How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy

Benda, Aleš; Beneš, Martin; Mareček, V.; Lhotský, Alexandr; Hermens, and W. Th.; Hof, Martin

doi:10.1021/la0270136

Cited by 227 publications

(298 citation statements)

References 28 publications

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“…As expected, addition of cholesterol decreased the diffusion coefficients by a factor of 4-5 [26,34]. In the presence of cholesterol, binding of the agonist antibody to SA ⁄ slightly decreased the mobility, which may indicate weak interactions between the protein and the lipid bilayer surface.…”

Section: Functionalized Slbs For Fas Mediated Apoptosissupporting

confidence: 64%

Agonist mobility on supported lipid bilayers affects Fas mediated death response

et al. 2015

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a b s t r a c tExtrinsic apoptosis is initiated by recognition and clustering of the single-pass transmembrane proteins Fas ligand and Fas expressed at the surface of closely apposed lymphocytes and target cells, respectively. Since Fas-mediated death response was mainly studied with soluble antibodies, the mobility constraints for receptor activation by a membrane embedded agonist is not well understood. We explored this influence by stimulating apoptosis on functionalized supported lipid bilayers, where we quantified agonist mobility by z-scan fluorescence correlation spectroscopy. Using different lipid compositions, we show that the apoptotic response correlates with increased lateral mobility of the agonist in the lipid bilayer.

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Section: Functionalized Slbs For Fas Mediated Apoptosissupporting

confidence: 64%

Agonist mobility on supported lipid bilayers affects Fas mediated death response

et al. 2015

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“…Most of the fluorescent lipid dye molecules were bleached so that a few excited molecules could be tracked over time. From this time-dependent measurement of lipid diffusion, a mean diffusion constant for a membrane on a single-membrane-coated silica sphere of D ¼ (1.32 5 0.20) mm 2 s À1 was calculated, which is comparable to a flat solid supported membrane deposited on a glass surface (15). Note that the distribution of diffusion coefficients obtained from various spheres is rather broad.…”

Section: A B C Dmentioning

confidence: 90%

Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion

Savić

Kliesch

Verbeek

et al. 2016

Biophysical Journal

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The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed.

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“…Here, the focal volume may be moving on a line (Petersen 1986;Petersen et al 1986), a circle in lateral axis (Petrasek and Schwille 2008;Petrasek et al 2010) or along z-axis (Benda et al 2003). The basic principle of scanning FCS perpendicular to the membrane is shown in Figure 6.…”

Section: Scanning Fcs (Sfcs)mentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy

Cited by 227 publications

References 28 publications

Agonist mobility on supported lipid bilayers affects Fas mediated death response

Agonist mobility on supported lipid bilayers affects Fas mediated death response

Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion

Fluorescence Techniques to Study Lipid Dynamics

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