Analysing plasma membrane asymmetry of lipid organisation by fluorescence lifetime and correlation spectroscopy

A fundamental feature of a eukaryotic cell membrane is the asymmetric arrangement of lipids in the two leaflets. A cell invests significant energy to maintain this asymmetry and utilizes it to regulate important biological processes such as apoptosis and vesiculation. Here, we employ fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the exofacial and cytoplasmic leaflet of live mammalian cells. We characterize the biophysical properties of fluorescent analogues of phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS) in two mammalian cell membranes. Due to their specific transverse membrane distribution, these probes allow leaflet specific investigation of the plasma membrane. We compare the results with regard to the different temporal and spatial resolution of the methods. Fluorescence lifetimes of fluorescent lipid analogues were found to be in a characteristic range for the liquid ordered phase in the outer leaflet and liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet is supported by free diffusion in the inner leaflet with high average diffusion coefficients. The liquid ordered phase in the outer leaflet is accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogues provides a powerful tool to investigate lateral and trans-bilayer characteristics of plasma membrane in live cells.


INTRODUCTION
The plasma membrane is a bilayer composed of a plethora of chemically diverse lipids and a range of proteins. The distinct physicochemical properties of the membrane components lead to the formation of transient assemblies such as cholesterol-dependent domains, cholesterol independent domains and protein oligomers which are typically below the diffraction limit (20-100 nm) and are in dynamic equilibrium with each other 1,2,3 . The transient assemblies exhibit unique lifespans governed by the mutual interactions of their components. In addition to the lateral heterogeneity, a fundamental feature of the plasma membrane is the asymmetry of lipid composition between the two leaflets 4-6 . The dynamic lateral heterogeneity of the membrane is essential for cell signalling 7 while the asymmetric arrangement of lipids across the plasma membrane is found to be critical in the regulation of several biological processes, including apoptosis 8 , cell-cell fusion 9 and signalling in immune cells 10 . The primary classes of lipids that constitute a typical mammalian plasma membrane are glycerophospholipids (e.g. PC, PS, PE, PA, PI), sphingolipids (e.g. SM, ceramide, GSLs) and cholesterol 11 . In the plasma membrane, PC and sphingolipids are prevalent in the outer leaflet while aminophospholipids such as PS, and PE are prevalent in the inner leaflet 4 . Since the outer-leaflet comprises more domain forming lipids and the inner-leaflet is in direct contact with the cytoskeleton, it is expected that the organization and dynamics of the two leaflets are different. However, the precise lateral and transbilayer organization of the membrane and how the two leaflets are dynamically coupled are unknown. Several studies have focused on understanding membrane asymmetry of organisation, however, due to experimental limitations they have mostly been conducted on model membranes with limited physiological relevance as it is difficult to recapitulate the structural complexity of an intact cell membrane [12][13][14] . A detailed analysis of plasma membrane asymmetry in physiological context requires a methodology that can probe dynamics and organization of both leaflets of an intact cell membrane separately with sufficient spatiotemporal resolution. In addition to the quantitative nature of the method, the other key points necessary to analyze the transbilayer membrane organization are 15 : (1) There should be a way of separating the information originating from the two leaflets. This can be achieved by using lipid probes that confine themselves predominantly to one particular leaflet or by selectively quenching the fluorescence signal from one leaflet using a membrane impermeable agent, e.g. sodium dithionite, TNBS, and Doxyl-PC. (2) The time taken to complete the assay should be less than what it takes for lipids to undergo transbilayer movement. (3) The quenching agent should not have any additional effects on the membrane integrity. (4) Certain chemical treatments can alter the membrane lipid composition by modulating the rates of endo-or exocytosis. During the analysis of plasma membrane asymmetry, any chemical treatment should not alter the membrane composition.
Based on the above-mentioned considerations, we perform a detailed analysis of the plasma membrane asymmetry by examining the outer and the inner leaflet of the membrane separately in live mammalian cells using a combination of fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). FLIM has been utilized to investigate the asymmetrical arrangement of the plasma membrane 16,17 . It can reliably detect and resolve microscopic domains present in model membranes as identified by their different lifetimes. However, in GPMVs and intact plasma membranes, where lateral heterogeneities are on the nanometer scale, FLIM is unable to resolve discrete membrane domains. Due to the domains, FLIM detects a mixture of lifetimes leading to a broad distribution of fluorescence lifetimes indicating the existence of a wide variety of transient assemblies 16 . In order to obtain complementary information to membrane organization as measured by FLIM we use ITIR-FCS, to measure membrane dynamics [18][19][20][21] . ITIR-FCS is an imaging modality that allows multiplexed FCS measurements on the whole region of interest simultaneously and provides spatially resolved diffusion coefficient and number of particle maps. And although ITIR-FCS is diffraction-limited, the use of the FCS diffusion law, which determines the change of diffusion with spatial scale, allows the investigation of sub-resolution membrane organization 22,23 . FLIM measurements require environment-sensitive fluorophores while for FCS measurements fluorophores with better photostability are ideal. Therefore, we use 1-palmitoyl-2-[6-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (NBD) labeled lipid analogues for FLIM as NBD lifetime is sensitive to the environmental polarity and molecular packing around the probe 17,24-26 ( Figure 1A). However, low photostability and brightness of NBD make it non-ideal for FCS measurements 27 . Therefore, we use dipyrrometheneboron difluoride (TopFluor) labeled lipids, which offer sufficient photostability, for FCS measurements ( Figure 1A). To evaluate cell type, domain, and leaflet specific information, we examine the biophysical properties of fluorescently labeled PC, SM, and PS analogues in the plasma membrane of CHO-K1 and RBL-2H3 cells. Our FLIM results show that the lifetime of all the tested probes is typically longer in the outer leaflet than in the inner leaflet most convincingly shown for NBD-PS which is predominantly localized in the inner leaflet of the membrane. In support of lifetime results, ITIR-FCS measurements on the same cell lines show a higher diffusion coefficient and less domain confined diffusion of lipid probes localized in the inner leaflet of the membrane and a slower diffusion accompanied with higher confinement in the outer leaflet of the membrane, with characteristic differences between cell lines. Overall, our results reveal that the outer leaflet exhibits a liquid-ordered environment while the inner leaflet of the membrane is more similar to the liquid disordered phase. Furthermore, we show cell line differences in the transbilayer organization of the plasma membrane.

Giant plasma membrane vesicle preparation
Giant plasma membrane vesicles (GPMVs) were prepared from CHO-K1 and RBL-2H3 cells by the formaldehyde-based induction of plasma membrane blebbing. The detailed protocol is described in Sezgin et al 40 .

Cell membrane labeling
The

Cholesterol depletion experiment
Cholesterol depletion was performed by treating the cells with 3 mM methyl-β-cyclodextrin (Sigma Aldrich, Germany). The cells were incubated for 30 minutes were then measured.

Fluorescence lifetime spectroscopy
where I(t) is the fluorescence intensity at time t, α i the pre-exponential factor representing the intensity of the time-resolved decay of the component with lifetime τ i . All intensity decays were fitted to bi-or triexponential model functions depending on the studied sample. We ensured the quality of fit by the χ 2 value, the distribution of residuals and the autocorrelation function of residuals. The fitting error was calculated using a support plane error analysis and included into the error estimation.

Instrumentation and data acquisition
Images were acquired by a confocal laser scanning microscope with an inverted Fluoview 1000 microscope (Olympus, Tokyo, Japan) and a 60X (NA 1.

Data analysis
For extracting the fluorescence lifetimes of NBD analogues, membrane regions are selected by applying an intensity threshold to exclude fluorescence from background or cytoplasm. The selection was further refined manually to exclude regions not associated with the membrane. Then overall fluorescence decay curve was calculated by adding up the photons registered from the selected region. The part of the decay curve that comprises instrument response function was removed, and the rest was used for the analysis.
A non-linear least squares iterative fitting procedure was used to fit the fluorescence decay curves as a sum of exponential terms, where F(t) denotes the fluorescence intensity at time t, and denotes a pre-exponential factor representing the intensity of the time-resolved decay of the component with lifetime . The quality of fits was evaluated by the distribution of the residuals and the χ 2 value.
A typical FLIM experiment yields a spatial distribution of lifetime (usually lifetime is mapped in a color-coded fashion) however since the size of membrane domains is below the spatial resolution individual images do not provide any additional insights. Thus, we represent the data in the form of lifetimes and amplitude averaged over the whole membrane.

Imaging total internal reflection fluorescence correlation spectroscopy
The experiments were done on TopFluor lipid analogue labeled cell membranes. The experiments are performed at 25 °C with 5% CO 2.   58 . Bleach correction is applied on the data using a 4 th order polynomial function. The ACF for each pixel was individually fitted with the following one-particle model for diffusion using the same software.
Here G(τ) represents the ACF as a function of correlation time (τ) and N, a, D and σ are the number of particles per pixel, pixel side length, diffusion coefficient and standard deviation of the Gaussian approximation of the microscope point spread function (PSF) respectively. G ∞ represents the convergence value of the ACF at long correlation times.
Fitting of experimentally obtained ACFs with theoretical models yields D and N. Since it is an imaging based FCS modality, we obtain spatially resolved diffusion coefficient (D), and the number of particles (N) maps 58 . In this study, the data are represented as mean ± standard deviation (SD). The SD is obtained from the measurements over 441 pixels per experiment. The SD of an ITIR-FCS measurement not only contains contributions from the measurement variability but also from the cell 2√Dτ + σ 2 membrane heterogeneity. Each data point for diffusion coefficient obtained using ITIR-FCS is an average of 441 measurements therefore, N = 5292 at least.

Imaging FCS diffusion laws
For the analysis of cell membrane organization below the diffraction limit, we combined ITIR-FCS with FCS diffusion laws. These laws allow probing mode of molecular diffusion i.e. free diffusion or is hindered by the trapping sites such as domains 22,23 . To achieve this, we plot the spatial dependence of the diffusion time of the probe molecules on the observation area size. Then these plots are fitted to a standard error of mean (SEM) weighted straight line which is mathematically expressed as:

RESULTS AND DISCUSSIONS
Here, we investigated the dynamics and organization of the outer and the inner leaflet in the plasma membrane of mammalian cells. For outer leaflet membrane dynamics, we performed measurements at room temperature immediately after labeling of cells with fluorescent lipid analogues at 4 °C. Both FLIM and ITIR-FCS measurements are done at room temperature within the first 1-5 minutes after staining. As this is less than the time needed for the transbilayer movement of glycerophospholipids, which typically ranges from several minutes to hours at 37°C 29 , measured values report essentially on the outer leaflet. Subsequently, we measure after an incubation of 2 hours at room temperature.  (Table   S1). The fluorescence decay of NBD in LUVs exhibiting a single phase is best fitted to a bi-exponential model ( Figure S1). Past studies have suggested that only the long lifetime is sensitive to the membrane environment while the short lifetime is not dependent on the membrane phase 16,24 . Lifetime data from LUVs with coexisting phases is best fitted to a tri-exponential model where the two long lifetimes

The higher domain fraction in RBL-2H3 cells compared to CHO-K1 cells has been addressed in our
previous studies 36,37 . Lower lipid mobility and more significant entrapment of the probe can be attributed to higher levels of sphingolipids in the RBL-2H3 plasma membrane which is known to form trapping sites in the cell membrane 39 . This is also supported by an overall longer lifetime of NBD lipid analogues in giant plasma membrane vesicles (GPMVs) derived from RBL-2H3 cells than CHO-K1 cells ( Figure S3) measured at 25 °C and 37 °C. Since GPMVs preserve membrane composition but not organization 40 , the long lifetime in RBL-2H3 GPMVs show that the RBL-2H3 cell membrane composition confers a more ordered environment and a higher domain fraction.

Leaflet specific analysis of sphingomyelin fluorescent analogues in CHO-K1 and RBL-2H3 cell membranes
The existence of cholesterol-sphingomyelin complexes in cell membranes has been demonstrated previously [41][42][43] . It is expected that the organization and dynamics of the molecules forming these nanoscale assemblies would differ from that of PC which is ubiquitously present in the cell membrane. To understand the asymmetric transbilayer organization of domain-specific molecules, we characterized the environment and dynamics of SM fluorescent lipid analogues.
Sphingomyelins are the most abundant sphingolipids present in the plasma membrane of mammalian cells 11 .
They consist of a ceramide backbone and phosphocholine headgroup exhibiting a narrower cylindrical geometry than phosphatidylcholine and a phase transition above room temperature and thus, exist in the gel phase at room temperature. Sphingomyelins interact with cholesterol to form lipid domains in the membranes, but can also exist freely 41,44,45 . They are predominantly present in the outer leaflet; however, there is some evidence suggesting a pool of SM localized in the inner leaflet 46,47 . Overall, this is consistent with our quenching experiments that show ~ 80% SM lipid analogues are present in the outer leaflet (Table S1).
The use of tail-labeled NBD lipid analogues for membrane studies is debatable as they may not behave like the endogenous lipids 48,49 Therefore, to validate that the NBD probes are sensitive to cholesterol content we performed methyl-β-cyclodextrin induced cholesterol depletion experiments. We observed that as expected, the effect of cholesterol depletion significantly affects the NBD-SM lifetime as indicated by a drop of 6 ns in both cell lines ( Figure S4).
However, the NBD-PC lifetime shows a decline of 1 ns only. This experiment verifies that the disruption of cholesterol domains directly influences the SM microenvironment but to a much lesser extent that of PC. Thus, NBD lipid analogues reside in environments similar to their endogenous forms and therefore, can be used to examine the domain-specific plasma membrane organization.
Next, we proceeded with the leaflet specific analysis of SM probes. The lifetime analysis of NBD-SM immediately after labeling i.e. in the outer leaflet shows a lifetime of around 10.5 ns in both cell lines (Figure 3 A, B)  In summary, besides demonstrating a lower degree of heterogeneity in the inner leaflet, our results also demonstrate that the asymmetric arrangement of the plasma membrane can vary for different cell types used. Furthermore, probe related differences suggest that different aspects of organisation and dynamics are recorded. This can be visualized by analysing the differences in the ratios of D measured in the inner leaflet and the outer leaflet which also demonstrate the probe and cell type specific plasma membrane asymmetry (Table S2).

Leaflet specific analysis of phosphatidylserine fluorescent analogues in CHO-K1 and RBL-2H3 cell membranes
We probed the organization and dynamics of fluorescent phosphatidylserine analogues, to characterize the inner leaflet organization 51 . Usually, it comprises the fluid fraction of the plasma membrane 52 however, there is evidence suggesting its direct interaction with the cytoskeleton due to which PS can exhibit lower mobility 53

CONCLUSIONS
The plasma membrane is structurally complex and dynamic with a diversity of lateral molecular movements ranging from directed diffusion of immobile protein-lipid clusters to free diffusion of molecules and transversal movements of lipids occurring at varying rates.
Besides, there are fluctuating nanoscale assemblies on the membrane scaling from 5 nm to 500 nm. To understand the organization and dynamics of such a system, it is essential to use methods with varying spatial and temporal resolutions. In this study, we performed a spatiotemporal analysis of the plasma