Mechanosensitive Fluorescent Probes to Image Membrane Tension in Mitochondria, Endoplasmic Reticulum, and Lysosomes

Article abstract of DOI:10.1021/jacs.8b13189

Measuring forces inside cells is particularly challenging. With the development of quantitative microscopy, fluorophores which allow the measurement of forces became highly desirable. We have previously introduced a mechanosensitive flipper probe, which responds to the change of plasma membrane tension by changing its fluorescence lifetime and thus allows tension imaging by FLIM. Herein, we describe the design, synthesis, and evaluation of flipper probes that selectively label intracellular organelles, i.e., lysosomes, mitochondria, and the endoplasmic reticulum. The probes respond uniformly to osmotic shocks applied extracellularly, thus confirming sensitivity toward changes in membrane tension. At rest, different lifetimes found for different organelles relate to known differences in membrane organization rather than membrane tension and allow colabeling in the same cells. At the organelle scale, lifetime heterogeneity provides unprecedented insights on ER tubules and sheets, and nuclear membranes. Examples on endosomal trafficking or increase of tension at mitochondrial constriction sites outline the potential of intracellularly targeted fluorescent tension probes to address essential questions that were previously beyond reach.

Full text of DOI:10.1021/jacs.8b13189

Communication
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Mechanosensitive Fluorescent Probes to Image Membrane Tension
in Mitochondria, Endoplasmic Reticulum, and Lysosomes
Antoine Goujon,†,‡,∥ Adai Colom,†,‡,∥ Karolína Strakova, Vincent Mercier,†,‡ Dora Mahecic,‡,§
†,‡
́
,†,‡
Suliana Manley,‡,§ Naomi Sakai,†,‡ Aurelien Roux,* and Stefan Matile*,†,‡
́
†School of Chemistry and Biochemistry and ‡National Centre of Competence in Research (NCCR) Chemical Biology, University of
Geneva, CH-1211 Geneva, Switzerland
§
́
́
́
Institute of Physics, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
S
* Supporting Information
the DTTs. Sulfides and sulfones in the DTT bridges supported
by a chalcogen-bonding8 ether donor and a cyano acceptor
serve as push and pull components.
ABSTRACT: Measuring forces inside cells is particularly
challenging. With the development of quantitative
microscopy, fluorophores which allow the measurement
of forces became highly desirable. We have previously
introduced a mechanosensitive flipper probe, which
responds to the change of plasma membrane tension by
changing its fluorescence lifetime and thus allows tension
imaging by FLIM. Herein, we describe the design,
synthesis, and evaluation of flipper probes that selectively
label intracellular organelles, i.e., lysosomes, mitochondria,
and the endoplasmic reticulum. The probes respond
uniformly to osmotic shocks applied extracellularly, thus
confirming sensitivity toward changes in membrane
tension. At rest, different lifetimes found for different
organelles relate to known differences in membrane
organization rather than membrane tension and allow
colabeling in the same cells. At the organelle scale, lifetime
heterogeneity provides unprecedented insights on ER
tubules and sheets, and nuclear membranes. Examples on
endosomal trafficking or increase of tension at mitochon-
drial constriction sites outline the potential of intra-
cellularly targeted fluorescent tension probes to address
essential questions that were previously beyond reach.
In lipid bilayers, Flipper-TR 1 responds to the increasing
membrane order by planarization in the ground state, which
results in red-shifted excitation maxima and longer fluores-
cence lifetimes.5 Such properties are unique9 among the
membrane probes that usually report off-equilibrium in the
excited state on viscosity or polarity.9−16 In cellular
membranes, the mechanophore 1 reports the increasing
membrane order upon application of membrane tension σ by
increasing fluorescence lifetime τ. This change was attributed
to the formation of more ordered microdomains under
membrane tension, due to the increased line tension between
the stretchable and unstretchable lipid phases (Figure 1B, dark
gray).5,17 Roughly linear positive τ−σ correlations found with
all tested cells demonstrated the applicability of 1 to measure
membrane tension change by fluorescence lifetime imaging
microscopy (FLIM).5,18
The headgroup of flipper 1 contains an essential triazole and
a carboxylate to target the plasma membrane (PM; Figure
2C).19 We have shown that the carboxylate can be replaced
without loss of function by a boronic acid20 and a biotin21 to
target ganglioside-enriched lipid domains and biotinylated
targets on cell surfaces, respectively. Thus, probes 2−5 were
designed incorporating established motifs from the respective
trackers (Figure 1A).12−16 Namely, Lyso Flipper 2 was
equipped with a basic morpholine (pKa 8.4) and a short
hydrophobic linker to produce membrane-binding cationic
amphiphiles only after protonation within the acidic late
endosomes and lysosomes.13,14 In Mito Flipper 4, the same
short linker is used to install a hydrophobic triphenyl-
phosphonium cation for selective targeting of mitochondria
with strong inside negative membrane potential.13,15 ER
Flipper 3 contains a pentafluorophenyl group to react with
thiols in ER-membrane proteins, and a different, long, and
hydrophilic linker to enable the partitioning of the protein
anchored probe in the membrane.13,16 The synthesis of all
probes is described in the SI (Schemes S1−S4).
he importance of mechanical forces in biological
T
processes is only starting to emerge.1−3 Plasma
membrane tension is a topic of particular current interest
because mounting evidence suggests its involvement in
regulating various biochemical processes in cells.2 Although
membrane tension should also regulate membranous organ-
elles’ functions, standard techniques of force measurements,
such as optical tweezers or force microscopes, are difficult to
apply inside of cells.3 Therefore, the role of membrane tension
in organelles is so far poorly explored and awaits the
development of noninvasive measurement methods.1−4
Responding to the need in mechanobiology, this study
aimed at developing organelle-specific mechanophores based
on the recently introduced fluorescent membrane tension
reporter 1 (referred to as Flipper-TR or FliptR, Figure 1).5
This planarizable push−pull probe is composed of two
dithienothiophene (DTT)6 “flippers”.7 They are twisted out
of coplanarity by repulsion between methyl groups and the σ
holes8 on the endocyclic sulfurs next to the bond connecting
Consistent with increasing planarization in the ground state,
flippers 2−4 gave the expected red-shifted excitation maxima
Received: December 10, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b13189
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 1. (A) Structure of Flipper-TR 1, Lyso, ER, and Mito Flippers 2−4, and Mito Control 5. (B) With increasing membrane tension (right), the
appearance of ordered domains (dark gray) causes an increase of fluorescence lifetimes, whereas the complementary domain disassembly (left)
accounts for decreasing lifetimes with decreasing tension.
a
Table 1. Mechanophore Characteristics
c
d
e
f
h
i
h
i
λSo
λLd
τLo
τLd
τi
τh
τi
τh
b
j
g
C
(nm) (nm) (ns) (ns) PCC
(ns) (ns) (ns) (ns)
S
1
2
3
4
5
LUVs GUVs
490 5.9
HeLa
4.8
3.7
3.4
3.2
COS7
435
437
436
439
2.8
3.1
3.0
2.9
−
4.2
3.3
3.0
3.0
3.2
−
−
475
475
475
5.6
5.8
5.5
−
0.9
0.9
0.9
0.8
3.9
3.5
3.3
−
3.6
3.3
3.1
−
k
l
l
br
br
−
b
3.2
a
Expanded version: Table S1. Conditions; probes 1−5: Figure 1; S:
Systems. Excitation maxima in So DPPC LUVs. Same in Ld DOPC.
Average fluorescence lifetime in SM/CL 7:3 GUVs. Same in DOPC.
Pearson’s correlation coefficients determined using CLSM against
LysoTracker (2), ER-Tracker (3), MitoTracker Red (4, 5). Average
τ under isotonic conditions. Same under hypertonic conditions. Data
from refs 5 and 19; average τ recalculated. Examples for
c
d
e
f
Figure 2. (A) Excitation spectra (λem 570 nm) of 1 (dashed) and 3
(solid) in Ld (blue, 1,2-dioleoyl-sn-glycero-3-phosphocholine: DOPC)
and So LUVs (red, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine:
DPPC). (B) FLIM images of 4 in Ld (top) and Lo GUVs (bottom,
sphingomyelin/cholesterol: SM/CL). (C−F) Confocal images of
HeLa Kyoto cells stained with PM 1 (C), Lyso 2 (D), ER 3 (E), and
Mito Flipper 4 (F) (top), the respective trackers (middle), and the
merged images (bottom); scale bars: 10 μm. (G) Fluorescence
lifetime of 2−4 in HeLa Kyoto cells before (black) and after
hypertonic osmotic shock (red).
g
h
i
j
k
suborganellar heterogeneity ignored in average lifetimes: tubular ER,
4.1 ns; ER sheets, 3.5 ns; nuclear membrane, 3.4 ns. Broad signals
without distinct maxima.
l
their specific trackers12−16 were excellent in both HeLa Kyoto
(Figures 2D−2F, S12−S13, Table 1) and COS7 cells (Figures
S14−S15). Characteristic staining patterns of organelles were
also visible in FLIM images of probes 2−4 in standard HeLa
(Figure S16) and more clearly in COS7 cells (Figure 3). COS7
cells treated with 3 revealed dense staining around the nucleus
attributed to ER sheets, a thin meshwork attributed to tubular
ER, and also the nuclear envelope (Figures 3B, S14, S19, S20).
The average lifetimes were different for all probes in HeLa
Kyoto cells (PM 1 > Lyso 2 > ER 3 > Mito 4, Table 1, Figure
2G, black) and COS7 cells (Figure 3, black histograms, Table
1). All values were in-between the extremes measured in pure
single-component model Ld and Lo membranes in GUVs. The
variations in lifetimes originate mainly from differences in
membrane organization (including contributions from lipid
composition, microdomains, maybe also proteins, viscosity,
etc.),5,9,10,23 and from the different mechanosensitivity of
probes 1−4 (Figure 2A, Table 1). Thus, lifetimes in organelles
in solid-ordered (So) compared to liquid-disordered (Ld)
membranes of large unilamellar vesicles (LUVs, Figures 2A,
S1−S9, Table 1).7,9,19−21 Also as expected were the higher
average lifetimes τ of 2−4 in liquid-ordered (Lo) compared to
Ld membranes in FLIM images of giant unilamellar vesicles
(GUVs, Figures 2B, S11, Table 1).5,19 Compared to the
meticulously optimized original 119 in ordered membranes (λSo
490 nm, τLo 6.4 ns), the new probes 2−4 showed an overall
smaller red shift of the excitation wavelength (λSo 475 nm) and
lifetime increase (τLo 5.5−5.8 ns), presumably due to less
perfect positioning along the lipid tails in the membrane
(Table 1).7,22 These results confirmed that substitution of the
carboxylate in the new probes 2−4 is well tolerated without
significant losses in mechanosensitivity.20,21
Intracellular targeting by probes 2−4 was studied using
confocal microscopy. Co-localization of targeted flippers with
B
DOI: 10.1021/jacs.8b13189
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Journal of the American Chemical Society
Communication
Figure 4. (A) Confocal image of Hela MZ cells incubated for 10 min
with far red EGF (red) and Lyso Flipper 2 (green; arrows indicate
lack of colocalization, top) and of cells rinsed and chased for 2 h
(arrows indicate colocalization, bottom); scale bar: 10 μm. (B) FLIM
images of COS7 cells coincubated with ER 3 and Mito Flipper 4
(top), with lifetime along the indicated cross section (bottom). (C)
FLIM (top) and intensity images (middle) of COS7 cells incubated
with Mito Flipper 3, with fluorescence intensity (black) and lifetime
(red) along the indicated cross-section (bottom).
Figure 3. FLIM images of Lyso 2 (A), ER 3 (B, arrow: sheets, framed:
tubules), and Mito 4 (C) in COS7 cells (1 μM, 20 min incubation)
before (top) and after (middle) hypertonic osmotic shock, with
corresponding lifetime histograms before (black) and after shock (red,
bottom); scale bars: 10 μM.
colocalization (around 20%) of the internalized EGF receptor
(EGFR) in endosomes with probe 2 was observed. After 2 h,
the fluorescent EGF was mainly located in late-endosomes and
lysosomes and showed increased overlapping with the Lyso
Flipper 2. This experiment indicated that probe 2 selectively
accumulates in late endosomes and lysosomes. Replacement of
morpholine in 2 by a more basic amine can be envisaged to
allow targeting of less acidic compartments such as early
endosomes.
As another example, the different lifetimes of probes 1−4
were exploited to simultaneously detect different organelles.
Co-incubation of ER Flipper 3 and Mito Flipper 4 revealed
their targets by the different fluorescence lifetime and intensity
contrast: Mitochondria yielded higher signal intensity, whereas
the fluorescence lifetime of tension probes was higher in the
tubular ER (Figures 4B, S19). Other cross sections revealed
different fluorescence lifetimes for the nuclear envelope, the
ER, and lysosomes (Figure S20).
A striking example for a long-standing question that could
not be answered without the new tension probes was provided
by Mito Flipper 4 in COS7 cells (Figure 4C). Measurements
of fluorescence intensity and lifetime along sites of
mitochondrial constrictions that precede fission26 revealed
that, in the constricted area, the intensity decreases due to
lower probe concentration, but the lifetime increases, thus
providing direct experimental evidence of the increased
membrane tension during mitochondrial constriction. A
complementary analysis on the role of membrane tension in
mitochondrial fission using Mito Flipper 4 will be reported
elsewhere.27
In conclusion, three organelle-selective fluorescent mem-
brane tension probes were designed, synthesized, and
characterized in both vesicles and living cells. As proven by
the sensitivity of their lifetime to osmotic shock, they report on
changes in membrane tension and reveal different lifetimes for
each target. These preliminary results promise important
discoveries in future toward understanding the intracellular
membrane dynamics during biological events.
at rest cannot be directly correlated to possible differences in
membrane tension. However, the reduction of membrane
tension by hypertonic shocks uniformly reduced lifetimes of
probes in HeLa Kyoto cells (Figure 2G, red) and in COS7 cells
(Figure 3, red histograms, Table 1). Decreasing lifetimes with
decreasing tension was as with Flipper-TR 15 and thus
consistent with operational probes reporting on tension-
induced lipid reorganization in the membranes of the
respective organelles (Figure 1B). Increasing counts in FLIM
histograms with decreasing tension might originate from the
increased amount of folded membrane in the focal plane upon
cell shrinkage. Nevertheless, such intensity changes do not
affect the fluorescence lifetime.
Hydrophilic Mito control 5 equipped with a mitochondria
targeting unit colocalized with MitoTracker Red in HeLa cells,
but failed to respond to the membrane tension change (Table
1, Figure S18). These results are consistent with the
accumulation of this probe in the mitochondrial lumen.
Further, viscosity-sensitive molecular rotors halo-tagged into
the lumen responded to the osmotic shock in the opposite way
to those of probes 2−4.12 These differences provided
corroborative evidence that flippers 2−4 indeed localize in
the organellar membranes and respond to changes in
membrane tension with changes in fluorescence lifetime.
All organelles showed heterogeneous lifetime distributions.
For instance, membranes of the tubular ER appeared more
ordered (τ = 4.1 ns) than those of the ER sheets (τ = 3.5 ns)
but responded equally to tension (Δτ ≈ 0.4 ns, Figures 3B,
S17). The lifetime of 3 in the nuclear membrane (τ ≈3.4 ns)
was similar to that in the ER sheets, consistent with their
continuous nature (Figure S20).
Selected experiments beyond probe characterization were
performed to outline the potential of intracellular tension
probes in promoting new discoveries. For instance, colabeling
of Lyso Flipper 2 with fluorescently labeled epidermal growth
factor24 provided insights on endosomal trafficking (Alexa
Fluor 647 EGF, Figures 4A, S13).25 After 10 min, poor
C
DOI: 10.1021/jacs.8b13189
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Journal of the American Chemical Society
Communication
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b13189.
■
S
Detailed experimental procedures (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*stefan.matile@unige.ch
*aurelien.roux@unige.ch
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ORCID
Stefan Matile: 0000-0002-8537-8349
Author Contributions
∥A.G. and A.C. contributed equally.
Notes
The authors declare the following competing financial
interest(s): Flipper-TR is commercially available from
Spirochrome, through the NCCR Store (https://nccr-chem-
bio.ch/technologies/nccr-store/). The NCCR receives 15% of
the revenues.
ACKNOWLEDGMENTS
■
We thank the NMR, MS, and bioimaging platforms for services
and the University of Geneva, the Swiss National Centre of
Competence in Research (NCCR) Chemical Biology, the
NCCR Molecular Systems Engineering and the Swiss NSF for
financial support.
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DOI: 10.1021/jacs.8b13189
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX