Fluorescent Cholesterol Analog Research Articles

Synthetic lipids and their role in defining macromolecular assemblies

Lipids have a variety of physiological roles, ranging from structural and biophysical contributions to membrane functions to signaling contributions in normal and abnormal physiology. This review highlights some of the contributions made by Robert Bittman to our understanding of lipid assemblies through the production of synthetic lipid analogs in the sterol, sphingolipid, and glycolipid classes. His contributions have included the development of a fluorescent cholesterol analog that shows strong functional analogies to cholesterol that has allowed live imaging of cholesterol distribution in living systems, to stereospecific synthetic approaches to both sphingolipid and glycolipid analogs crucial in defining the structure–activity relationships of lipid biological targets.

Design of new fluorescent cholesterol and ergosterol analogs: Insights from theory

Cholesterol (Chol) and ergosterol (Erg) are abundant and important sterols in the plasma membrane of mammalian and yeast cells, respectively. The effects of Chol and Erg on membrane properties, as well as their intracellular transport, can be studied with use of fluorescence probes mimicking both sterols as closely as possible. In the search for new and efficient Chol and Erg probes, we use a combination of theoretical methods to explore a series of analogs. The optical properties of the analogs (i.e. excitation energies, emission energies and oscillator strengths) are examined using time-dependent density functional theory (TDDFT) and their ability to mimic the effects of Chol and Erg on membranes is investigated with molecular dynamics (MD) simulations of each analog in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. From the set of analogs we find two probes (3a and 3b) to display favorable electronic transition properties as well as strong condensing abilities. These findings can lead to the use of new efficient probes and aid in the understanding of the structural features of Chol and Erg that impart to them their unique effects on lipid membranes.

A Combined Effect of Proteins SP-B and SP-C and Membrane Curvature on Cholesterol Partition in Lung Surfactant Membranes: Answers from Fluorescence

Cholesterol is the major neutral lipid present in lung surfactant, a lipid-protein complex responsible for the stabilization of the air-liquid interface in mammalian lungs, which is essential for the breathing function. It has been proposed that cholesterol content is tightly regulated in surfactant and that protein SP-C could have a role in this respect. To further analyse the affinity of surfactant membranes for cholesterol in the absence or presence of proteins, we have used cyclodextrin as a cholesterol solubilising agent and a fluorescent analog of cholesterol, cholestatrienol (CTL), whose partition can be followed in membrane vesicles with different lipid/protein compositions and sizes. Our results suggest that there is a direct effect of vesicle size/curvature on the affinity of membranes for CTL. This is translated into lower partition coefficients (Kx) for membranes with higher curvatures, possibly caused by an increased exposure of CTL in more curved bilayers. Surfactant hydrophobic proteins SP-B and SP-C were found to have a curvature-independent effect on CTL mobility, as well as in the mobility of other probes such as transparinaric acid-sphingomyelin (tPA-SM) or diphenylhexatriene-phosphatidylcholine (DPH-PC) in membranes of the same composition. Furthermore, in the absence of SP-B, SP-C-containing liposomes were found to spontaneously split into 30 nm vesicles increasing membrane curvature regardless lipid composition, which correlated with a reduction of Kx compared to pure lipid systems. Taken all together, our results indicate that curvature alters cholesterol accessibility in surfactant lipid bilayers and that lung surfactant proteins disturb membranes in cholesterol containing domains, which may have important physiological consequences for cholesterol mobilization in lung surfactant membranes and films.

Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells.

The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.

Differential basolateral-apical distribution of scavenger receptor, class B, type I in cultured cells and the liver.

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral–apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-014-1251-9) contains supplementary material, which is available to authorized users.

Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm2/second, which was ∼10 times greater than that of non-raft phospholipid molecules (0.33 µm2/second), despite Bdp-Chol's probable association with raft domains. Furthermore, Bdp-Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp-Chol and Cy3-conjugated dioleoylphosphatidylethanolamine (Cy3-DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin-based membrane skeleton reduces the D of Bdp-Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3-DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin-based membrane-skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp-Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3-DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane-skeleton-induced compartments and are contained within them.

The Effect of Hydrophobic Matching between Lipids and Transmembrane Peptides on Sterol Bilayer Affinity

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. It is therefore of interest to study the influence of lipid-protein interactions on membrane trafficking to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. We have measured the equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin to determine the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers. The sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without the peptides WALP19, WALP23 or WALP27. The results showed that hydrophobic matching will affect the affinity of the sterol for the bilayers. Stronger sterol binding to the bilayers was achieved by an increase in positive hydrophobic mismatch (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. Peptide insertion into the phospholipid bilayers was also observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid-protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This may well be of importance in membrane trafficking.

NBD-Labeled Cholesterol Analogues in Phospholipid Bilayers: Insights from Molecular Dynamics

Nitrobenzoxadiazole (NBD)-labeled sterols are commonly used as fluorescent cholesterol analogues in membrane biophysics. However, some experimental reports have questioned their ability to emulate the behavior of cholesterol in phospholipid bilayers. For the purpose of a detailed clarification of this matter, atomistic molecular dynamics simulations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, containing either cholesterol or one of two fluorescent cholesterol analogues, 22-NBD-cholesterol or 25-NBD-cholesterol, were carried out. It is found that these sterol probes tend to adopt conformations in which their tail-labeled fluorophore is oriented toward the lipid/water interface, with a location similar to that observed in molecular dynamics simulations of other NBD probes. This implies that in these molecules the long sterol axis is no longer aligned with the membrane normal, and preferentially adopts orientations approximately parallel to the bilayer plane. In turn, these stretched conformations, together with NBD-POPC atomic interactions, lead to slowed-down lateral diffusion of both fluorescent sterols, compared to cholesterol. From computation of the deuterium order parameter and acyl chain tilts of POPC chains for varying POPC-sterol distance, it is observed that the local ordering effect of sterol is altered in both fluorescent derivatives. In agreement with reported experimental data, both fluorescent sterols are able to increase the order of POPC at 20 mol % concentration (as some molecules adopt an upright conformation, possibly related to formation of transbilayer aggregates), albeit to a smaller extent to that of cholesterol. Altogether, this study indicates that both 22- and 25-NBD-cholesterol are unable to mimic the most important features of cholesterol's behavior in lipid bilayers.

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (α-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity.

Abstract 56: Macrophage Reverse Cholesterol Transport in Mice Relies on The Lymphatic Vasculature

Reverse cholesterol transport (RCT) refers to mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used two models of disrupted lymphatic drainage from skin -one surgical and the other genetic- to quantitatively track RCT following injection of [3H]cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analogue (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aorta of donor atherosclerotic apoE-/- mice with [2H]6-labeled cholesterol and surgically transplanting these aortas into recipient apoE-/- mice that were treated with anti-VEGFR3 mAb to block lymphatic regrowth or with control mAb to allow such regrowth. [2H]-Cholesterol was retained in aortas of anti-VEGFR3 treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. Therefore, supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

Organization of fluorescent cholesterol analogs in lipid bilayers — Lessons from cyclodextrin extraction

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.

HPLC‐FLD determination of NBD‐cholesterol, its ester and other metabolites in cellular lipid extracts

22-[N(-7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), a fluorescent cholesterol analog, was an extragenous cholesterol tracer used to study cholesterol absorption and metabolism in cultured cells. In order to measure free intracellular cholesterol and its esters, a precise and sensitive method employing high-performance liquid chromatography/fluorescence detection (HPLC-FLD) was developed for the first time. Method validation showed a limit of detection at 30 ng/mL. The calibration curve was linear within the range of 0.0625-10.0 µg/mL (r(2) = 0.999). Accuracy and precision were highlighted by good recovery and low variations. Apart from NBD-cholesteryl oleate, two additional cellular metabolites of NBD-cholesterol, probably an isomer and an oxidation product, were determined in the lipid extracts of Caco-2 human colon adenocarcinoma cells according to mass spectrometry. In AC29 mouse malignant mesothelioma cells overexpressing acyl-CoA:cholesterol acyltransferase-1 (ACAT1) or ACAT2, only the oxidized metabolite was detected. Using the newly developed method, YIC-C8-434, a known ACAT inhibitor, was shown to inhibit ACAT activity in Caco-2 cells, as well as in AC29/ACAT1 or AC29/ACAT2 cells. In conclusion, the sensitive and specific HPLC-FLD method is a powerful tool for simultaneous quantification of intracellular NBD-cholesterol and its oleoyl-ester.

Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice

Reverse cholesterol transport (RCT) refers to the mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used 2 models of disrupted lymphatic drainage from skin--1 surgical and the other genetic--to quantitatively track RCT following injection of [3H]-cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aortae of donor atherosclerotic Apoe-deficient mice with [2H]6-labeled cholesterol and surgically transplanting these aortae into recipient Apoe-deficient mice that were treated with anti-VEGFR3 antibody to block lymphatic regrowth or with control antibody to allow such regrowth. [2H]-Cholesterol was retained in aortae of anti-VEGFR3-treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. These results suggest that supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: Plasma membrane change induced by cholesterol depletion

HEK293 cells stably expressing δ-opioid receptor were labeled first with fluorescent analog of cholesterol, 22-NBD-cholesterol, exposed to cholesterol-depleting agent β-cyclodextrin (β-CDX) and analyzed by fluorescence lifetime imaging microscopy (FLIM). In accordance with chemical analysis of cholesterol level, the total cellular signal of this probe was decreased to half. Distribution of lifetime (τtot) values of 22-NBD-cholesterol, however, when screened over the whole cell area indicated no significant difference between control (τtot=4.9±0.1ns) and β-CDX-treated (τtot=4.8±0.1ns) cells. On the contrary, comparison of control (τtot=5.1±0.1ns) and β-CDX-treated (τtot=4.4±0.1ns) cells by analysis of 25-NBD-cholesterol fluorescence implied highly significant decrease of lifetime values of this probe. The observation that 22-NBD-cholesterol appears to be indifferent to the changes in the membrane packing in living cells is in agreement with previous studies in model membranes. However, our data indicate that the alternation of plasma membrane structure induced by decrease of cholesterol level by β-CDX makes the membrane environment of NBD moiety of 25-NBD-cholesterol probe a significantly more hydrated. This finding not only encourages using 25-NBD-cholesterol in living cells, but also demonstrates that previously drawn discouraging conclusions on the use of 25-NBD-cholesterol in model membranes are not valid for living cells.

Lipid Exchange between Borrelia burgdorferi and Host Cells

Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or 3H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.

Differential Interaction of Bovine Alpha-Lactalbumin with Membranes: Interplay of Negatively Charged Lipids and Cholesterol

Many soluble proteins are known to interact with membranes and the mechanism of such interactions in cellular processes is beginning to be understood. Bovine alpha-lactalbumin (BLA) is a soluble protein and yet it interacts with membranes. We recently reported the specific binding of apo-BLA with negatively charged membranes using a variety of fluorescence approaches [1]. A novel finding is that BLA exhibits an enhanced binding to negatively charged membranes in the presence of cholesterol and it possesses cholesterol recognition/interaction amino acid consensus (CRAC), a motif recognized for preferential association with cholesterol in many proteins. We monitored the specificity of sterol interaction by replacing cholesterol with 7-dehydrocholesterol (an immediate biosynthetic precursor of cholesterol), and observed that BLA-sterol interaction is specific to membrane cholesterol. Significant Fluorescence Resonance Energy Transfer (FRET) was observed between the tryptophan residues of BLA and dehydroergosterol (a naturally occurring fluorescent analog of cholesterol) in presence of negatively charged membranes, indicating close proximity between them. Dipole potential measurements upon BLA binding to membranes and docking studies of cholesterol with BLA provide useful insight into the lipid selectivity of BLA binding to membranes. Depth analysis by the parallax approach provides evidence for interfacial localization of tryptophans of BLA when bound to membranes. Taken together, our results assume significance in the light of tumoricidal and antimicrobial functions of α-lactalbumin [2,3].

Sterol affinity for phospholipid bilayers is influenced by hydrophobic matching between lipids and transmembrane peptides

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, a good starting point for studying the influence of lipid–protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid–protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking.

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

BackgroundCholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport.ResultsWe found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t > ~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%.ConclusionsThe mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport.

Cholesterol-Analoga für die Forschung — nichts ist wie das Original!

Cholesterol is a major component of eukaryotic cells. For membrane biophysics and cell biology, fluorescent and ESR-active cholesterol analogues have been synthesized that should mimic the basic properties of cholesterol. However, data reported here shows that these molecules do not fully carry the important properties of cholesterol. In particular, the analogues fail to reproduce the condensation effect that is key for membrane fluidity and lateral organization of the lipids in the membrane.

22-NBD-cholesterol, a new fluorescent substrate of bacterial cholesterol-oxidases

Fluorescent analogs of sterols are convenient agents for studying the transport, distribution, and protein—ligand interaction of natural sterols in cells [1]. However, the effect of the fluorescent moiety on the interaction parameters with cholesterol-transforming enzymes such as bacterial cholesterol-oxidases or mammalian steroid-hydroxylating cytochrome P450 has not been addressed in the literature. Herein we report data for the interaction of the fluorescent cholesterol analog 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3 -ol [22-NBD-cholesterol (1)] with cholesteroloxidases (EC 1.1.3.6). Cholesterol-oxidases are FAD-dependent enzymes that catalyze the transformation of 3 -hydroxy5steroids and are widely used for laboratory and clinical cholesterol determinations [2]. We calculated the interaction of 1 with the enzyme based on the three-dimensional structure of Brevibacterium sterolicum cholesterol-oxidase (code PDB 1COY) using the Autodock 4.0 programs and MGLTools. It was found with the help of molecular docking that 1 can bind to the active center of cholesterol-oxidase such that its 3 -hydroxy group is situated in close proximity to the functionally important His447 and Glu361 residues [3] and the redox-active groups of the FAD molecule. The calculated binding energies for cholesterol and 1 were practically the same (–11.43 and –11.94 kcal/mol, respectively). Next selective transformation of 1 was demonstrated using cholesterol-oxidase in vitro. After biotransformation of fluorescent substrate 1 with purified cholesterol-oxidase, the formation of 2, which retained the fluorescent properties of 1 (Scheme 1) was detected by HPLC (Shimadzu LC10-AD chromatograph with an RF-10A 1 fluorimetric detector and SPD-10A spectrophotometric detector; CH3CN:H2O:i-PrOH, 84:16:5; LiChroCART C18 column, Merck, 250 4 mm).