Fluorescent Membrane Tension Probes for Early Endosomes

Article abstract of DOI:10.1002/anie.202016105

Fluorescent flipper probes have been introduced recently to image membrane tension in live cells, and strategies to target these probes to specific membranes are emerging. In this context, early endosome (EE) targeting without the use of protein engineering is especially appealing because it translates into a fascinating transport problem. Weakly basic probes, commonly used to track the inside of acidic late endosomes and lysosomes, are poorly retained in EE because they are sufficiently neutralized in weakly acidic EE, thus able to diffuse out. Here, we disclose a rational strategy to target EE using a substituted benzylamine with a higher pK_a value as a head group of the flipper probe. The resulting EE flippers are validated for preserved mechanosensitivity, ready for use in biology, particularly to elucidate the mechanics of endocytosis.

Full text of DOI:10.1002/anie.202016105

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International Edition: doi.org/10.1002/anie.202016105
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Fluorescent Probes
Hot Paper
Fluorescent Membrane Tension Probes for Early Endosomes
Francesca Piazzolla+, Vincent Mercier+, Lea Assies, Naomi Sakai, Aurelien Roux,* and
Stefan Matile*
Abstract: Fluorescent flipper probes have been introduced
recently to image membrane tension in live cells, and strategies
to target these probes to specific membranes are emerging. In
this context, early endosome (EE) targeting without the use of
protein engineering is especially appealing because it translates
into a fascinating transport problem. Weakly basic probes,
commonly used to track the inside of acidic late endosomes
and lysosomes, are poorly retained in EE because they are
sufficiently neutralized in weakly acidic EE, thus able to
diffuse out. Here, we disclose a rational strategy to target EE
using a substituted benzylamine with a higher pKa value as
a head group of the flipper probe. The resulting EE flippers are
validated for preserved mechanosensitivity, ready for use in
biology, particularly to elucidate the mechanics of endocytosis.
to increase intensity and lifetime.[5] Contrary to most other
small-molecule membrane probes[6,7] such as molecular
rotors[6] that respond off-equilibrium in the excited state
and report on viscosity, planarizable push-pull flipper probes
thus respond at equilibrium in the ground state to physical
forces from a confining environment.
In organic solvents, the planarizable push–pull probes
absorb in the near-UV region, and do not fluoresce in water.
The ordered environment of lipid bilayers increases their red
shifts and the fluorescence lifetimes as expected from polar-
izing planarization. According to fluorescence lifetime imag-
ing microscopy (FLIM), membrane tension applied with
micropipettes or osmotic stress to mixed membranes, includ-
ing biomembranes, increases lifetimes due to membrane
reorganization with the dominant response from fully pla-
narized flippers in highly-ordered microdomains composed of
out-sorted un-stretchable lipids (Figure 1 f),[3] together with
decreasing membrane deformations.
For local membrane tension measurements in living cells,
flipper probes must be placed in the membrane of interest
(MOI). We already reported flipper localization strategies
using either the more empirical chemistry known from
trackers[8] or the general, rational design known from fusion
proteins, e.g., HaloFlippers targeting HaloTags expressed in
the MOI.[9] In this context, early endosomes (EE) attracted
our attention. They are the first endosomal compartment
reached by molecules endocytosed in vesicles (Figure 1d). At
this sorting station, they can be recycled to the surface or sent
toward late endosome (LE) and lysosome (LY), the latter
depending on the endosomal sorting complex required for
transport III (ESCRT-III). The importance of membrane
tension in endolysosomal dynamics,[10,11] including the regu-
lation of ESCRT-III, called for a precisely localized imaging
of membrane tension in the EE. EE targeting is possible by
engineering fusion proteins or labeling receptors at the cell
surface and then following their endocytosis (vide infra),[11,12]
but rarely by small-molecule probes. From EE to LE and LY,
the acidity in the organelle increases (Figure 1d). This
gradually decreasing pH has been much used for halochromic
imaging,[13] endocytosis inhibition,[14] and endosomal
escape[15,16] (proton sponge effect[16]). The pH gradients are
also routinely used for targeting LE and LY[17] but not EE.[18]
This question interested us because it translates EE targeting
into a fascinating transport problem,[19–21] that is irreversible
penetration by unidirectional translocation along a weak pH
gradient. In the following, we tackle this subtle transport
challenge and introduce EE flipper 1 (Figure 1e).
T
he imaging of physical forces in biology with small-
molecule fluorescent probes that can be added to unmodified
living systems is a scientific challenge that calls for solutions
from chemistry. The use of standard physics tools is mostly
limited to the exterior and is quite perturbing to the system.
Bioengineered tension sensors mostly use FRET pairs that
report on structural changes of protein or DNA constructs in
specific model systems and are not mechanosensitive by
themselves.[1] These approaches do not satisfy the demand
from mechanobiology for small-molecule fluorescent probes
that are intrinsically mechanosensitive and work in unmodi-
fied cells at the location of interest. As a possible strategy to
uncover such chemistry tools, we have introduced flipper
probes.[2,3] They are constructed around dithienothiophene[4]
dimers that are twisted out of co-planarity by repulsion
between methyls and sulfurs next to the twistable bond
(Figure 1e). Their planarization shifts absorption and excita-
tion maxima to the red and increases fluorescence intensity
and lifetime. The redshift originates from the mechanochem-
ical increase of the ground-state energy and the lowering of
the less twisted Franck-Condon (FC) excited-state energy.[5]
The less twisted FC state preferentially relaxes to the emissive
planar intramolecular charge transfer (PICT) state rather
than to the more distorted non-emissive relaxation pathways
[*] Dr. F. Piazzolla,[+] Dr. V. Mercier,[+] L. Assies, Dr. N. Sakai,
Prof. A. Roux, Prof. S. Matile
School of Chemistry and Biochemistry, National Centre of Compe-
tence in Research (NCCR) Chemical Biology, University of Geneva,
Geneva (Switzerland)
E-mail: aurelien.roux@unige.ch
stefan.matile@unige.ch
Homepage: https://www.unige.ch/sciences/chiorg/matile/
[+] These authors contributed equally to this work.
LysoTrackers function with an ammonium cation with
a pKa ꢀ 7.4, often morpholinium, also used in flipper 2
(Figure 1b).[17,8] In the cytosol, around pH 7,[22] these ammo-
nium cations stay mostly protonated. However, since pKa
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016105.
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Figure 1. Labeling of a) early endosomes with weakly acidic ammonium cations 1 by unidirectional penetration along a weak pH gradient,
b) coinciding with conventional LE tracking along a stronger pH gradient and c) preceded by directionless plasma membrane (PM) penetration;
compared to more acidic control 2 for LE tracking (b) together with directionless penetration of EE (a) and PM (c). d) The increase of acidity
within organelles during endocytosis from PM to EE, LE, and LY. e,f) Structure and mode of action of EE flippers 1. g) Structure of EE flipper
1 compared to LE flipper 2 and 3–12, and flipper-free model head groups 1’-10’ with pKa values from NMR titrations. FL=flipper, as in (e). See
Figure S1 for full structures of flippers. The exchangeable counterions of cationic flippers, synthesized as TFA salts, are omitted. [a] Models 5’ and
8’ available, flippers 5 and 8 not (unstable). NBn=p-nitrobenzyl.
values depend significantly on the polarity of the environ-
ment,[23,19] the ammonium cation in 2 can temporarily release
its proton to diffuse across the hydrophobic barrier as the
neutral conjugate amine base of 2, and re-protonate at the
other side. Inside LE and LYat pH ꢁ 5.5 with a pKa ꢀ 7.4, this
temporary deprotonation becomes impossible. As a result,
the ammonium cation 2 is trapped, cannot move back to the
cytosol. With time, this irreversible unidirectional penetration
causes the cation 2 to accumulate in LE and LY, and thus label
both. Similar mechanisms account for the loading of weakly
basic drugs in liposomal delivery systems.[24] Temporary
changes of pKa by up to four orders of magnitude also allow
carboxylates and fluoride to move across bilayer membrane
as neutral conjugate acids.[19] Related proximity effects are
ubiquitous in enzymes to produce amines and carboxylic acids
in neutral water for base and acid catalysis, respectively,
including aldolases, glycosidase, cholesterol cyclase, HIV
protease, and so on.[25] However, in a biological context, pKa
changes beyond four orders of magnitude are rare.[26] The best
example is the guanidinium cation with pKa ꢀ 12.5, which
accounts for the translocation of arginine-rich cell-penetrat-
ing peptides through repulsion-driven ion pairing rather than
transient deprotonation.[21]
LY. However, the acidity within EE is insufficient to prevent
deprotonation and inhibit the return into the cytosol, i.e.,
achieve the irreversible penetration needed for retention and
labeling. These considerations suggested that what would be
needed to target EE are less acidic ammonium cations.
However, low acidity should also prevent deprotonation in
the cytosol and thus inhibit translocation across any mem-
brane.[21] The rare use of pKa finetuning in EE trackers[18]
suggested that the involved gradients are too weak, and the
challenge cannot be met. In the following, we show that this
impression is incorrect.
We selected benzyl substituted ammonium cations as head
groups of potential EE flippers 1, 3–12 (Figure 1g). The
acidity of these ammonium cations can be readily modulated
by the attached electron-donating or withdrawing substitu-
ents on the phenyl rings. The number of nitrogen substituents
and the tethers length were also expected to influence the
pKa. Further, phenyl groups are suited for a membrane probe
since aromatic-lipid bilayer interactions are well known from
membrane proteins to assist their translocation and anchoring
at the interface,[27,28] presumably by cation-p interactions with
the choline headgroup of many lipids.[27]
Flippers 1, 3, 4, 6, 7, 9–12, and controls 1’-10’ used in this
study were prepared by multistep synthesis similar to that of
LY-flipper 2 (Schemes S1–S4). Reductive amination reactions
between the substituted benzaldehydes and amines provided
EE are poorly labeled by this method because the pH
gradient is insufficient (Figure 1a). The morpholino flipper 2
enters the EE by temporary deprotonation, just like LE and
2
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different mono- and di-benzyl amines easily. The obtained
as outlined in Figure 1a–c, and not by endocytosis, which
benzylamines with an azide terminus then underwent azide-
alkyne click reactions with a common flipper intermediate
with an alkyne terminus to give desired flippers. Among the
EE-flipper candidates, p-thiomethyl- and p-nitro-benzyl flip-
pers 5 and 8 were inaccessible due to their instability caused
by photoinduced oxidation and debenzylation (Figure S28),
respectively. The pKa values of hydrophobic models 1’–10’
without the flipper mechanophore were estimated to range
between 5.1 and 9.8 by pH titration using 1H NMR spectros-
copy (Figures 1g, S2–S27).
EE labeling was evaluated by co-localization with the
epidermal growth factor (EGF) labeled with Alexa-647 (Far-
Red, FR) to avoid overlap with flipper fluorescence (Fig-
ures 2, S29–S33, S37b). In this standard pulse-chase endocy-
tosis assay,[11,29] EGF-FR mainly labels EE after a 10 minute
incubation of cells (pulse) and LE and LY when pulse is
followed by a 2 hour incubation in a medium without EGF-
FR (chase). During the last 10 minutes of the above process,
cells were incubated with specific flipper probes because they
rapidly label the compartments compatible with their pKa.
Complete labeling of LE and LY within ꢀ 6 minutes as well as
the absence of plasma membrane staining[8] confirmed that
flippers enter cells and organelles by directional penetration
would take ꢀ 2 hours for LE and LY, rather than 6 minutes
(Figure 1d, 2).
Confocal laser scanning microscopy (CLSM) images of
HeLa MZ cells labeled with EGF-FR (red) and flipper
1 (green) for 10 minutes were taken (Figure 2a). Since flipper
1 also labels LE and LY, the global co-localization with EGF-
FR cannot be high (green vs. yellow). However, the propor-
tion of EGF-FR co-localized with flipper 1 was significant
(red vs. yellow). This result contrasted with LY-flipper 2,
which was practically excluded from EGF-FR labeled EE
(Figure 2b). After a 2 hour chase, EGF-FR in LE co-localized
equally well with both flippers (Figure 2c,d).
Co-localization of EGF-FR and various flippers was
quantified using automated high-content microscopy (Fig-
ure 2e,f).[30] This method allowed us to analyze thousands of
cells in a short time. Dibenzylamino-flippers 9, 10, and 12
were excluded from the analysis because of their poor
staining. Also abandoned was the SO2Me substituted flipper
7 because it was unstable in the media due to debenzylation
(Figure S28). For the remaining six flippers, co-localization
ratios were determined from the ratio of co-labeled organ-
elles and all organelles labeled with EGF-FR (Figure 2e,f).
The results obtained after 10 minutes of pulse showed a clear
correlation between co-localization ratios and the pKa of the
ammonium cations, as expected for EE labeling (Figure 2e).
After a 2 hour chase, this correlation vanished, and co-
localization ratios were high and constant, independent of the
pKa of the ammonium cations as expected for LE/LY labeling
(Figure 2 f). A comparison of benzylamine flippers with
*
^
)
a long (Figure 2e, 6 red ) or short tether (11 grey
revealed poor reproducibility with the latter and thus
confirmed that the former, as in the original morpholino
flipper 2, is preferable for acidity screening.
Overall less than perfect co-localization is intrinsic to
EGF-FR labeling, which produces a broader distribution
along the endocytic pathway with, for instance, a few LE
already labeled when most EGF-FR is still in the EE and
a few EE remaining labeled when most EGF-FR has reached
the LE (Figure 2). The use of classical LysoTrackers instead
was not meaningful because EEs remain invisible. According
to the mean fluorescence intensities, the efficiency of probe
internalization in cells did not correlate with the pKa of the
ammonium cations (Figure S34). We also have not observed
PM staining with this series of flippers. Thus, these results
indicated that the higher limit of pKa to prevent the
deprotonation in the membrane is still not reached with
flipper 1.
The mechanosensitivity of EE flipper 1 was explored with
FLIM. Very weak and diffuse background fluorescence with
flipper 1, at least one order of magnitude less intense than the
tiny endosomes, was removed by adjusting the threshold of
the number of photons per pixel. Average lifetimes (t1) of all
labeled organelles by flipper 1, including EE, LE, and LY,
were around t = 4.42 ns. This was significantly lower than for
LY flipper 2 (t = 4.60 ns), suggesting that probe 1 is in a more
liquid-disordered membrane, as expected from its EE local-
ization (Figure 3a,c, blue, S37a).[8,11] The application of
hyperosmotic stress lowered the lifetime to t = 4.21 ns (Fig-
Figure 2. a–d) Merged CLSM images of HeLa MZ cells labeled with
1 (a,c), or 2 (b,d, 1 mM, green) and EGF-FR (red) after 10 min
pulse (a,b) or 10 min pulse followed by 2 h chase (c,d). Insets:
zoomed portions of images with arrows pointing at the EGF-FR labeled
endosomes (note how with their small size, yellow dots can appear
red without magnification; (a), top left, compare (e,f)). Scale
bars=10 mm. e) Fractions of EGF-FR labeled organelles co-localized
*
*
*
*
*
with 1 (blue ), 2 (black ), 3 (green ), 4 (violet ), 6 (red ), or 11
^
(grey , 1 mM) after 10 min chase (mean Æ SD). f) As (e), after 2 h
chase.
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and identify the maximum. Meanwhile, the here introduced
EE flipper 1 is ready for use in biology, without the need for
cellular engineering or possible interference from proteins
used for targeting. The simple addition of the fluorescent
probe to unmodified cells is particularly attractive to study
the mechanics of endocytosis.[10,11]
Acknowledgements
We thank D. Moreau and S. Vossio (University of Geneva) for
help with high-content microscopy and analysis, the NMR,
the MS, and the Bioimaging and ACCESS platforms for
services, and the University of Geneva, the Swiss National
Centre of Competence in Research (NCCR) Molecular
Systems Engineering, the NCCR Chemical Biology, and the
Swiss NSF for financial support.
Figure 3. a,b,d,e) FLIM images of 1 (1 mM) in HeLa MZ cells befo-
re (a,d) and after hypertonic shock (b,e) without (a,b) and with EE
filtering using the 10-min internalized A647-Dextran image (d,e). Scale
bar=10 mm. c) Fluorescent lifetimes under isotonic (iso) and hyper-
tonic (hyper) conditions, all pixels (blue) or only pixels corresponding
to EE (black). Symbols with error bars represent the mean values
ÆSD, dashed lines the individual fields from 3 independent experi-
ments. Two-tailed paired Student’s t-tests gave P<0.0001 (****) for
the two set of data.
Conflict of interest
The University of Geneva has licensed four Flipper-TRꢀ
probes to Spirochrome for commercialization, including 2,
but not 1.
ure 3b,c, blue). Originating from the tension-induced disas-
sembly of ordered microdomains or increasing membrane
deformations, this decrease was consistent with flipper
deplanarization in response to decreasing membrane tension
under hyperosmotic conditions.[3,8,9] It thus validated EE
flipper 1 as an operational membrane tension probe.
Keywords: directionality · early endosomes ·
fluorescent probes · mechanosensitivity ·
membrane penetration · pH gradients
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Manuscript received: December 3, 2020
Revised manuscript received: January 18, 2020
Accepted manuscript online: February 3, 2021
Version of record online: && &&, &&&&
6
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These are not the final page numbers!

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Communications
Fluorescent Probes
Unidirectional penetration along mini-
malist pH gradients is developed as
a strategy to label the membrane of early
endosomes with mechanosensitive flip-
per probes, by simple addition to
unmodified cells. Substituted benzyl-
amines offer the right acidity, pKa =10,
not too strong to allow reversibility, not
too weak to inhibit penetration from
neutral water in the cytosol, and, perhaps,
extra returns from interfacial aromatic
anchoring.
F. Piazzolla, V. Mercier, L. Assies,
N. Sakai, A. Roux,*
S. Matile*
&&&& — &&&&
Fluorescent Membrane Tension Probes
for Early Endosomes
Angew. Chem. Int. Ed. 2021, 60, 1 – 7
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7
These are not the final page numbers!