Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

., ira; Zou, Shan; Ramirez, Daniel M. Carter; Vanderlip, Sapphire; Ogilvie, William W.; Jakubek, Zygmunt J.; Johnston, Linda J.

doi:10.1016/j.jsb.2009.03.014

Cited by 45 publications

(55 citation statements)

References 52 publications

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“…A new method has also been applied to detect lipid rafts, called LG-TIRF (Sohn et al 2010). Other applications were to elucidate the role of ceramide in membrane restructuring (Ira et al 2009), the organization of bacterial light harvesting complex 2 (Dewa et al 2006), the role of cholesterol in antibody binding ), EGFR activation by EGF (Sako et al 2000;Cannon et al 2005;Teramura et al 2006), and the phase preference of peptides (Choucair et al 2007). Membrane curvature, exocytosis, and endocytosis are some other topics in which TIRF is successfully applied (Merrifield et al 2002(Merrifield et al , 2005Byrne et al 2008;Nagamatsu and Ohara-Imaizumi 2008;Joselevitch and Zenisek 2009;Ohara-Imaizume et al 2009;Aoki et al 2010;Gorg et al 2010;Lam et al 2010).…”

Section: Tirf Applications On Membrane Dynamicsmentioning

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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Section: Tirf Applications On Membrane Dynamicsmentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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“…Elevated Cer levels have been found in the brain in patients affected by Alzheiincreasing acyl chain order and altering membrane permeability ( 25 ). Its exogenous addition or generation through SMase activity leads to lateral phase separation and the enlargement and clustering of preexistent nanodomains, promoting raft fusion ( 12,(26)(27)(28) and giving rise to Cer-enriched platforms that facilitate the clustering of receptor molecules and their ligands. Thus, Cer-induced clustering of the CD95/Fas death receptor ( 29,30 ) precedes the induction of apoptosis, evidencing the close relationship between Cer's structural role and modulation of cell death.…”

Section: The Sphingolipids: a Vast Familymentioning

confidence: 99%

Regulating survival and development in the retina: key roles for simple sphingolipids

Rotstein

Miranda

Abrahan

et al. 2010

Journal of Lipid Research

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tions as structural membrane components and energetic fuels. However, the fi ndings of the previous half century have extended these roles, shedding light on their indisputable relevance as signaling molecules controlling key aspects of cellular life and development. The identifi cation of diacylglycerol and inositol 1,4,5 triphosphate ( 1 ) as second messengers seemed at the time a peculiarity of these lipids. The subsequent fi ndings of the roles of arachidonic acid metabolites ( 2 ), platelet activating factor ( 3 ), and other minor inositol lipids ( 4 ) in cell signaling began to establish that being intracellular messengers was not an oddity but a habitual task for lipids in cells. The family of lipid signaling molecules largely expanded in the last two decades, when several sphingolipids were successively shown to regulate a multiplicity of cell functions. Sphingosine (Sph) was the fi rst sphingolipid to be established as a bioactive lipid, involved in the regulation of protein kinase (PK)C activity ( 5 ). Ceramide (Cer) and sphingosine-1-phosphate (S1P) were next shown to signal a myriad of cell functions and the number of sphingolipid molecules with bioactive properties has gone on enlarging in recent years ( 6, 7 ).Though a vast literature exists on the functions of sphingolipids in several tissues, less is known concerning its roles in the eye, and in the retina in particular. Accumulation of sphingolipids originated in defects in the lysosomal Abstract Many sphingolipids have key functions in the regulation of crucial cellular processes. Ceramide (Cer) and sphingosine (Sph) induce growth arrest and cell death in multiple situations of cellular stress. On the contrary, sphingosine-1-phosphate (S1P), the product of Sph phosphorylation, promotes proliferation, differentiation, and survival in different cell systems. This review summarizes the roles of these simple sphingolipids in different tissues and then analyzes their possible functions in the retina. Alterations in proliferation, neovascularization, differentiation, and cell death are critical in major retina diseases and collective evidence points to a role for sphingolipids in these processes. Cer induces infl ammation and apoptosis in endothelial and retinal pigmented epithelium cells, leading to several retinopathies. S1P can prevent this death but also promotes cell proliferation that might lead to neovascularization and fibrosis. Recent data support Cer and Sph as crucial mediators in the induction of photoreceptor apoptosis in diverse models of oxidative damage and neurodegeneration, and suggest that regulating their metabolism can prevent this death. New evidence proposes a central role for S1P controlling photoreceptor survival and differentiation. Finally, this review discusses the ability of trophic factors to regulate sphingolipid metabolism and transactivate S1P signaling pathways to control survival and development in retina photoreceptors. When asked about the roles of cellular lipids, the fi rst thought usually coming to our minds re...

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“…[21][22][23] CRPs have been visualized by electron microscopy and fluorescence microscopy in the plasma membrane of cells as macrodomains with diameters of 200 nm up to several microns. [7,24,25] To fully understand 'ceramide biology' we have to develop refined methods to study their distribution in the plasma membrane ideally quantitatively with molecular resolution. The limited knowledge about the distribution of sphingolipids in plasma membranes is mainly due to experimental hurdles.…”

Section: Graphical Abstractmentioning

confidence: 99%

Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

Burgert

Schlegel

Bécam³

et al. 2017

Angewandte Chemie

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The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane which segregates membrane receptors and affect membrane curvature and vesicle formation, fusion and trafficking. Here, we label ceramides by immunocytochemistry and visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50-60% of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of ~ 75 nm which are composed of at least ~ 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold. Graphical Abstractd STORM reveals that independent of the cell type membrane ceramides form nanodomains (CRPs) with a diameter of ~ 75 nm containing 50-60% of all ceramides. Treatment with sphingomyelinase increases the quantity of ceramides, the size and density of CRPs as well as the ceramide concentration in CRPs.Correspondence to: Markus Sauer. Supporting information for this article is given via a link at the end of the document. The plasma membrane is mainly composed of glycerophospholipids, sphingolipids and cholesterol. Sphingolipids are natural lipids comprised of the sphingoid base backbone sphingosine, which when N-acylated with fatty acids forms ceramide, a central molecule in sphingolipid biology. De-novo synthesis of ceramide occurs in the endoplasmatic reticulum followed by conversion into complex sphingolipids in the Golgi apparatus. In addition, sphingomyelinases (SMases) can generate ceramide from sphingomyelin in the plasma membrane. [1,2] Interactions of sphingolipids with one another and with cholesterol typically result in membrane microdomains or 'rafts' which segregate membrane-associated proteins and compartmentalize signaling components within the plasma membrane. [3][4][5][6] Activation of SMases by cellular stress or ligation of receptors results in ceramide synthesis and subsequent self-association within the plasma membrane resulting in ceramide-rich platforms (CRPs). [7,8] The formation of CRPs rearranges the organization of the plasma membrane including clustering of diverse receptors and facilitates vesicle formation and fusion. [8,9] These changes induce differentiation, proliferation, growth arrest, and cell death. [1,2] Moreover, SMases and ceramides have been shown to be critically involved in the internalization of pathogens. [10][11][12][13][14][15] The recent observation, that CRP formation is essential in controlling the metabolic activity of regulatory T cells demonstrates that ceramides play also an essential role in regulating immune functions and i...

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Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

Cited by 45 publications

References 52 publications

Fluorescence Techniques to Study Lipid Dynamics

Fluorescence Techniques to Study Lipid Dynamics

Regulating survival and development in the retina: key roles for simple sphingolipids

Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

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