Imaging approaches for analysis of cholesterol distribution and dynamics in the plasma membrane

Wüstner, Daniel; Modzel, Maciej; Lund, Frederik Wendelboe; Lomholt, Michael A.

doi:10.1016/j.chemphyslip.2016.03.003

Cited by 25 publications

(22 citation statements)

References 256 publications

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“…Due to space limitations, this review focuses on reports that contextualize the development of current models of plasma membrane organization, and the results that that have led some to question or even reject the raft hypothesis (Shaw, 2006; Kenworthy, 2008; Kraft, 2013; Sevcsik and Schütz, 2016; Wüstner et al, 2016). Emphasis is placed on the findings acquired with a new approach for chemically mapping isotope-labeled lipids in the plasma membrane with high-resolution, which were reported by the author and collaborators.…”

Section: Introductionmentioning

confidence: 99%

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

Kraft

2017

Front. Cell Dev. Biol.

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Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed.

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Section: Introductionmentioning

confidence: 99%

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

Kraft

2017

Front. Cell Dev. Biol.

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“…Different studies showed that the performance of several fluorescent cholesterol derivatives were different depending on the experimental modalities. For example, TopFluor-cholesterol was more adapted than NBD-cholesterol derivatives to study the cholesterol partition in giant unilamellar vesicles (GUVs) by imagery, and a NBD-cholesterol derivative was more suitable in cellular trafficking studies [ 33 , 34 ]. To our knowledge, the cholesterol-pyrene probe described by Le Guyader and collaborators [ 35 ] is the less disturbing cholesterol-modified probe available.…”

Section: Introductionmentioning

confidence: 99%

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths

Almeida¹,

Wreede²,

Lamazière³

et al. 2018

PLoS ONE

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Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as individual fluorophore. However, no efficient formulation of a cholesterol probe has been available so far. Herein, we described a cholesterol-pyrene probe behaviour in heterogeneous membranes. We characterised the pyrene fluorescence spectra in liquid-ordered (Lo) and liquid-disordered (Ld) membranes. Using statistical multivariate analysis, we found out the most appropriate wavelengths for membrane domains studies. 373 nm and 379 nm were the most discriminant wavelengths to follow the liquid-ordered and the liquid-disordered environments. Cholesterol clustering behaviour was quantified by the modulation of the cholesterol-pyrene excimers peak (474 nm). In liquid-ordered membranes at low temperature, cholesterol-pyrene was found as multimers and as monomers. At high temperature, the liquid-ordered status of the membrane decreases and cholesterol-pyrene tends to cluster. In liquid-disordered membranes, cholesterol-pyrene was present mostly as monomers and the small quantity of excimers increased with temperature. Cholesterol-pyrene was used to test the ceramide effect on membranes, and presented a behaviour in agreement with the cholesterol behaviour reported in the literature. Overall, the presented data show that cholesterol-pyrene is an efficient sensor to study liquid ordered and liquid disordered organisation in membranes.

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“…Analysis of cellular cholesterol trafficking requires minimal invasive monitoring technology, and one particularly suitable approach is optical microscopy of suitable cholesterol analogues (Solanko et al, 2016;Wüstner et al, 2016). The high sensitivity of optical methods allows for minimal chemical alterations of the sterol structure, either as intrinsic fluorescence in the steroid backbone, as extrinsic, covalently linked organic dyes, such as Raman active functional groups or as linker for click-reaction of dyes after cellular uptake of alkyne sterols (see references in (Solanko et al, 2016)).…”

Section: Introductionmentioning

confidence: 99%

Live‐cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

Modzel

Solanko

Szomek

et al. 2018

Journal of Microscopy

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Analysis of intracellular cholesterol transport by fluorescence microscopy requires suitable fluorescent analogues of cholesterol. Most existing cholesterol analogues contain lipophilic dyes which can compromise the sterol properties in membranes. An alternative strategy is to introduce additional double bonds into the sterol ring system resulting in intrinsic fluorescence, while at the same time keeping the cholesterol-like properties of the analogues. Existing polyene sterols, such as dehydroergosterol (DHE) or cholestatrienol (CTL), however, contain only three double bonds and suffer from low brightness, significant photobleaching and excitation/emission in the ultraviolet region. Thus, special equipment is required to image such sterols. Here, we describe synthesis, characterization and intracellular imaging of new polyene sterols containing four conjugated double bonds in the sterol ring system. We show that such analogues have red-shifted excitation and emission by ∼20 nm compared to DHE or CTL. The red shift was even more pronounced when preventing keto-enol tautomer equilibration by protecting the 3'-hydroxy group with acetate. We show that the latter analogue can be imaged on a conventional wide field microscope with a DAPI/filipin filter cube. The new polyene sterols show reduced photobleaching compared to DHE or CTL allowing for improved deconvolution microscopy of sterol containing cellular membranes.

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Imaging approaches for analysis of cholesterol distribution and dynamics in the plasma membrane

Cited by 25 publications

References 256 publications

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

Cholesterol-pyrene as a probe for cholesterol distribution on ordered and disordered membranes: Determination of spectral wavelengths

Live‐cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

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