10.1002/anie.201804743
Angewandte Chemie International Edition
COMMUNICATION
A Ratiometric Two-Photon Fluorescent Probe for Tracking the
Lysosomal ATP Level: Direct in cellulo Observation of Lysosomal
Membrane Fusion Processes
Yong Woong Jun[a], Taejun Wang[b], Sekyu Hwang[a], Dokyoung Kim[c,d], Donghee Ma[a], Ki Hean Kim[b],
Sungjee Kim[a], Junyang Jung[c], and Kyo Han Ahn*[a]
Abstract: Vesicles exchange its contents through membrane fusion
processes—kiss-and-run and full-collapse fusion. Indirect
observation of these fusion processes using artificial vesicles
To unravel the mechanism of membrane fusion, in vitro
fusion assays based on artificial vesicles, mostly focused on the
synaptic vesicle fusion, have been evolved in recent years.[4–12]
enhanced our understanding on the molecular mechanisms involved. These artificial vesicle systems equipped with fluorescent dyes
Direct observation of the fusion processes in a real biological system, and proteins have contributed to our understanding on the
however, remains a challenge owing to many technical obstacles.
We disclose ratiometric two-photon probe offering real-time
tracking of lysosomal ATP with quantitative information for the first
molecular mechanisms of the vesicle fusion processes.[13,14]
Although many details are still unknown, it is suggested that the
vesicle fusion occurs through sequential steps aided by the
a
time. By applying the probe to two-photon live-cell imaging technique, fusion proteins: tethering and docking of a vesicle to another or
lysosomal membrane fusion process in cells has been directly to plasma membrane, followed by fusion-pore opening through a
observed along with the concentration of its content—lysosomal ATP. hemifusion state, and then full collapse into merged
vesicle/plasma membrane (full collapse fusion) or retrieval (kiss-
and-run).[1,2]
Examples of vesicle fusion assays, either in vitro or in cellulo,
a
Results show that the kiss-and-run process between lysosomes
proceeds through repeating transient interactions with gradual
content mixing, whereas the full-fusion process occurs at once.
Furthermore, it is confirmed that both the fusion processes proceed
with conservation of the content. Such a small-molecule probe
exerts minimal disturbance and hence has potential for studying
various biological processes associated with lysosomal ATP.
are based on the visualization of the fusion process under
stimulus conditions by tracking pH-dependent luminescence for
synaptic vesicles loaded with single quantum dots[15] or GFP
(green fluorescent protein),[16] the measurement of vesicle-
SNARE (soluble N-ethylmaleimide-sensitive factor activating
protein receptor) recycling in synapse,[17] and also the tracking of
multiple rounds of kiss-and-run by artificial SNARE-reconstituted
vesicle.[13,14] However, a direct tracking of endogenous contents
during the membrane fusion processes in living cells remains as
a challenge. Furthermore, tracking of the processes with
quantitative information remains as a formidable task. Here, we
disclose an in cellulo assay that enables direct monitoring of the
membrane fusion processes between vesicles (lysosomes),
together with quantitative information.
We focused on lysosomes that degrade the debris and also
release endogenous signaling molecules to extracellular matrix
through the membrane fusion processes.[18,19] Specifically, we
targeted the lysosomal ATP, a ubiquitous cellular element, since
it is not only an indispensable intracellular energy source in
living organisms but also plays distinct roles as a signaling unit
for such as immunogenic cell death, apoptosis and neuro-
transmission[20–23]. Monitoring of the lysosomal ATP is itself an
important research subject due to the lack of techniques to
visualize it selectively. Our approach is in cellulo real-time
monitoring of the lysosomal ATP with a novel two-photon
fluorescent probe that allows long-term imaging together with
high spatiotemporal resolution and, moreover, quantitative
information.
Vesicles, lipid bilayer-bound small compartments, are
involved in the secretion and uptake of cellular substances
through membrane fusion processes. The membrane fusion
processes are essential in viral infection, fertilization, and
releasing neurotransmitters and hormones into the extracellular
milieu through exocytosis.[1,2] The fusion process occurs between
cells, intracellular compartments, and the plasma membrane
through either of “kiss-and-run” or “full-collapse fusion”
process.[1,3] The former process indicates reversible fusion of
vesicles, involving transient pore opening and retrieval of the
vesicles, whereas the latter indicates irreversible fusion of
vesicles to form merged ones or to be collapsed into the plasma
membrane.
[a]
Dr. Y. W. Jun, S. Hwang, Dr. D. Ma, Prof. S. Kim, Prof. K. H. Ahn
Department of Chemistry
Pohang University of Science and Technology (POSTECH)
77 Cheongam-Ro, Nam-Gu, Pohang 37673 Rep. of Korea
E-mail: ahn@postech.ac.kr
[b]
[c]
[d]
Dr. T. Wang, Prof K. H. Kim
Division of Integrative Biosciences and Biotechnology
Pohang University of Science and Technology (POSTECH)
77 Cheongam-Ro, Nam-Gu, Pohang 37673 Rep. of Korea
Prof. D. Kim, Prof. J. Jung
Department of Anatomy and Neurobiology
College of Medicine, Kyung Hee University
26 Kyungheedae-Ro, Dongdaemun-Gu, Seoul 02447 Rep. of Korea
Prof. D. Kim
The size of lysosomes varies up to ~1.2 μm and their pH
ranges from 4.5 to 5.5. On the basis of the acidic environment of
lysosomes, we aimed at the development of a fluorescent probe
that can selectively detect the ATP molecules only in acidic pH.
Furthermore, to obtain long-term/high-resolution images and
quantitative information of lysosomal ATP, the probe is designed
to be two-photon excitable as well as ratiometric responding
through fluorescence resonance energy-transfer (FRET). To this
end, we have investigated rhodamine-based fluorescent probes,
inspired by the rhodamine-B based turn-on type probe reported
Center for Converging Humanities
College of Medicine, Kyung Hee University
26 Kyungheedae-Ro, Dongdaemun-Gu, Seoul 02447 Rep. of Korea
Supporting information for this article is given via a link at the end of
the document.
the author(s) of this article can be found under:
DOI: 10.1002/anie.201804743
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