Fluorescence Correlation Spectroscopy Relates Rafts in Model and Native Membranes

Bacia, Kirsten; Scherfeld, Dag; Kahya, Nicoletta; Schwille, Petra

doi:10.1529/biophysj.104.040519

Cited by 301 publications

(293 citation statements)

References 46 publications

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“…Additionally, microfilament depolymerization can disrupt lipid rafts (Bacia et al, 2004). The latter finding is of particular interest, since it has been reported that 16E5 induces complete disassembly of microfilaments in mouse fibroblasts (Thomsen et al, 2000).…”

Section: E5-induced Redistribution Of Ganglioside Gm1 Is Not Mediatmentioning

confidence: 96%

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

et al. 2007

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High-risk human papillomaviruses (HPVs), especially HPV-16, play a primary role in the pathogenesis of cervical cancer. HPV-16 encodes the E5, E6 and E7 oncoproteins. Although the biological functions of E5 are poorly understood, recent studies indicate that its expression correlates with papillomavirus oncogenicity. In this study we demonstrate that the HPV-16 E5 oncoprotein increases plasma membrane expression of caveolin-1, which is a constituent of lipid rafts and regulator of cell signaling, and that this phenotype is mediated by the C-terminal 10 amino acids of E5. Moreover, E5 (but not mutant E5) induces a 23-to 40-fold increase in the lipid raft component, ganglioside GM1, on the cell surface and mediates a dramatic increase in caveolin-1/GM1 association. Since gangliosides strongly inhibit cytotoxic T lymphocytes, block immune synapse formation and are expressed at high levels on the surface of many tumor cells, our results suggest a potential mechanism for immune evasion by the papillomaviruses. Additionally, surface gangliosides are known to enhance proliferative signaling by the epidermal growth factor (EGF) receptor, providing a possible mechanistic basis for observations that EGF signaling is enhanced in E5-expressing cells. Finally, the upregulation of caveolin-1 and ganglioside GM1 at the plasma membrane of E5-expressing cervical cells provides potential new therapeutic targets and diagnostic markers for high-risk HPV infections.

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Section: E5-induced Redistribution Of Ganglioside Gm1 Is Not Mediatmentioning

confidence: 96%

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

et al. 2007

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“…From a variety of possible probes we stringently selected those that at steady state showed mainly apical membrane localization on polarized MDCK cells with no staining of the endoplasmic reticulum or other organelles close to the plasma membrane. FRAP experiments were routinely performed at room temperature (Ϸ25°C) to minimize membrane deformations caused by temperature gradients and the influence of membrane traffic events on fluorescence recovery (31). The apparent diffusion coefficient D and the percentage recovery were derived from the fluorescence intensity at ''infinite'' time after photobleaching, I ϱ , calculated by the theoretical fit (25,26) and not from a visual examination of the experimental curves that were rarely followed until recovery was complete.…”

Section: Resultsmentioning

confidence: 99%

Phase coexistence and connectivity in the apical membrane of polarized epithelial cells

Meder

Moreno

Verkade

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

158

125

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Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature (Ϸ25°C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37°C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12°C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phaseseparated system with a continuous (percolating) raft phase <25°C in which isolated domains of the nonraft phase are dispersed, whereas at 37°C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.fluorescence recovery after photobleaching ͉ rafts ͉ paurdan ͉ liquid-ordered ͉ Madin-Darby-canine kidney T he domain organization of biological membranes is presently under intense scrutiny. In particular, the existence and role of sphingomyelin-and cholesterol-rich lipid bilayer phases, commonly known as rafts, have drawn much attention (1-5). Phase diagrams of model membrane systems made from ternary mixtures of sphingomyelin, 1-palmitoyl-2-oleoylphosphatidylcholine, and cholesterol show regions of fluid-fluid phase coexistence (6). The two fluid phases of special relevance are a liquid-ordered phase, characterized by high conformational and low translational order, and a liquid-disordered phase, characterized by low conformational and translational order (7,8). The coexistence of these two phases has been visualized by several laboratories in giant unilamellar vesicles and supported lipid bilayers prepared from synthetic lipids. More importantly, giant unilamellar vesicles prepared from cell membrane lipid extracts (9) also show visible fluid-fluid phase coexistence. However, there was no direct evidence for phase separation in native cell membranes (2, 5). Glycosylphosphatidylinositol (GPI)-anchored proteins in the exoplasmic leaflet (10, 11) and lipid-anchored proteins in the cytoplasmic leaflet (12-14) of cell membranes were shown to b...

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“…FCS is a method that has been extensively used and further developed by our group, being introduced and established as a very suitable approach to characterize model and cellular membranes (Schwille et al 1999a;Bacia et al 2004). It is, in a way, a single-molecule method, but provides sufficient statistical significance to also use it for general characterization of membranes, mainly through the diffusion properties of their constituents.…”

Section: Fluorescence Correlation Spectroscopy (Fcs)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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Fluorescence Correlation Spectroscopy Relates Rafts in Model and Native Membranes

Cited by 301 publications

References 46 publications

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells

Phase coexistence and connectivity in the apical membrane of polarized epithelial cells

Fluorescence Techniques to Study Lipid Dynamics

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