uk; john.mccarron@strath.ac.uk
■
ACKNOWLEDGMENTS
■
C.Q. and A.G.C. acknowledge BBSRC and University of
Glasgow. S.C., S.T.C., R.C.H., and J.G.M. acknowledge The
Wellcome Trust (092292/Z/10/Z and 092292/B/10/Z).
́
Helena M. Cocheme is acknowledged for artwork.
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