Simultaneous in situ quantification of two cellular lipid pools using orthogonal fluorescent sensors

Article abstract of DOI:10.1002/anie.201408153

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid-binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems-level investigation of lipid-mediated cell signaling and regulation.

Full text of DOI:10.1002/anie.201408153

Angewandte
Chemie
DOI: 10.1002/anie.201408153
Lipid Membranes
Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using
Orthogonal Fluorescent Sensors**
Shu-Lin Liu, Ren Sheng, Matthew J. OꢀConnor, Yang Cui, Youngdae Yoon, Svetlana Kurilova,
Daesung Lee,* and Wonhwa Cho*
Abstract: Lipids regulate a wide range of biological activities.
Since their local concentrations are tightly controlled in
a spatiotemporally specific manner, the simultaneous quantifi-
cation of multiple lipids is essential for elucidation of the
complex mechanisms of biological regulation. Here, we report
a new method for the simultaneous in situ quantification of two
lipid pools in mammalian cells using orthogonal fluorescent
sensors. The sensors were prepared by incorporating two
environmentally sensitive fluorophores with minimal spectral
overlap separately into engineered lipid-binding proteins. Dual
ratiometric analysis of imaging data allowed accurate, spatio-
temporally resolved quantification of two different lipids on
the same leaflet of the plasma membrane or a single lipid on
two opposite leaflets of the plasma membrane of live mamma-
lian cells. This new imaging technology should serve as
a powerful tool for systems-level investigation of lipid-medi-
ated cell signaling and regulation.
Under physiological conditions, multiple regulatory lipids
are metabolically and functionally linked to one another.[3]
Also, a single lipid species can exist disproportionally in
opposite faces of lipid bilayers, performing distinct func-
tions.[4] Most notably, the tightly controlled trans-bilayer
asymmetry of lipids in the plasma membrane (PM) of
mammalian cells is crucial for cell survival, function, and
regulation.[4] Thus, a new technology is needed for the
simultaneous in situ quantification of multiple lipids in the
same membrane leaflet or a lipid species in opposite leaflets
of cell membranes. As a first step toward the simultaneous
quantification of multiple lipids, we developed a new strategy
for dual in situ lipid quantification in live cells.
Simultaneous dual-lipid quantification would require
orthogonal lipid sensors that allow robust dual ratiometric
analysis. Unfortunately, the limited availability of amphiphilic
ESFs greatly hampers the development of orthogonal lipid
sensors. We therefore searched for an ESF that can be used
orthogonally with the most commonly employed thiol-reac-
tive amphiphilic ESF, acrylodan (6-acryloyl-2-dimethylami-
nonaphthalene).[5] When coupled to a cysteine residue of
a protein, the 2-dimethylaminonaphthaloyl (DAN) group
undergoes a green-to-blue spectral shift with a large increase
in fluorescence emission intensity as the protein binds its
cognate lipid in the membrane, allowing robust ratiometric
quantification of cellular lipids through in vitro calibration.[2]
An ideal orthogonal ESF partner for DAN would be an
amphiphilic fluorescence dye that shows a spectral shift from
red to orange fluorescence upon lipid binding. Among
reported red fluorophores, Nile Red possesses such properties
and its maleimide derivatives have been prepared for cysteine
labeling.[6] These Nile Red derivatives are, however, highly
lipophilic and have extremely low water solubility. This not
only lowers the yield of protein-labeling reactions in aqueous
solution but also adversely affects the structure, stability, and
membrane-binding properties of the labeled proteins. To
overcome these major limitations, we designed and synthe-
sized several cysteine-specific acrylate derivatives of Nile Red
with a varying degree of lipophilicity.
M
embrane lipids are among the most important and
ubiquitous regulatory molecules that control the localization,
activity, and mutual interactions of a wide variety of cellular
proteins.[1] Because local lipid concentrations are highly
variable and may serve as activation thresholds for myriad
biological processes mediated by these proteins, spatiotem-
porally resolved lipid quantification is essential for elucidat-
ing the diverse and complex mechanisms of biological
regulation.[2] We recently developed a chemical strategy for
in situ quantification of a single lipid species in live
mammalian cells using a hybrid sensor constructed with an
engineered lipid-binding protein and an environmentally
sensitive (or solvatochromatic) fluorophore (ESF).[2] Quan-
tification of cellular phosphatidylinositol-4,5-bisphosphate
(PIP2) by ratiometric imaging analysis demonstrated the
spatiotemporal dynamics of this important signaling lipid in
unprecedented detail and provided new insight into how it
regulates such diverse biological processes.[2]
[*] Dr. S.-L. Liu, Dr. R. Sheng, M. J. O’Connor, Y. Cui, Dr. Y. Yoon,[+]
S. Kurilova, Dr. D. Lee, Dr. W. Cho
Department of Chemistry, University of Illinois at Chicago
845 W. Taylor St., Chicago, IL (USA)
E-mail: dsunglee@uic.edu
The 2-hydroxy Nile Red benzophenoxazinone core
2-HONR was synthesized starting from 5-diethylamino-2-
nitrosophenol hydrochloride and 1,6-dihydroxynaphthalene
(Scheme 1).[7] The first-generation fluorophore NR1 was
prepared by treatment of 2-HONR with acryloyl chloride
and triethylamine. Although NR1 displayed favorable spec-
tral properties, its solubility in water was still extremely low
and thus its protein-labeling efficiency was low. We thus
introduced more oxygen atoms and an additional hydroxy
group into the linker to increase the water solubility. NR2
wcho@uic.edu
[+] Present address:
Department of Environmental Health Science, Konkuk University
1 Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea)
[**] We thank Dr. Mingjie Zhang for a kind gift of myosin X PH domain.
The work was supported by NIH grants, GM68849 and GM110128.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408153.
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tively. Both DAN-eLactC2 and NR3-eLactC2 showed char-
acteristic spectral changes upon binding to vesicles in a PS
concentration-dependent manner (Figure 1a,b). After per-
forming in vitro ratiometric calibration of these PS sensors
using giant unilamellar vesicles containing varying concen-
trations of PS (Figure 1c,d; see the Supporting Information
for details), we microinjected DAN-eLactC2 into NIH 3T3
cells and added NR3-eLactC2 to the medium (or vice versa).
We then triggered the cell apoptosis with 1 mm doxorubicin,
and simultaneously monitored DAN-eLactC2 and NR3-
eLactC2 bound to the PM by a four-channel two-photon
microscope equipped with two femtosecond-pulsed laser
sources.
No current light microscopy, including super-resolution
microscopy, allows spatial resolution of the two lipid layers of
cell membranes (ca. 5 nm thick).[10] Because our PS sensors
Scheme 1. Synthesis of Nile Red derivatives with an acrylate linker:
a) DMF, reflux, 69%; b) acryloyl chloride (1.2 equiv), Et3N (1.5 equiv),
08C, CH2Cl2, 30 min, 85%; c) NaH (1.25 equiv), DMF, 08C, 30 min;
allyl bromide (2.5 equiv), 08C to RT, 3 h, 72%; d) OsO4 (cat.), NMO
(2 equiv), acetone/H2O/MeOH (4:1:0.5), 65%; e) acryloyl chloride
(0.33 equiv), Et3N (1.5 equiv), À308C, CH2Cl2, 10 min, 25%.
DMF=N,N-dimethylformamide, NMO=N-methylmorpholine N-oxide.
(bisacrylate) and NR3 (monoacrylate) were synthesized from
the common precursor 2-HONR by a three-step sequence
involving allylation, dihydroxylation, and acrylation.
Among these derivatives, NR3 afforded the highest
coupling yield to the engineered ENTH domain (eENTH)
that was previously used for the PIP2 sensor.[2] Also, the
eENTH labeled with NR3 (NR3-eENTH) had the most
favorable properties, including the lowest tendency to self-
associate in solution and to nonspecifically adsorb to lipid
membranes and glass surfaces. Furthermore, NR3-eENTH
showed a large PIP2 concentration-dependent spectral shift
from red to orange-red when it bound to PIP2-containing
vesicles (Figure S1a in the Supporting Information). A
minimal spectral overlap between NR3-eENTH and DAN-
eENTH (Figure S1b in the Supporting Information) suggests
that DAN and NR3 may be used for preparing orthogonal
lipid sensors.
To test this notion, we prepared DAN- and NR3-labeled
phosphatidylserine (PS) sensors and simultaneously mea-
sured the concentrations of PS in the inner (cytofacial) and
the outer (exofacial) leaflets of the PM of mammalian cells.
PS is a major lipid component of PM and is predominantly
found in the inner leaflet under normal conditions.[8] It is,
however, translocated to the outer leaflet by the action of
a Ca2+-dependent scramblase during apoptosis and blood
vessel damage and triggers phagocytosis and the blood
coagulation cascade, respectively.[8] Currently, little is known
about the spatiotemporal dynamics of PS across the PM under
physiological conditions mainly because current optical
imaging technologies do not allow simultaneous in situ
quantification of PS in the two leaflets of the PM.
To prepare orthogonal PS sensors, we first engineered the
lactadherin C2 domain (eLactC2), the fluorescence-protein-
tagged forms of which have been widely used as a specific
cellular PS probe,[9] to introduce a single cysteine in its
membrane-binding surface. We then labeled it with acrylodan
and NR3 to obtain DAN-eLactC2 and NR3-eLactC2, respec-
Figure 1. a–d) Spectral properties of and ratiometric calibration for
orthogonal PS sensors. a,b) Fluorescence emission spectra of DAN-
eLactC2 (a) and NR3-eLactC2 (b) (both 500 nm) in the presence of
phosphatidylcholine (PC)/PS (100Àx:x) large vesicles measured spec-
trofluorometrically. Numbers (x) indicate mole% of PS. The excitation
wavelength was 392 nm for DAN-eLactC2 and 520 nm for NR3-
eLactC2. Both exhibited a dramatic blue shift upon PS binding.
c,d) Ratiometric calibration curves of DAN-eLactC2 (c) and NR3-
eLactC2 (d) for PS quantification. The PS sensors were monitored by
a two-photon microscope in the presence of PC/PS (100Àx:x) giant
vesicles. Nonlinear least-squares analysis of the plot using the
equation (for DAN-eLactC2); FB/FG =(FB/FG)max/(1+Kd/[PS])+C yields
Kd, (FB/FG)max, and C values and the calibration curves are constructed
using these parameters. FB, FG, Fo, and FR indicate fluorescence
intensities of the blue, green, orange, and red channels, respectively.
Kd, (FB/FG)max, and C are the equilibrium dissociation constant (in
mole%), the maximal FB/FG value, and the arbitrary instrumental
parameter. Blue and orange channels depict membrane-bound sensors
whereas green and red channels show membrane-bound plus free
sensors. Error bars indicate standard deviations calculated from at
least three independent sets of measurements. 20 mm Tris buffer,
pH 7.4, containing 0.16m KCl was used for all measurements.
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cannot cross the PM,[2] however, we were able to unambig-
uously distinguish the signals from the outer and the inner PM
by analyzing fluorescence signals from four discrete optical
channels in a time-dependent manner (Figure 2a). The slow
rate of apoptosis and major changes in cell size and shape
correlation was seen between the increase in PS in the outer
PM and its decrease in the inner PM (both spatially averaged)
at a given time (Figure 2c), showing that the PS concentration
increase in the outer PM is due to direct trans-bilayer
translocation of PS from the inner PM. Collectively, these
results not only demonstrate the feasibility of our orthogonal
lipid sensor technology but also shed new light on the trans-
bilayer movement of PS and its local distribution. The same
experimental approach can be applied to the real-time
quantification of other lipid species (e.g., cholesterol, sphin-
gomyelin, etc.) across the PM, which should provide impor-
tant new insight into the trans-bilayer dynamics and the
location-specific functions of many vital cellular lipids.
Having established the feasibility of our orthogonal lipid
sensor methodology, we then performed simultaneous quan-
tification of two metabolically linked signaling lipids, PIP2 and
phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 is pro-
duced from PIP2 in the cytofacial leaflet of the PM by
phosphoinositide 3-kinase (PI3K) in response to stimuli, such
as growth factors,[11] and is converted back to PIP2 by a tumor
suppressor lipid phosphatase, PTEN.[12] PIP2 mediates a wide
range of cellular processes, including cell signaling,[3a,13] while
PIP3 activates myriad of other cellular activities, including cell
growth.[11] Under normal conditions, PIP3 is thought to exist in
much lower concentration than PIP2 and only transiently
because PI3K activation is tightly regulated.[14] Interestingly,
it has been recently reported that some mammalian cells may
have significant basal levels of PIP3 in the PM.[15] This in turn
suggests that the PIP3-to-PIP2 ratio may significantly vary
among different mammalian cells, greatly affecting their basal
cell activities and responses to various stimuli. The PIP3-to-
PIP2 ratio, however, has not been accurately measured in live
cells.
We thus simultaneously quantified PIP2 and PIP3 in
different mammalian cells using orthogonal sensors for PIP2
and PIP3. As a PIP3 sensor, we engineered the PIP3-selective
tandem pleckstrin homology (PH) domains of myosin X
(eMyoXPH)[16] for single-site cysteine labeling (see the
Supporting Information for details), labeled the engineered
protein with NR3 (NR3-eMyoXPH), and measured its
spectral properties upon binding to PIP3-containing vesicles
(Figure S2 in the Supporting Information). This protein was
selected over other PIP3-selective PH domains, including
BTK and Grp1 PH domains,[15b] because none of the latter
exhibited desired spectral properties when labeled with
acrylodan or NR3. NR3-labeled eMyoXPH and DAN-
eENTH were then co-microinjected into NIH 3T3 cells and
PIP2 and PIP3 were simultaneously quantified by dual
ratiometric analysis of images (Figure 3a) before and after
stimulation by insulin that activates PI3K.[17]
Figure 2. a–c) Simultaneous in situ quantification of PS in the inner
and outer PM of NIH 3T3 cells. a) Four-channel images of representa-
tive apoptotic cells at two time points. PS in the inner and outer PM
was monitored using microinjected DAN-eLactC2 (green and blue
channels) and media-added NR3-eLactC2 (red and orange channels)
sensors, respectively. Blue and orange channels depict membrane-
bound sensors whereas green and red channels show membrane-
bound plus free sensors. b) Time-lapse PS quantification in the outer
PM at 14 h and 24 h. After apoptosis was induced by 1 mm doxorubi-
cin, PS in the outer and the inner PM was quantified from the blue/
green and orange/red ratios, respectively, using the ratiometric calibra-
tion curves (Figure 1c,d). c) Time-dependent changes of spatially
averaged PS concentrations in the outer and inner PM. Scale bars
indicate 5 mm.
during the process (Figure 2a) made continuous imaging
analysis impractical. We therefore performed time-lapse
imaging and data analysis. Figure 2b depicts PS quantification
in the outer PM of apoptotic NIH 3T3 cells after 14 and 24 h
of doxorubicin treatment. The analysis reveals for the first
time pronounced local heterogeneity of PS in the outer leaflet
of the PM. This implies that PS locally enriched beyond
a threshold level in the outer PM may constitute hot spots for
the recognition of apoptotic cells by phagocytes as seen with
PIP2.[2] This notion is also consistent with the finding that
although PS started to appear in the outer PM after 8 h
(Figure 2c), the cells showed apoptotic phenotypes (e.g.,
shape changes) only after 20 h. Furthermore, an excellent
At a given time, both PIP2 and PIP3 exhibited a high
degree of local heterogeneity (Figure 3b). Interestingly,
a good spatial correlation was detected between the decrease
in PIP2 concentration and the increase in PIP3 concentration
(Figure 3c), indicating localized conversion of PIP2 to PIP3
and segregation of the two lipids. When spatially averaged at
a given time, the PIP3-to-PIP2 ratio was 0.25 Æ 0.05 (average Æ
standard deviation (S.D) in 15 cells) in the resting NIH 3T3
cells, demonstrating the significant basal PI3K activity in
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Figure 4. a,b) The PIP3-to-PIP2 ratio in PC cell lines. a) Time-dependent
changes of spatially averaged PIP2 and PIP3 concentrations in the PM
of PC3 cells lacking PTEN. b) Time-dependent changes of spatially
averaged PIP2 and PIP3 concentrations in the PM of PC3 cells with
transiently expressing PTEN.
treatment (Figure 4b), verifying that PTEN is directly
responsible for the PIP3-to-PIP2 transition under these con-
ditions. Taken together, these results show that PIP2 and PIP3
are locally metabolized by PI3K and PTEN at the PM and
that the same stimulus can induce different changes in the
PIP3-to-PIP2 ratio, depending on the cell type. This informa-
tion should be essential for understanding the basis of
differential regulatory mechanisms for PIP3 signaling in
mammalian cells.
In this study, we demonstrate the feasibility of our new
orthogonal lipid sensor technology. Our method allows robust
and simultaneous in situ quantification of two lipid pools in
Figure 3. a–d) Simultaneous in situ quantification of PIP2 and PIP3 in
the inner PM of NIH 3T3 cells. a) Four-channel images of representa-
tive NIH 3T3 cells before and 5 min after insulin treatment. PIP2 and
PIP3 were monitored using microinjected DAN-eENTH and NR3-
eMyoXPH, respectively. Blue and orange channels depict membrane-
bound sensors whereas green and red channels show membrane-
bound plus free sensors. b) PIP2 and PIP3 were quantified through
ratiometric calibration (see Figure S2). c) Angular profiles of PIP2 and
PIP3 in the PM 3 min after insulin treatment. PIP2 and PIP3 show
pronounced spatial heterogeneity but a good reciprocal spatial correla-
tion was observed between them. d) Time-dependent changes of
spatially averaged PIP2 and PIP3 concentrations in the PM. Scale bars
indicate 5 mm.
living mammalian cells in
a spatiotemporally resolved
manner. The study also shows that simultaneous quantifica-
tion of two lipid pools provides physiologically important data
and new insight unattainable by conventional methodologies.
Collectively, it represents an important technical advance
toward the systems-level analysis and understanding of
complex lipid-mediated cell regulation.
Received: August 11, 2014
Published online: October 24, 2014
Keywords: imaging · lipids · membranes · sensors
.
these cells. The ratio reached 0.5 within 5 min of insulin
stimulation and rapidly returned to the basal level (Fig-
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