Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics

Article abstract of DOI:10.1002/anie.201402030

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

Full text of DOI:10.1002/anie.201402030

Angewandte
Chemie
DOI: 10.1002/anie.201402030
Vesicular Imaging
Acidic-pH-Activatable Fluorescence Probes for Visualizing Exocytosis
Dynamics**
Daisuke Asanuma, Yousuke Takaoka, Shigeyuki Namiki, Kenji Takikawa, Mako Kamiya,
Tetsuo Nagano, Yasuteru Urano,* and Kenzo Hirose*
Abstract: Live imaging of exocytosis dynamics is crucial for
a precise spatiotemporal understanding of secretion phenom-
ena, but current approaches have serious limitations. We
designed and synthesized small-molecular fluorescent probes
that were chemically optimized for sensing acidic intravesic-
ular pH values, and established that they can be used to
sensitively and reliably visualize vesicular dynamics following
stimulation. This straightforward technique for the visualiza-
tion of exocytosis as well as endocytosis/reacidification pro-
cesses with high spatiotemporal precision is expected to be
a powerful tool for investigating dynamic cellular phenomena
involving changes in the pH value.
a vesicle-resident protein-tagged green fluorescent protein
(GFP)[2] or a fluorescent styryl dye[3] in combination with total
internal reflection fluorescence microscopy made it possible
to monitor exocytosis phenomena in terms of diffusely
diminished fluorescence; however, the practical utility of
this approach is limited by the noisy background due to
fluorophores in the nonvesicular region. In another study,
a pH-sensitive GFP analogue, ecliptic synapto-pHluorin, was
used to detect intravesicular neutralization upon exocytosis;[4]
however, the pre-exocytic vesicular distribution and dynamics
could not be tracked well, since synapto-pHluorin is non-
fluorescent at the acidic intravesicular pH value (generally
pH ꢀ 5[4]). Therefore, for the sensitive visualization of vesic-
ular dynamics, we focused on selective sensing of the
characteristic acidic pH value inside the secretory vesicles
by fluorescent probes. Such probes can be used to monitor
intracellular vesicular dynamics, and their signals are turned
off upon exocytosis (Figure 1). Several fluorescent probes
E
xocytosis plays fundamental roles in critical cellular events,
such as inflammatory response, neuronal transmission, and
the release of hormones.[1] The molecular mechanisms of
exocytosis have been intensively studied with biochemical
and electrophysiological methods, and are well understood at
the level of the whole cell.[1] However, precise regulatory
mechanisms, including the spatiotemporal dynamics of indi-
vidual secretory vesicles, remain unclear owing to the lack of
suitable tools for their investigation. For example, the use of
[*] D. Asanuma,[+] Y. Takaoka,[+] S. Namiki, K. Takikawa, M. Kamiya,
Prof. Y. Urano, Prof. K. Hirose
Graduate School of Medicine, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo (Japan)
E-mail: uranokun@m.u-tokyo.ac.jp
Figure 1. Visualization of vesicular dynamics with probes activated
under acidic conditions.
kenzoh@m.u-tokyo.ac.jp
Prof. T. Nagano, Prof. Y. Urano
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo (Japan)
Y. Takaoka[+]
Department of Synthetic Chemistry and Biological Chemistry
Kyoto University
Katsura, Nishikyo-Ku, Kyoto (Japan)
activated at acidic pH values have been developed and
applied to biological systems,[5–7] but these probes are not
suitable for selective vesicular imaging from the viewpoints of
signal activatability and photostability. For example, com-
mercially available CypHer5E[5] and pHrodo[6] have high
backgrounds at the physiological pH value of 7.4 (see Fig-
ure S1 in the Supporting Information), whereas our previ-
ously developed DiEtNBDP[7] proved to be insufficiently
photostable (see Figure S2). To sensitively visualize vesicular
dynamics, we need new fluorescent probes with both high
signal activatability at the acidic intravesicular pH value of 5.0
versus pH 7.4, and high photostability.
Our strategy for the development of fluorescent probes
that can be activated at acidic pH values, RhPs, was based on
the selection of rhodamine as a bright and photostable
fluorophore and piperazines as pH-sensitive switches that
operate by a mechanism of photoinduced electron transfer
(Figure 2a).[7,8] To examine the feasibility of this approach, we
synthesized RhP-H as a pilot compound. Although Wu and
Prof. T. Nagano
Open Innovation Center for Drug Discovery, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo (Japan)
[+] These authors contributed equally.
[**] This research was supported in part by SENTAN, JST (grant to K.H.),
KAKENHI (Grant no. 24750155 to D.A., 24115502 and 25115704 to
S.N., 22000006 to T.N., 20117003 and 23249004 to Y.U., and
24116004 to K.H.), The Tokyo Society of Medical Sciences (grant to
D.A.), and the Research Foundation for Opto-Science and Tech-
nology (grant to D.A.).
Supporting information for this article, including detailed exper-
imental procedures for the synthesis of the probes, the photo-
chemical measurements, and the imaging experiments, is available
on the WWW under http://dx.doi.org/10.1002/anie.201402030.
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Table 1: Properties of fluorescent probes activated under acidic con-
ditions.
Activation[a] [-fold]
Photostability[b] [%]
RhP-EF
pHrodo
CypHer5E
DiEtNBDP
58
1.1
4.6
12
98Æ1.9
98Æ0.3
74Æ1.6
8.8Æ1.4
[a] Activation was calculated by dividing the fluorescence intensity of
probes at pH 5.0 by that at pH 7.4. [b] Photostability was evaluated as
the percentage fluorescence intensity of probes after irradiation at
50 JcmÀ2 as compared with that before irradiation (taken as 100%).
Values are mean Æ standard deviation (n=3).
offers both high activatability at acidic intravesicular pH val-
ues and excellent photostability (Table 1). Also, RhP-EF was
extremely bright under acidic conditions (Ffl > 0.6; see Fig-
ure S3), and its fluorescence was not influenced by biologi-
cally relevant substances, including other cations and gluta-
thione (see Figure S10). Further investigation revealed rapid
and complete reversibility of the response to environmental
changes in the pH value (Figure 2e). We therefore concluded
that RhP-EF should enable real-time monitoring of intra-
vesicular pH-value changes, including multiple cycles. In
short, these in vitro results demonstrated that RhP-EF is
a promising scaffold for the selective sensing of acidic
intravesicular pH values, with superior photostability and
excellent reversibility.
For the visualization of exocytosis dynamics, an acid-
otropic fluorescent probe, named aRhP-EF (Figure 3), was
Figure 2. Design and properties of fluorescent probes activated under
acidic conditions, RhPs. a) Acid–base equilibrium and b) pH profiles
of RhPs. c) Photograph of cuvettes containing RhP-EF excited at
365 nm at pH values from 4 (left) to 8 (right) in increments of 0.5
pH units. d) Emission spectra of RhP-EF excited at 534 nm at pH 5.0
and 7.4. e) Fluorescence reversibility of RhP-EF during pH cycles
between pH 7.2 and 3.1.
Figure 3. Chemical structures of fluorescent probes for exocytosis
imaging.
Burgess reported a similar molecule with pH-dependent
properties, including fluorescence off–on reversibility,[9] its
detailed properties, such as its pKa values, were not evaluated.
We found that the background fluorescence of RhP-H at
pH 7.4 was unacceptably high, reaching approximately 30%
of the maximum fluorescence (Figure 2b). Thus, RhP-H is
unsuitable for the selective detection of the acidic vesicular
environment. Next, to chemically optimize the pH sensitivity,
we prepared a series of derivatives of RhP-H by introducing
N-substituents (Figure 2a). Dependently upon the electron-
withdrawing properties of the N-substituents, these RhPs
showed acidic shifts of the fluorescence curves (Figure 2b; for
details, see Table S1 and Figure S3 in the Supporting Infor-
mation). From these derivatives, we selected RhP-EF as
a fluorescent scaffold that could distinguish intravesicular
pH values of 4–6 from the physiological pH value of 7.4
(Figure 2c) owing to its potent fluorescence activation upon
putative acidification in the vesicles (58-fold; Figure 2d; see
also Table S1). Unlike previously reported probes, RhP-EF
developed by introducing an N,N-dimethylamino (dMA)
group into RhP-EF to promote localization to the acidic
vesicles, in line with pH-partitioning theory.[10] Unlike RhP-
EF, aRhP-EF sensitively stained granules in RBL-2H3 cells,
which are a well-studied mast-cell model[11] (see Figure S4).
Moreover, we demonstrated that only aRhP-EF enabled
selective granular staining with sufficiently high photostabil-
ity for continuous observation; it also showed superior
activation at acidic pH values (95-fold at pH 5.0 versus
pH 7.4) as compared to other granule-targeted pH-activat-
able probes (Cy-pHer5E-dMA, pHrodo-dMA, and
DiEtNBDP-dMA, all prepared in-house, and commercially
available LysoSensor dye; see Table S2 and Figures S5 and
S6). As a proof of concept (Figure 1), we performed time-
lapse confocal imaging of degranulation induced by stimulat-
ing aRhP-EF-stained cells with the Ca2+ ionophore ionomy-
cin in the presence of an extracellular fluid-phase marker
2
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dynamics at the level of indi-
vidual vesicles. Furthermore,
the simple technique of stain-
ing vesicles by incubating cells
with the probe should enable
this method to be readily
applied to other cell types,
including
cells.
nontransfectable
In secretory cells, exocyto-
sis and compensatory endocy-
tosis are tightly coupled, so
that the releasing functionality
is restored.[1] To further
address the visualization of
exo- and endocytosis process-
es, we developed hRhP-EF by
coupling a HaloTag ligand to
RhP-EF (Figure 3). Our imag-
ing strategy was based on the
covalent tethering of RhP-EF
to granule-localizing vesicle-
associated membrane pro-
tein 2 (VAMP2)[14] by using
the HaloTag labeling technol-
ogy[15] (Figure 5a). To test the
performance of hRhP-EF, we
incubated it with RBL-2H3
cell lines expressing or not
expressing VAMP2-HaloTag
protein. We confirmed that
granular labeling occurred
selectively
VAMP2-HaloTag
in
RBL-2H3/
cells,
Figure 4. Exocytosis dynamics in RBL-2H3 cells visualized with aRhP-EF. a) Schematic representation of the
imaging of exocytosis by our proposed method.b,c) Degranulation in ionomycin-stimulated RBL-2H3 cells.
A fluid-phase marker was used in (b). Scale bars: 1 mm. The time course of normalized fluorescence
intensity at numbered granules is shown.
whereas only minimal nonspe-
cific staining was observed in
the original RBL-2H3 cells,
presumably because of the
chemically introduced poly(-
ethylene glycol) (PEG) spacer
(Figure 4a). Intriguingly, the fluorescence of some granules
suddenly disappeared at different time points (Figure 4b,
“Granules”; see also Movie S1 in the Supporting Informa-
tion), and we observed synchronized influx of the fluid-phase
marker into W-shaped membrane structures formed upon
degranulation (Figure 4b, “Fluid phase”; see also Movies S2
and S3). These results indicate that the disappearance of the
granular fluorescence corresponds to exocytosis events. To
our knowledge, the dynamics of individual exocytosis events
in RBL-2H3 cells has not been visualized previously without
the need to artificially enlarge the granules with serotonin.[12]
We also succeeded in visualizing an intriguing process of
“sequential exocytosis”, in which successive vesicular releases
occurred around an initial event (Figure 4c; see also
Movie S4), thereby providing direct evidence for a focal
release mechanism that was originally proposed on the basis
of the electron-microscopic observation of hormone-treated
pancreatic acinar cells.[13] Thus, our technique was confirmed
to enable the sensitive and reliable visualization of exocytosis
in hRhP-EF (see Figure S7). We also confirmed that the
granular fluorescence reversibly responded to the environ-
mental pH cycle (see Figure S8). Moreover, we found that
only hRhP-EF enabled selective granular labeling of
VAMP2-HaloTag-expressing cells and continuous observa-
tion, in contrast to the other HaloTag-ligand-coupled pH-
activatable probes prepared in-house, CypHer5E-halo and
pHrodo-halo (see Table S3 and Figures S7 and S9). Next,
hRhP-EF-labeled cells were sensitized with anti-dinitro-
phenyl (DNP) IgE and stimulated with DNP-conjugated
bovine serum albumin (DNP-BSA) to induce degranulation.
Intriguingly, some granules showed cyclic “off” and “on”
fluorescence changes (Figure 5b; see also Movie S5). By
using a fluid-phase marker, we also revealed that this type of
off–on phenomenon occurs at the identical vesicle (see
Figure S11). This result is direct evidence of “kiss-and-run”
events that consist of granular exocytosis in the “off” phase
and endocytosis followed by intragranular reacidification in
the “on” phase. Fernandez et al. reported the existence of
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Figure 6. Exocytosis and endocytosis/reacidification dynamics in cul-
tured neurons visualized with hRhP-EF. a) Synaptic-vesicle dynamics in
cultured neurons: 200 action potentials (APs) at 10 Hz were applied at
20–40 s; intravesicular neutralization (NH4Cl) and immunostaining
(VGLUT) were also performed. Scale bar: 5 mm. b) Time course of
normalized fluorescence intensity at synapses numbered in (a).
Figure 5. Exocytosis and endocytosis/reacidification dynamics in RBL-
2H3 cells visualized with hRhP-EF. a) Schematic representation of the
visualization of vesicular dynamics. b) Representative vesicular dynam-
ics in anti-DNP IgE/DNP-BSA-stimulated RBL-2H3/VAMP2-HaloTag
cells. Scale bar: 5 mm. The time course of normalized fluorescence
intensity at numbered granules is shown.
In conclusion, we have developed fluorescent probes that
are chemically optimized for sensing the acidic intravesicular
environment. When conjugated with an acidotropic moiety or
used in combination with protein-labeling technology, our
chemical probes, aRhP-EF and hRhP-EF, enabled the visual-
ization of vesicular dynamics in living cells with high intra-
vesicular acidic activation of fluorescence, high staining
specificity, and good photostability; they were greatly supe-
rior to other existing probes in these respects. Our results
demonstrate that the dynamic fluorescence changes reflect
exocytosis as well as endocytosis/reacidification processes.
Because the background is minimal with these probes,
vesicular imaging can be performed simply with confocal or
conventional fluorescence microscopy. This study has pro-
vided a versatile and powerful tool for understanding
vesicular events as spatiotemporally dynamic cellular phe-
nomena, with substantial advantages over electron microsco-
py and electrophysiology. Furthermore, the technique has
potential application in high-throughput screening systems.
Secretion phenomena, such as degranulation during allergic
response and synaptic transmission during neuronal function,
certainly involve potential drug targets, and cell-based screen-
ing systems for compound libraries are under construction
with our probes.
exo- and endocytosis events at whole-cell resolution by
measuring the membrane capacitance of RBL-2H3 cells.[16]
In comparison with these capacitance measurements, our
technique represents a major technical advance that permits
a fine spatiotemporal understanding of individual vesicular
events, including exocytosis and endocytosis/reacidification
processes, at single-granule resolution.
To further confirm the applicability of our technique, we
applied it to neurons to visualize the exo- and endocytosis
dynamics of synaptic vesicles during neuronal transmission.
Cultured rat hippocampal neurons expressing VAMP2-Hal-
oTag protein were labeled with hRhP-EF and electrically
stimulated to release synaptic vesicles. The intensity of
punctate fluorescence decreased by 30–40% during the
electrical stimulation and subsequently recovered to the
initial level (Figure 6; see also Movie S6). These changes in
fluorescence probably correspond to synaptic-vesicle exocy-
tosis and subsequent endocytosis/reacidification. We also
confirmed that synaptic labeling was successfully performed
with hRhP-EF on the basis of colocalization with a synaptic
marker of vesicular glutamate transporter 1 (VGLUT1; Fig-
ure 6a “0 s” versus “VGLUT”), and we further confirmed
that the synaptic fluorescence diminished in a pH-dependent
manner by intravesicular neutralization with NH4Cl (Fig-
ure 6a, “NH4Cl”). Thus, our technique is applicable to
neuronal studies. It may be primarily useful for exocytosis
studies to reveal vesicular dynamics with high spatiotemporal
precision. However, it would also be suitable for endocytosis
studies, such as receptor internalization,[7] because proteins of
interest (e.g. receptors) can be readily tracked with the probe
by the use of protein-labeling technology.
Received: February 2, 2014
Revised: March 25, 2014
Published online: && &&, &&&&
Keywords: acidity · exocytosis · fluorescent probes ·
.
spatiotemporal dynamics · vesicular imaging
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[9] L. Wu, K. J. Burgess, J. Org. Chem. 2008, 73, 8711.
[10] C. de Duve, T. de Barsy, B. Poole, A. Trouet, P. Tulkens, F.
Van Hoof, Biochem. Pharmacol. 1974, 23, 2495.
[1] E. D. Gundelfinger, M. M. Kessels, B. Qualmann, Nat. Rev. Mol.
Cell Biol. 2003, 4, 127.
[2] D. Liu, J. A. Martina, X. S. Wu, J. A. Hammer III, E. O. Long,
Immunol. Cell Biol. 2011, 89, 728.
[3] D. Zenisek, J. A. Steyer, W. Almers, Nature 2000, 406, 849.
[4] G. Miesenbçck, D. A. De Angelis, J. E. Rothman, Nature 1998,
394, 192.
[11] E. Passante, N. Frankish, Inflammation Res. 2009, 58, 737.
[12] R. M. Williams, J. B. Shear, W. R. Zipfel, S. Maiti, W. W. Webb,
Biophys. J. 1999, 76, 1835.
[13] A. Ichikawa, J. Cell Biol. 1965, 24, 369.
[14] U. Blank, B. Cyprien, S. Martin-Verdeaux, F. Paumet, I. Pombo,
J. Rivera, M. Roa, N. Varin-Blank, Mol. Immunol. 2002, 38, 1341.
[15] G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N.
Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohana,
M. Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G.
Vidugiris, J. Zhu, A. Darzins, D. H. Klaubert, R. F. Bulleit, K. V.
Wood, ACS Chem. Biol. 2008, 3, 373.
[5] A. Beletskii, M. Cooper, P. Sriraman, C. Chiriac, L. Zhao, S.
Abbot, L. Yu, Biotechniques 2005, 39, 894.
[6] M. Ogawa, N. Kosaka, C. A. Regino, M. Mitsunaga, P. L.
Choyke, H. Kobayashi, Mol. Biosyst. 2010, 6, 888.
[7] Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M.
Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, H.
Kobayashi, Nat. Med. 2009, 15, 104.
[16] J. M. Fernandez, E. Neher, B. D. Gomperts, Nature 1984, 312,
453.
[8] Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose, T. Nagano,
J. Am. Chem. Soc. 2005, 127, 4888.
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Communications
Vesicular Imaging
Going live: Live imaging of the dynamics
of exocytosis is crucial for a precise
spatiotemporal understanding of secre-
tion phenomena. Small-molecular fluo-
rescent probes designed to be activated
under acidic intravesicular conditions
enabled the sensitive and reliable visual-
ization of vesicular dynamics with high
spatiotemporal precision (see picture).
D. Asanuma, Y. Takaoka, S. Namiki,
K. Takikawa, M. Kamiya, T. Nagano,
Y. Urano,* K. Hirose*
&&&& — &&&&
Acidic-pH-Activatable Fluorescence
Probes for Visualizing Exocytosis
Dynamics
6
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!