Fluorescent Probe for Transmembrane Dynamics during Osmotic Effects

Article abstract of DOI:10.1021/acs.analchem.9b05390

Membrane tension pores determine organelle dynamics and functions, giving rise to physical observables during the cell death process. While fluorescent organelle-targeted probes for specific chemical analytes are increasingly available, subcellular dynamic processes involving not only chemical parameters but also physicochemical and physical parameters are uncommon. Here, we report a mitochondrial chemical probe, named RCN, rationally designed to monitor osmotic effects during transmembrane tension pore formation by using local mitochondrial polarity and a subcellular localization redistribution property of the probe. Utilizing fluorescence spectroscopy, high-resolution confocal imaging, and spectrally resolved confocal microscopy, we provide a new correlation between mitochondrial dynamics and bleb vesicle formation using osmotic pressure stimuli in the cell, where the mitochondrial local polarity was found to drastically increase. The RCN provides a reliable protocol to assess transmembrane pore formation driven by osmotic pressure increments through local polarity variations and is a more robust physicochemical parameter allowing the health and decease status of the cell to be measured.

Full text of DOI:10.1021/acs.analchem.9b05390

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Article
Fluorescent Probe for Transmembrane Dynamics During Osmotic Effects
Eva Palacios-Serrato, Daniela Araiza Olivera, and Arturo Jiménez-Sánchez
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b05390 • Publication Date (Web): 30 Jan 2020
Downloaded from pubs.acs.org on January 31, 2020
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Fluorescent Probe for Transmembrane Dynamics During
Osmotic Effects
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Eva Palacios-Serrato, Daniela Araiza-Olivera and Arturo Jiménez-Sánchez*
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Instituto de Química - Universidad Nacional Autónoma de México.
Circuito Exterior s/n, Coyoacán 04510. Ciudad Universitaria, Ciudad de México, México. E-mail:
arturo.jimenez@iquimica.unam.mx
ABSTRACT: Membrane tension pores determine the organelles dynamics and function giving rise to
physical observables during cell death process. While fluorescent organelle-targeted probes for
specific chemical analytes are increasingly available, subcellular dynamic processes involving not
only chemical but physicochemical and physical parameters are uncommon. Here we report a
mitochondrial chemical probe, named RCN, rationally designed to monitor the osmotic effects
during the transmembrane tension pore formation by using local mitochondrial polarity and a
subcellular localization redistribution property of the probe. Utilizing fluorescence spectroscopy,
high-resolution confocal imaging and spectrally-resolved confocal microscopy we provide a new
correlation between mitochondrial dynamics and bleb vesicles formation with osmotic pressure
stimuli in the cell, where the mitochondrial local polarity resulted to be drastically increased. The
RCN provides a reliable protocol to assess the transmembrane pore formation driven osmotic
pressure increments though local polarity variations, a more robust physicochemical parameter
allowing to measure the health and decease status of the cell.
Cells and subcellular structures are delimited by lipid bilayers which determine organelle
dynamics such as motility, fusion/fission of the organelle framework, morphology and volume, and
inter-organelle contacts.1-7 Membrane tensional status as well as tension pores are then major
regulators of the organelle dynamics.8,9 Although the mechanical forces governing subcellular
interactions is an emerging area,8-11 now the fact that membrane tension regulates membranous
organelles’ function, is well established. Examples reporting the organelle tensional status have
been reported.8-13 However, the way tensional status of an organelle membrane modifies subcellular
function and dynamics is still an unmeet need. In fact, the appearance of transmembrane tension
pores is determined by the influx of water molecules into the cell, giving rise to an augmented
membrane tension and local polarity (in terms of dielectric constant).14-16 The organelle shape
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variations and different osmotic phenomena such as the formation of transient tension pores and
vesicle rupture17 comprise a common physical observation during cell death process.18 Monitoring
such tension stimuli-responsive scenario is challenging since the transmembrane tension pores, with
their effective radii that are comparable to the size of small molecules and ions, have size selective
properties19-21 which increase the plasma membrane permeability, specially for ions and small
molecules, thus disbalancing the electrochemical gradients of the cell. For this reason, the
functional visualization and monitoring of transmembrane dynamics upon tensional stimuli using
common fluorescent organelle localizers is tremendously difficult as they exhibit signal
interferences and artefacts due to the local membrane potential depolarization during tension
stimuli. Deservingly so, the correlation of transmembrane tension pores with dynamic properties
such as local polarity helps to understand the tensional status in terms of membrane function.8 The
local polarity monitoring serves as a general reporter of all the osmotically driven ions and
molecules for which any membrane is selective. Then, local polarity is a more useful bioanalitycal
parameter of osmotically perturbed membrane function during a specific cell stage, i.e. cell
proliferation or cell death process. More importantly, mitochondrial dynamics is directly affected
during the osmotic variations inside the cell where both, the tensional and local polarity status of the
organelle is substantially altered. In fact, the inflow of K+ ion increases the mitochondrial osmotic
pressure, which results in the formation of the so-called donut-shaped mitochondria.22,23 Also, the
osmotic energy gained from K+ ion inflow leads to an increase of mitochondrial volume. Then, fast-
response fluorescent probes able to monitor mitochondrial dynamics and function during tensional
and osmotic stimuli could be helpful to understand cell death process, cell bleb formation and how
mechanical forces regulate cell membrane status.
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Herein, we describe a mitochondrial fluorescent probe, called RCN (Scheme 1), that follows a
sensing mechanism aimed to monitor the osmotic effects on the transmembrane tension pore
formation by using local mitochondrial polarity and a subcellular localization redistribution
property of the probe. The RCN consists of a chromenylium-cyanine structure derived from a
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cinnamic-chromenylium-cyanine (RCin fragment) and a naphthalimide (Naph fragment), Figure 1.
By itself, the RCin exhibits highly tunable optical properties, i.e. high solvatochromic effects, large
Stoke shifts, a well-defined dual-fluorescent behavior which can be promoted either by Kasha rule
breaking, intramolecular proton transfer or intramolecular charge transfer.24 However, the
chromenylium-cyanine structure usually results in very low fluorescent quantum yields (Φf of RCin
= 0.01). On the other side, the Naph fragment exhibits a high Φf of 0.63 (Supplementary
Information file) avoiding a versatile photophysical scheme, thus acting as a fluorescent signal
enhancer. The solvatochromic, polarity-dependent RCin fragment shows mitochondria specific
localization in the red confocal channel as a consequence of its chromenylium ion identity while the
Naph fragment equilibrates the cytosol and nucleoli in the green confocal channel. When tether
these fluorophores into RCN, the whole construct shows a clean mitochondrial localization under
green and red confocal channels with bright emission (Φf of 0.45) and excellent photostability (see
Figure S1, SI). The mitochondrial co-localization analysis using MitoLite™ Blue stain as well as
functional mitochondrial co-localization dynamic using mitochondrial membrane potential
uncoupling stimulus are shown in Figure S2.
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Scheme 1. Structures and synthetic methodology to obtain RCN probe: (a) Toluene, 120 °C, 24 h;
then NaOH (30% aq), 90 °C, 12 h; (b) H2SO4, 60 °C, 2.5h, then HClO4; (c) acetic anhydride, 60 °C,
3 h; (d) 120 °C, 6h; (e) Cs2CO3, HBTU, DMF, 90 °C, 24 h.
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Figure 1. Chemical structure of the RCN probe and its localization. (A) The solvatochromic,
polarity-dependent RCin fragment shows a mitochondria specific localization in the red confocal
channel. (B) With high fluorescence quantum yield, the Naph fragment equilibrates the cytosol and
nucleoli in the green confocal channel. (C) The whole construct RCN exhibiting mitochondrial
localization under green and red confocal detection. Mitochondrial co-localization analysis is shown
in Figure S2, SI file. Scale bars represent 20 µm.
EXPERIMETAL SECTION
Materials and Methods. All starting reagents were obtained from commercial suppliers and used
as received.
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Synthesis. The RCN probe was synthesized according to Scheme 1. Detailed description and
chemical characterization are presented in the Supplementary Information file.
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Optical spectroscopy. Absorption spectra were acquired in a 10 mm path-length quartz-cell
Cary-50 (Varian) spectrophotometer, the emission and excitation spectra were obtained in an
Edinburgh Instruments FS500 fluorimeter at room temperature (20 ± 1 °C) under aerated
conditions.
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Cell Culture and Confocal Microscopy. HeLa cells as well as live human pulmonary
adenocarcinoma epithelial cells (SK-LU-1) were cultured in RPMI-1640 medium (RPMI Medium
1640 (1x), Gibco, Gaithersburg MD) supplemented with 10% fetal bovine serum (FBS, Invitrogen,
Carlsbad CA), L- glutamine (2 µM), penicillin G (100 u/mL), streptomycin sulfate (100 µg/ mL) at
37°C with 5% v/v CO2. Live SK-LU-1 cells were seeded on 8 Petri dishes of 5 cm diameter with
glass bottom for 36 hours before experiments using RPMI-1640 medium supplemented. Then,
specific concentrations of RCN from 5 to 8 µM were used. Commercial specific organelle
localizers were added on each Petri dish 45 minutes before imaging experiments. All dishes were
washed two times with RPMI. During confocal imaging, microscope parameters were maintained
constant and excitation light was fully-shielded to prevent laser artefacts. Live HeLa cells were
seeded in 8 well μ–slides (iBidi, Germany) at a density of 20000 cells per well one day prior to
experiments in MEM alpha with 10% FBS. On treatment day, cells were washed once in MEM
alpha with no FBS and incubated with 5 to 8 μM probe RCN for 30 minutes. For experiments with
MitoLite® Blue (Thermo Fisher Scientific), 10 nM were added 10 minutes before RCN. Cells were
then washed twice in MEM alpha with no FBS and imaged maintaining 5% CO2 and 37°C during
the experiments using an inverted Zeiss LSM 880 microscope or a Nikon A1R upgraded with a
spectral detector unit. On treatment day for fluorescence time course experiments, cells were
incubated with 7 μM probe RCN for 30 minutes in MEM alpha with 5% FBS for the indicated time
at 37°C with 5% CO2, then imaged at the same conditions.
RESULTS AND DISCUSSION
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Spectroscopic features of the RCN probe. As mentioned, the RCin contribution to RCN
emission profile allows a strong solvatochromic effect, i.e. the emission maximum (λem) of the RCin
in hexane is 400 nm while in methanol is 795 nm, a difference of ca. 12,500 cm-1 (400 nm) with
intermediate emission maximum wavelengths for other solvents, Figure 2A and S3. However, such
extremely high and unusual solvatochromic effect turns out to be diminished to some extent in
RCN since the main emission feature is the fluorescence intensity coming from the Naph fragment
although the solvatochromic effect is still high for the whole RCN construct. In fact, a significant
sensitivity to solvent polarity was observed, Figure 2B-C. In order to calibrate such fluorescence
response under variable polarity, we performed the calibration of polarity effect using organic
solvents with different dielectric constant for RCN and RCin fragment, Figure 2B-C. Also, dioxane
: water system calibration gave similar calibration curves, Figure S4, SI file. The RCN probe
exhibits a dual-band fluorescence profile with a strong enhancement in the visible region as the
polarity of the solvent decreased while the NIR emission was quenched, Figure 2B. The ratio of
fluorescence intensity at 515 nm versus 715 nm upon variable dielectric constant was then
quantified observing a linear relationship with a regression coefficient of R2 = 0.987. Importantly,
the 200 nm difference of fluorescence wavelength recordings for the calibration plot is large enough
to be suitable for confocal ratiometric imaging correlations of subcellular environments with no
confocal channel interference. A similar result was observed for the isolated RCin fragment, here
the fluorescence ratio at 750 nm versus 500 nm reproduce a similar dielectric constant dependence
with a linear relationship of R2 = 0.995. In fact, using ratiometric confocal imaging, both isolated
fluorophores (RCN and RCin) can be used as local polarity reporters on mitochondria where
comparison of those polarity calibrations can afford a more robust measurement. Then,
mitochondrial local polarity was monitored with the fluorescent profile of RCN under conditions
promoting osmotic pressure variations through transmembrane pore formation in order to evaluate
the probe to track dynamic changes that eventually cause strong morphological changes and
pronounced physicochemical variations in the organelle microenvironment.25,26
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Figure 2. UV-Vis (black lines) and fluorescence (red lines, λex = 380 nm) spectra in methanol for
the RCin (solid lines) and the Naph (dashed lines) fluorophore units (A). Calibration of the polarity
effect using organic solvents with different dielectric constant for RCN (B) and RCin (C). Water :
dioxane calibration as a function of dielectric constants is presented in Figure S4, SI file.
On the other hand, the viscosity effect on the RCN probe was also assessed by the acetone :
glycerol continuous variation, which is a solvent mixture having a larger dielectric constant
difference compared to methanol : glycerol. Fluorescence spectra are presented in Figure S3-C,
where no significant fluorescence increment was observed in both, green and red channels. Then, a
Catalán scale analysis was also implemented, which comprises a more quantitative solvent
interaction scheme. Solvents of different viscosity and dielectric constant were used, Figure S3-B.
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The correlation coefficients for fluorescence maximum observable did not show significant changes
when considering the viscosity parameter of the solvents compared to the correlation without
considering the viscosity effect, this indicates that solvent viscosity has no influence on the polarity
(Table S1, SI file).
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Mitochondrial dynamics and bleb formation upon tension stimuli in epithelial cells. The
membrane tension was stimulated by osmotic pressure increments. If the pressure in the cell is
being continuously increased at a point disturbing the membrane binding to the cytoskeleton, the
membrane detaches from it, then starts stretching and a bleb vesicle is formed.27,28 While the bleb
formation can be initiated by a cell volume increment, the mechanism necessarily involves an
osmotic pressure increment by the simultaneous formation of transmembrane pores.18 Then, to
carefully initiate the bleb formation process we used nystatin, a transmembrane pore-forming agent,
especially acting on the plasma and mitochondria. However, the mitochondrial dynamics is
consequently altered by this process and to the best of our knowledge, no fluorescent probes able to
follow bleb formation and mitochondrial dynamics by staining both entities have been reported to
date. Our studies on intracellular communication using fluorescent probes indicated that RCN
probe continuously localized mitochondria during nystatin treatment, thus allowing functional
visualization of bleb formation, Figure 3. As can be seen, the donut-shape mitochondrial network is
clearly formed after 30 minutes treatment as shown in the green and red confocal channels.
However, the RCN fluorescence profile indicate two different probe distribution patterns. In the
green- and red-channels, the mitochondrial network and bleb membrane were observed, while a
clean and structured nuclear distribution was only observed in the green-channel, Figure 3A. This
phenomenon suggests the RCN probe to be fragmented into the Naph- and RCin-type entities
promoted by the nystatin treatment since the green and red emission signal is the same as the one
observed for RCin, and the Naph green emission is located in the nuclei. In fact, Figure 3A shows a
brighter green emission in the nuclei (similar to Naph signal) compared to the RCin low intensity
green-channel emission located in the mitochondrial network.
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Figure 3. Confocal images of 5 µM RCN in SK-Lu-1 cells under 1 hour 100 µM nystatin treatment
in the (A) green and (B) red-channels. (C) Merge image with insets showing mitochondrial donut
formation with major red emission contribution and green signal redistribution into the nucleus.
Mitochondrial co-localization analysis is shown in Figure S2, SI. Scale bars represent 20 µm.
Furthermore, not only the mitochondrial shape and dynamics is altered during transmembrane
pore formation, but such osmotic variations induced a homogeneous increment in the cell volume
observed in few minutes (5 to 15 minutes time scale) giving rise to the vesicle leakage prior to cell
lysis. Figure 4 shows a time-course experiment taken between 0 and 25 minutes after low-dose
nystatin addition using high-resolution confocal microscopy. Firstly, the bright field allows to
follow the general cell-shape alterations and the green and red-channels evidencing the RCN
emission profile located in mitochondria.
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Figure 4. Confocal images of 5 µM RCN in SK-Lu-1 cells under 25 minutes low-dose 50 µM
nystatin treatment observed in the (A) bright field, (B) green- and (C) red-channels taken at time t0
= 0 min, then each 5 min until t5 = 25 min. Insets show the indicated expansion to visualize a
mitochondrial fragmentation and motility due to swelling process. Excitation light was fully
shielded between recordings to prevent artefacts and photobleaching. Organelle morphology (form
factor) analysis is presented in Figure S5. Scale bar represents 20 µm.
Although both, mitochondrial and bleb swelling were observed, the mitochondrial dynamics was
altered to a greater extent. A form factor (F) analysis indicated a relatively larger F value (defined
as P2/4πA, where P is the perimeter and A is the area of a single mitochondrion) of ca. 50% after
nystatin treatment. This is because the increment in the osmotic pressure achieved by the inflow of
K+ ions results in both, mitochondrial donut formation and swelling. As the fluorescent staining
observed for the membrane blebs required a higher laser potential to be properly observed, we
utilized a higher concentration of RCN before nystatin treatment in order for the blebs to be well-
contrasted under the green channel setup using low laser power conditions, Figure 5. These
conditions also allowed a slight nucleoli visualization and the in-plane bleb illumination provided a
bright and clean emission on these membranes, while the mitochondrial framework was observed in
the red-channel setup, the channels overlap is shown in Figure 5D.
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Figure 5. Confocal images of 8 µM RCN in SK-Lu-1 cells under 25 min 100 µM nystatin
treatment in the (A) green- (B) red- and (C) bright-channels. (D) Green and red merged image
showing bleb formation and morphologically altered mitochondrial framework, respectively. Insets
show the indicated expansions. Scale bar represents 20 µm.
As this probe fragmentation was observed after treatment of high-dose nystatin up to the cell
death process is initiated, we hypothesized that proteases were responsible for such probe rupture.29
Then, effector proteases can break peptide bonds such as the one present in RCN.30 Structural
analysis of RCN interaction with protease k by high resolution mass spectrometry and fluorescence
spectroscopic revealed the formation of Naph, RCin and an RCin-derived oxonium ion, the
fluorescence titration of proteinase k to RCN probe also suggested the activation of the isolated
Naph emission, Figures S6-S8.
Ratiometric confocal imaging experiments were then correlated before and after nystatin
treatment, i.e. using the RCN and RCin polarity calibration profiles. Before nystatin treatment,
using the RCN profile the local polarity (in terms of dielectric constant) was determined to be ε =
16.1 ± 0.6. To note, a similar result was found when using the RCin profile, with a ε = 18.8 ± 0.5.
Such difference could be a consequence of the higher feasibility of the RCin to be solvated by polar
species in its surrounding, giving a high dependency in the polar region since the dipole moment is
located on the longitudinal π-conjugated axis (from the dimethylamino-cinnamic to the
dimethylamino-chromenylium moieties) which is in-plane. The RCN probe, on the other hand, has
this dipole moment in contact with the Naph electronic density, thus relocalizing it out of the
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Figure 6. Ratiometric confocal imaging to study the effect of osmotic pressure increments through
transmembrane pore formation by nystatin agent on the mitochondrial polarity of live cells stained
with 5 μM RCN. Pseudo-color images show changes in polarity: (A) HeLa cells before and after 30
min treatment; (B) SK-Lu-1 cells before (left) and after (right) 30 min treatment and (C) SK-Lu-1
during donut-shape formation at 45 min (left) and 60 min (right) treatment. Scale bars are 20 µm.
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molecular plane. Significant polarity increments were obtained after nystatin treatment, finding the
dielectric constants of 30.7 ± 0.8 and 33.5 ± 0.6 with RCN and RCin probes, respectively, Figure 6.
To corroborate the ratiometric analysis we implemented spectrally-resolved confocal microscopy
experiments since one of the main drawbacks in the confocal ratiometric analysis is the loss of the
probe structural information and local spectral-resolution during time-course experiments,
particularly when the fluorophore chemically interacts with a given stimuli or is being fragmented.
In this sense, spectrally-resolved imaging gives analytical bioimaging the ability to perform in-situ
calibration, thus allowing continuous structural information of the probe response profiles. Using a
confocal array coupled with a spectral detector unit, accurate spectral unmixing enabled RCN to be
spectrally resolved in real time with 32- channel images of 512x512 pixels, Figure 7.
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Figure 7. Spectrally-resolved confocal microscopy for real-time local fluorescence spectra of RCN.
From left to right: non-stained SK-Lu-1 cells, 30 min incubation with 5 μM RCN and 50 μM
nystatin treatment for 30 minutes. Cells were washed three-times before experiments. Excitation
wavelength is 488 nm.
Then, spectral-resolution at the single-cell level revealed that nystatin-induced transmembrane
pore formation leads to a strong enhancement of the green-channel emission band while a slight
fluorescence increment in the red confocal channel thus providing a real-time local structural
information of the Naph and RCin formation. Spectroscopic fluorescence titrations and imaging
microscopy using nystatin and a series of transmembrane pore formation agents such as digitonin,
etoposide and amphotericin A as well as oxidation, pH and lysis buffer controls is presented in the
Supplementary Information file (Figures S7-S9), although similar effects were observed with other
pore forming agents, nystatin produced stronger influences on both, fluorescence and subcellular
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localization profiles of the RCN, observing no probe interference with the pH or oxidation.
Interestingly, the found polarity variation associated with osmotic pressure increments also indicate
the mitochondrial membrane to be more hydrated after transmembrane pore formation by nystatin
treatment, which is in agreement with recently described studies using optical super-resolution
microscopic techniques.31-33
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Finally, to correlate the morphological changes of the whole cell with the mitochondrial-bleb
formation dynamics as a consequence of the transmembrane pore formation and osmotic pressure
increments, the reorganization of the actin cytoskeleton upon nystatin dose was assessed. Cells
expressing GFP-tagged actin were then imaged, Figure 8A. Actin framework distribution was
successfully observed before the leakage of the cell content caused by lysis. After that, the in-situ
addition of 5 µM RCN along with 100 µM nystatin (Figure 8B-C) allowed the observation of the
actin microfilaments evidencing that those filaments were assembled on the bleb surroundings.
While slight actin-cytosol equilibration is being initiated, the leakage of cell content is efficiently
blocked by the cytoskeleton for the first few minutes (ca. 25 min). However, since the bleb
formation continues and larger vesicle sizes are forming, the actin microfilaments and blebs start to
lose their physical contacts, gaining thus a larger mobility as indicated by the bright green dots
present in Figures 8C vs. 8B. Notably, for larger periods of time, the loss of actin organization
during nystatin treatment also causes cell detachment which can lead to the spatial recording of
different signals, thus interfering the analysis.
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Figure 8. Confocal images of 5 µM RCN in SK-Lu-1 cells under 1 hour 100 µM nystatin treatment
(A) prior transmembrane pore and bleb formation. (B) Small bleb vesicles formation after 25 min
and (C) 45 min nystatin and RCN addition. Scale bar represents 10 µm.
CONCLUSION
The RCN probe provides a reliable protocol to assess the transmembrane pore formation driven
osmotic pressure increments though polarity variations, a more robust physicochemical parameter
allowing to measure the health and decease status of the cell. As strong morphology variations
during bleb formation occur, the fluorescent probe described here was successfully utilized to
analyze such complex subcellular scenario since the RCN provided a clear response to image the
time-course distribution and associated mitochondrial-bleb dynamics, a challenging phenomenon
whose monitoring could provide useful information of the physical implications of mitochondrial-
bleb communication during cell death process. Using this new fluorophore architecture sensitive to
membrane local polarity and super-resolution microscopy will provide a powerful tool for cell
characterization using chemical, physical and physicochemical signaling.
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge at http://pubs.acs.org spectroscopic 1H and
13C NMR data, HRMS data, UV-Vis and emission spectrophotometry, confocal microscopy figures,
are provided in the supporting information file.
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AUTHOR INFORMATION
Corresponding Author
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arturo.jimenez@iquimica.unam.mx
ACKNOWLEDGMENT
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Financial support by PAPIIT-UNAM IA200320 is greatefully acknowledge. We acknowledge the
assistance of Evarardo Tapia Mendoza (MS), Ruth Rincón Heredia (PhD. Unidad de Imagenología,
IFC-UNAM), Miguel Tapia Rodríguez (PhD. IIBO, UNAM) in imaging microscopy. Salvador
Ramírez Jiménez (Biol. IIBO, UNAM) and Daniela Araiza-Olivera (PhD.) are aknowledge for
assistance in tissue culture.
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