FCS Analysis of Protein Mobility on Lipid Monolayers.
ABSTRACT: In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.
Project description:Diffusion obstacles in membranes have not been directly visualized because of fast membrane dynamics and the occurrence of subresolution molecular complexes. To understand the obstacle characteristics, mobility-based methods are often used as an indirect way of assessing the membrane structure. Molecular movement in biological plasma membranes is often characterized by anomalous diffusion, but the exact underlying mechanisms are still elusive. Imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is a well-established mobility-based method that provides spatially resolved diffusion coefficient maps and is combined with FCS diffusion law analysis to examine subresolution membrane organization. In recent years, although FCS diffusion law analysis has been instrumental in providing new insights into the membrane structure below the optical diffraction limit, there are certain exceptions and anomalies that require further clarification. To this end, we correlate the membrane structural features imaged by atomic force microscopy (AFM) with the dynamics measured using ITIR-FCS. We perform ITIR-FCS measurements on supported lipid bilayers (SLBs) of various lipid compositions to characterize the anomalous diffusion of lipid molecules in distinct obstacle configurations, along with the high-resolution imaging of the membrane structures with AFM. Furthermore, we validate our experimental results by performing simulations on image grids with experimentally determined obstacle configurations. This study demonstrates that FCS diffusion law analysis is a powerful tool to determine membrane heterogeneities implied from dynamics measurements. Our results corroborate the commonly accepted interpretations of imaging FCS diffusion law analysis, and we show that exceptions happen when domains reach the percolation threshold in a biphasic membrane and a network of domains behaves rather like a meshwork, resulting in hop diffusion.
Project description:Many efforts have been undertaken over the last few decades to characterize the diffusion process in model and cellular lipid membranes. One of the techniques developed for this purpose, fluorescence correlation spectroscopy (FCS), has proved to be a very efficient approach, especially if the analysis is extended to measurements on different spatial scales (referred to as FCS diffusion laws). In this work, we examine the relevance of FCS diffusion laws for probing the behavior of a pure lipid and a lipid mixture at temperatures below, within and above the phase transitions, both experimentally and numerically. The accuracy of the microscopic description of the lipid mixtures found here extends previous work to a more complex model in which the geometry is unknown and the molecular motion is driven only by the thermodynamic parameters of the system itself. For multilamellar vesicles of both pure lipid and lipid mixtures, the FCS diffusion laws recorded at different temperatures exhibit large deviations from pure Brownian motion and reveal the existence of nanodomains. The variation of the mean size of these domains with temperature is in perfect correlation with the enthalpy fluctuation. This study highlights the advantages of using FCS diffusion laws in complex lipid systems to describe their temporal and spatial structure.
Project description:In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane.
Project description:Only a limited number of noninvasive techniques are available to directly measure the dynamic behavior of lipids in model and cell membranes. Here, we explored whether a commercial instrument could be used for fluorescence correlation spectroscopy (FCS) under pulsed stimulated emission depletion (STED). To overcome issues with photobleaching and poor distinction between confocal and STED signals, we implemented resonant line-scan STED with filtered FCS, which has the additional benefit of autocalibrating the dimensions of the point-spread function and obtaining spatially resolved molecular mobility at subdiffraction resolution. With supported lipid bilayers, we achieved a detection spot radius of 40 nm, although at the expense of decreased molecular brightness. We also used this approach to map the dynamics of Atto646N-labeled sphingomyelin and phosphatidylethanolamine in the plasma membrane. Despite the reliability of the method and the demonstration that photobleaching and the photophysical properties of the dye did not influence diffusion measurements, we found great heterogeneities even within one cell. For both lipids, regions of high local density correlated with slow molecular diffusion, indicating trapping of Atto646N-labeled lipids. Future studies with new dyes are needed to reveal the origin of the trapping.
Project description:The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). Here, we compare the results of recovered diffusion coefficients and mean-square displacements of the two methods by simulations of free, domain-confined, or meshwork diffusion. We introduce, to our knowledge, a new method for the determination of confinement radii from ITIR-FCS data. We further establish and demonstrate simultaneous SPT/ITIR-FCS for direct comparison within living cells. Finally, we compare the results obtained by SPT and ITIR-FCS for the receptor tyrosine kinase MET. Our results show that SPT and ITIR-FCS yield complementary information on diffusion properties of biomolecules in cell membranes.
Project description:Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.
Project description:Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations.
Project description:Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signaling and are suggested to be strongly associated with the actin cytoskeleton. Here we use superresolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live-cell plasma membrane and in actin cytoskeleton-free, cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids is abolished in the GPMVs, whereas transient nanodomain incorporation of ganglioside lipid GM1 is apparent in both the live-cell membrane and GPMVs. For GPI-APs, we detect two molecular pools in living cells; one pool shows high mobility with transient incorporation into nanodomains, and the other pool forms immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules and highlight a powerful experimental approach to decipher specific influences on molecular plasma membrane dynamics.
Project description:Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.
Project description:Ion pairing between the major phospholipids of the Staphylococcus aureus plasma membrane (phosphatidylglycerol - PG and lysyl-phosphatidylglycerol - LPG) confers resistance to antimicrobial peptides and other antibiotics. We developed 3adLPG, a stable synthetic analogue which can substitute for the highy-labile native LPG, in biophysical experiments examining the membrane-protecting role of lipid ion pairing, in S. aureus and other important bacteria. Here we examine the surface charge and lipid packing characteristics of synthetic biomimetic mixtures of DPPG and DP3adLPG in Langmuir monolayers, using a combination of complementary surface-probing techniques such as infrared reflection-absorption spectroscopy and grazing-incidence x-ray diffraction. The resultant phase diagram for the ion paired lipids sheds light on the mixing behavior of lipids in monolayer models of resistant phenotype bacterial membranes, and provides a platform for future biophysical studies.