On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements

Macháň, Radek; Foo, Yong Hwee; Wohland, Thorsten

doi:10.1016/j.bpj.2016.06.001

Cited by 47 publications

(34 citation statements)

References 30 publications

(39 reference statements)

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“…To gain structural insight, EVs and POPC liposomes were fluorescently labeled with 0.01% w/w of Liss Rhod PE and their SLBs investigated with FCS . FCS, together with FRAP, is commonly used to monitor the bidimensional diffusion of species embedded in lipid membranes and it is particularly suitable to investigate lipid membranes with complex composition and structure, even native cell membranes . The normalized autocorrelation function (ACF) profiles of the EVSLB and of the POPC SLB are compared in Figure 2 a.…”

Section: Resultsmentioning

confidence: 99%

Biogenic Supported Lipid Bilayers from Nanosized Extracellular Vesicles

Montis

Busatto

Valle³

et al. 2018

Adv. Biosys.

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biotechnological and medical potential of synthetic SLBs still awaits to be fully realized, because of the limitations and open challenges in obtaining functional 2D analogues of complex natural biomembranes with satisfying robustness and fidelity, achievable with methods amenable to large-scale production. So far, bottom-up synthesis has been the fabrication criteria for biomimetic SLBs, based on adding complexity to plain synthetic membranes, for example, by combining supramolecular strategies with adaptive chemistry or molecular biology. [1,2] This route, often studded with challenging and sophisticated chemistry, may though suffer from limited fidelity and/or severe technical hurdles, besides cost limitations when extended beyond the batch laboratoryscale. Fewer but remarkable "top-down" attempts to derive SLBs from cell plasma membranes were also proposed. They all made use of blebs (proteoliposomes) chemically or mechanically pulled out from plasma membranes and further modified with synthetic compounds and/or fused with liposomes, [2][3][4][5][6][7] which are though not naturally related to the functions of the originating cell. We propose here to exploit extracellular vesicles (EVs) to move a step forward.EVs are micro-and nanosized vesicles secreted by all cells, for specific intercellular transfer of multiple information in the form of proteins, RNA, lipids, and metabolites. They include microvesicles, budded from the plasma membrane with a size from 100 to 2000 nm, and exosomes, of endosomal membrane origin, with a size from 30 to 120 nm. EVs participate in human physiological and pathological processes as well as in crossorganism communication, encompassing viruses, bacteria, parasites, and plants. [8][9][10] Such properties and functions pose on EVs ever increasing interest as means for precision (nano) medicine [11,12] and for their broader impact in food safety and public health. Based on these considerations, SLBs from EVs (hereafter referred as EVSLBs) can be straightforward and accurate mimics of membranes of native cells laced with a richer link with their biology.The paper explores this idea and investigates the formation pathway by fusion route and the properties of EVSLBs from nanosized EVs of TRAMP-C2 cells, a murine cell line used as a model for prostate cancer. The peculiar properties of these EVSLBs are determined by synergistically leveraging stateof-the art techniques, such as quartz crystal microbalance with Fabrication of synthetic surfaces that reproduce structure and function of biological membranes is an open challenge. This work reports the first example of supported lipid bilayers obtained from extracellular vesicles (EVSLBs). EVSLBs harness and pattern in two dimensions key properties of extracellular vesicle (EV) membranes, which present intermediate complexity between synthetic mimics and natural membranes and innate link to phenotype and function of the originating cells. Silica-supportedEVSLBs are formed from nanosized EVs-separated from culture media of prostate cancer mod...

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Section: Resultsmentioning

confidence: 99%

Biogenic Supported Lipid Bilayers from Nanosized Extracellular Vesicles

Montis

Busatto

Valle³

et al. 2018

Adv. Biosys.

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“…FRAP analysis of molecules associated with the plasma membrane has been used extensively to characterize their dynamics [37][38][39][40][41]. Compared with intracellular molecules, using FRAP to estimate diffusion coefficients in membranes is simplified due to the two-dimensional nature of the membrane, as well as the slower dynamics of the molecules.…”

Section: Membrane Frapmentioning

confidence: 99%

Understanding boundary effects and confocal optics enables quantitative FRAP analysis in the confined geometries of animal, plant, and fungal cells

Kingsley

Bibeau

Mousavi

et al. 2016

Preprint

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Fluorescence Recovery After Photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus or cell membrane. While many FRAP models have been developed over the past decades, the influence of the complex boundaries of three-dimensional cellular geometries on the recovery curves, in conjunction with ROI and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a three-dimensional computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorescent protein, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, S. pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite, DCMS-can also perform fluorescence dynamics assays, such as Number and Brightness (N&B), Fluorescence Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy (RICS), and could help shape the way these techniques are interpreted.

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“…S 2 E -G). These differences which are inherent to such methodologies have been highlighted previously (38) and were taken into consideration in our experiments by using the same measurement and labelling mode when comparing mobility between individual drug treatments.…”

Section: The Cortical Actin Network Restricts Lyve-1 Lateral Mobilitymentioning

confidence: 95%

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

Stanly

Fritzsche

Banerji

et al. 2020

Journal of Biological Chemistry

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Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

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On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements

Cited by 47 publications

References 30 publications

Biogenic Supported Lipid Bilayers from Nanosized Extracellular Vesicles

Biogenic Supported Lipid Bilayers from Nanosized Extracellular Vesicles

Understanding boundary effects and confocal optics enables quantitative FRAP analysis in the confined geometries of animal, plant, and fungal cells

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

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