scholarly journals Single-lipid dynamics in phase-separated supported lipid bilayers

2020 ◽  
Author(s):  
Xinxin Woodward ◽  
Christopher V. Kelly
Keyword(s):  
Lipid Bilayers ◽  
Fluorescence Correlation Spectroscopy ◽  
Cholesterol Content ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Supported Lipid Bilayers ◽  
Membrane Composition ◽  
Fluorescence Correlation ◽  
Diffusion Rates ◽  
Lipid Diffusion

ABSTRACTPhase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases.HIGHLIGHTSLipid diffusion varies with the lipid phases, temperature, and aggregationAggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopyMembrane with higher cholesterol content or at low temperature have more aggregatesA more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content

Quantifying Lipid Diffusion by Fluorescence Correlation Spectroscopy: A Critical Treatise

Langmuir ◽  
2012 ◽  
Vol 28 (37) ◽  
pp. 13395-13404 ◽  
Author(s):  
Fabian Heinemann ◽  
Viktoria Betaneli ◽  
Franziska A. Thomas ◽  
Petra Schwille
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Lipid Diffusion

scholarly journals Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx

2007 ◽  
Vol 293 (2) ◽  
pp. L328-L335 ◽  
Author(s):  
Andrew P. Stevens ◽  
Vladimir Hlady ◽  
Randal O. Dull
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Endothelial Glycocalyx ◽  
Concentration Profiles ◽  
Fluorescence Correlation ◽  
Structure Function Relationship ◽  
Diffusion Rates ◽  
Function Relationship

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 μm above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2–3 μm in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.

scholarly journals Complementary studies of lipid membrane dynamics using iSCAT and super-resolved Fluorescence Correlation Spectroscopy

2017 ◽  
Author(s):  
Francesco Reina ◽  
Silvia Galiani ◽  
Dilip Shrestha ◽  
Erdinc Sezgin ◽  
Gabrielle de Wit ◽  
...  
Keyword(s):  
Gold Nanoparticle ◽  
Fluorescence Correlation Spectroscopy ◽  
Membrane Dynamics ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Molecular Organization ◽  
Complementary Information ◽  
Fluorescence Correlation ◽  
Apparent Diffusion Coefficients ◽  
Fluorescent Tag

AbstractObservation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag – gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 ≤ t ≤ 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag – gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

scholarly journals Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

2015 ◽  
Vol 2 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Cornelia Junghans ◽  
Franz-Josef Schmitt ◽  
Vladana Vukojević ◽  
Thomas Friedrich
Keyword(s):  
Membrane Proteins ◽  
Fluorescence Correlation Spectroscopy ◽  
Chemical Properties ◽  
Cholesterol Content ◽  
Correlation Spectroscopy ◽  
Molecular Crowding ◽  
Diffusion Behavior ◽  
Temporal Precision ◽  
Fluorescence Correlation ◽  
Quenching Process

AbstractMeasurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1– 100 ms). Since the origin of this quenching process is currently unclear, care has to be taken when the Dreiklang label is intended to be used in FCS applications.

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Diffusion Behavior ◽  
Fluorescence Correlation ◽  
Apparent Diffusion ◽  
Diffusion Enhancement

ABSTRACTTheoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. Experimental observations of catalysis-enhanced enzyme diffusion, to date, have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an Anti-Brownian ELectrokinetic (ABEL) trap and in-solution spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an Anti-Brownian ELectrokinetic (ABEL) trap and in-solution single-particle tracking (SPT), we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ~20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte-Carlo simulations, we establish that pNPP-induced dye blinking at the ~10 ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution SPT and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.SIGNIFICANCE STATEMENTRecent experiments have suggested that the energy released by a chemical reaction can propel its enzyme catalyst (for example, alkaline phosphatase, ALP). However, this topic remains controversial, partially due to the indirect and ensemble nature of existing measurements. Here, we used recently developed single-molecule approaches to monitor directly the motions of individual proteins in aqueous solution and find that single ALP enzymes do not diffuse faster under catalysis. Instead, we demonstrate that interactions between the fluorescent dye and the enzyme’s substrate can produce the signature of apparent diffusion enhancement in fluorescence correlation spectroscopy (FCS), the standard ensemble assay currently used to study enzyme diffusion and indicate that single-molecule approaches provide a more robust means to investigate diffusion at the nanoscale.

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