The imaging FCS diffusion law in the presence of multiple diffusive modes

Veerapathiran, Sapthaswaran; Wohland, Thorsten

doi:10.1016/j.ymeth.2017.11.016

Cited by 36 publications

(38 citation statements)

References 72 publications

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“…A positive intercept (τ 0 > 0.1s) represents transient entrapment of the probe, e.g., in cholesterol sphingomyelin complexes, and a negative intercept (τ 0 < -0.1s) implies a meshwork organization and hop diffusion of probe molecules, e.g., molecules hindered by the cytoskeleton. In situations where a molecule is transiently trapped and hindered by a meshwork simultaneously, the intercept represents a weighted average of both diffusion modes 33 . In this case, additional measurements that influence either one of the diffusion modes are necessary to disentangle the contributions by hop diffusion and transient trapping.…”

Section: Resultsmentioning

confidence: 99%

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

Gupta

Korte

Herrmann

et al. 2019

Preprint

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Asymmetric organization of plasma membrane lipids 2 ABSTRACT 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.

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Section: Resultsmentioning

confidence: 99%

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

Gupta

Korte

Herrmann

et al. 2019

Preprint

Self Cite

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“…This has enabled to directly identify deviations from pure Brownian motion in the plasma membrane of cells [41] or anomalous diffusion occurring, either during first order lipid phase transition [42] or in non-homogeneous fluids, gels and crowded solutions [43,44]. It has been recently extended to the line-scanning STED-FCS [45] and to Im-FCS [46].…”

Section: Fluorescence Correlation Spectroscopymentioning

confidence: 99%

Anomalous Subdiffusion in Living Cells: Bridging the Gap Between Experiments and Realistic Models Through Collaborative Challenges

et al. 2020

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The life of a cell is governed by highly dynamical microscopic processes. Two notable examples are the diffusion of membrane receptors and the kinetics of transcription factors governing the rates of gene expression. Different fluorescence imaging techniques have emerged to study molecular dynamics. Among them, fluorescence correlation spectroscopy (FCS) and singleparticle tracking (SPT) have proven to be instrumental to our understanding of cell dynamics and function. The analysis of SPT and FCS is an ongoing effort, and despite decades of work, much progress remains to be done. In this paper, we give a quick overview of the existing techniques used to analyze anomalous diffusion in cells and propose a collaborative challenge to foster the development of state-of-the-art analysis algorithms. We propose to provide labelled (training) and unlabelled (evaluation) simulated data to competitors all over the world in an open and fair challenge. The goal is to offer unified data benchmarks based on biologically-relevant metrics in order to compare the diffusion analysis software available for the community. Diffusion in cells | continuous-time random walks | fractional Brownian Motion | fluorescence correlation spectroscopy | single-particle tracking.

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“…Cell surface receptors can diffuse independently in the plane of the cell membrane providing insights of the molecular interactions between receptors, receptor–ligand complexes, neighboring membrane proteins and lipids, the underlying cytoskeleton, and other structures in the extracellular environment (Alenghat & Golan, ). In order to understand the kinetics of protein interactions involved in cell signaling and transduction, it is necessary not only to focus on the binding affinities and the concentration of the ligands, but also it is crucial to study the dynamics on the cell membrane and the factors that influence it (Veerapathiran & Wohland, ). For this purpose, different methodologies and techniques have been developed over the past few decades to describe membrane protein dynamics.…”

Section: Introductionmentioning

confidence: 99%

“…It can show how the diffusion changes due to various conditions providing simultaneous access to several critical parameters of the diffusing object, such as its protein diffusion law and also its particle size. The protein diffusion law is a plot of diffusion time versus the observation area (Veerapathiran & Wohland, ). From the analysis of this plot, it is possible to identify and determine whether the movement of molecules under observation undergoes pure isotropic, confined, or transiently confined diffusion (Moens et al, ).…”

Section: Introductionmentioning

confidence: 99%

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Planes

Vanderheyden²,

Gratton

et al. 2019

Microscopy Res & Technique

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The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT1) and Endothelin 1 type A (ETA) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT1 receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ETA receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.

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The imaging FCS diffusion law in the presence of multiple diffusive modes

Cited by 36 publications

References 72 publications

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

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

Anomalous Subdiffusion in Living Cells: Bridging the Gap Between Experiments and Realistic Models Through Collaborative Challenges

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

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