Cholesterol In Biological Membranes Research Articles

Nanoscale dynamics of cholesterol in the cell membrane

Cholesterol constitutes ∼30–40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.

Facilitated and Non-Gaussian Diffusion of Cholesterol in Liquid Ordered Phase Bilayers Depends on the Flip-Flop and Spatial Arrangement of Cholesterol.

The diffusion of cholesterol in biological membranes is critical to cellular processes such as the formation of cholesterol-enriched domains. The cholesterol diffusion may be complicated especially when cholesterol flip-flops and/or stays at the membrane center. Understanding the diffusion mechanism of cholesterol at a molecular level should be, therefore, a topic of interest. We perform molecular dynamics simulations up to 100 μs for lipid bilayers with various concentrations of cholesterol. We find that cholesterol diffusion in the liquid ordered phase depends on whether it is within leaflets or at the bilayer center, is non-Gaussian for several microseconds, and is enhanced significantly compared to that of lipids. Cholesterol at the bilayer center diffuses fast, while cholesterol in the hydrocarbon region with upright orientation diffuses relatively slowly. Such position-dependent dynamics of cholesterol leads to facilitated and non-Gaussian diffusion.

Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives.

BackgroundStreptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann–Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield.ResultsA region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1′ respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1′-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1′-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1′-hydroxyfilipin I.ConclusionsThis report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0307-4) contains supplementary material, which is available to authorized users.

The effect of cholesterol on the intrinsic rate of lipid flip–flop as measured by sum-frequency vibrational spectroscopy

Cholesterol is a major constituent of biological membranes in mammalian cells. Experiments have shown that cholesterol influences the physical properties of the plasma membrane, such as lateral diffusion and phase equilibrium. In addition to controlling the 2-dimensional phase behaviour and mobility of lipids in membranes, cholesterol has also been implicated in the transbilayer diffusion of lipids across the bilayer. Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the presence of cholesterol using planar supported lipid bilayer (PSLB) model membranes. Asymmetric PSLBs were prepared using the Langmuir-Blodgett (LB) method by placing a perdeuterated lipid analogue in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of DSPC within the membrane by measuring the decay in the CH3 vs intensity at 2875 cm(-1) with time and as a function of temperature. A complete kinetic analysis of DSPC flip-flop and the effect of cholesterol on the DSPC dynamics are presented. An analysis of the kinetic data in the framework of Eyring theory provides important insight into the transition state enthalpy (deltaH(double dagger)), entropy (deltaS(double dagger)) and free energy (deltaG(double dagger)) for this important biological process. In addition, the transmembrane migration of cholesterol molecules was also explored by SFVS. These combined studies are aimed at providing new insight in to the transbilayer migration of phospholipids and cholesterol in biological membranes and the effects cholesterol plays in membrane dynamics.

Cholesterol Tuning of BK Ethanol Response Is Enantioselective, and Is a Function of Accompanying Lipids

In the search to uncover ethanol's molecular mechanisms, the calcium and voltage activated, large conductance potassium channel (BK) has emerged as an important molecule. We examine how cholesterol content in bilayers of 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE)/sphingomyelin (SPM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) affect the function and ethanol sensitivity of BK. In addition, we examine how manipulation of cholesterol in biological membranes modulates ethanol's actions on BK. We report that cholesterol levels regulate the change in BK channel open probability elicited by 50 mM ethanol. Low levels of cholesterol (<20%, molar ratio) supports ethanol activation, while high levels of cholesterol leads to ethanol inhibition of BK. To determine if cholesterol affects BK and its sensitivity to ethanol through a direct cholesterol-protein interaction or via an indirect action on the lipid bilayer, we used the synthetic enantiomer of cholesterol (ent-CHS). We found that 20% and 40% ent-CHS had little effect on the ethanol sensitivity of BK, when compared with the same concentration of nat-CHS. We accessed the effects of ent-CHS and nat-CHS on the molecular organization of DOPE/SPM monolayers at the air/water interface. The isotherm data showed that ent-CHS condensed DOPE/SPM monolayer equivalently to nat-CHS at a 20% concentration, but slightly less at a 40% concentration. Atomic force microscopy (AFM) images of DOPE/SPM membranes in the presence of ent-CHS or nat-CHS prepared with LB technique or vesicle deposition showed no significant difference in topographies, supporting the interpretation that the differences in actions of nat-CHS and ent-CHS on BK channel are not likely from a generalized action on bilayers. We conclude that membrane cholesterol influences ethanol's modulation of BK in a complex manner, including an interaction with the channel protein. Finally, our results suggest that an understanding of membrane protein function and modulation is impossible unless protein and surrounding lipid are considered as a functional unit.

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis : Conformational Flexibility and Histidine Ligation State

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1'-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate.

Determination of cholesterol in erythrocyte membrane

Quantification of cholesterol in biological membranes is an important step toward understanding of metabolism of intracellular cholesterol, composition of cell membrane and plasma lipid profile. The aim of our study was to optimize the method for determination of cholesterol in erythrocyte membrane and then to use this method in the determination of cholesterol concentration in erythrocyte membrane in the studied group of blood samples. Cholesterol in erythrocyte membrane was determined in dry lipid extract of erythrocyte membrane by the enzymatic manual CHOD-PAP method. Cholesterol in erythrocyte membrane and plasma lipid parameters (total cholesterol HDL-cholesterol, LDL-cholesterol and triglycerides) were determined in blood samples of 58 females, obtained by routine health control. Lipid parameters were determined by standard biochemical methods. We examined the relationship between cholesterol in erythrocyte membrane and other plasma lipid parameters as well as other atherogenic risk factors (BMI, blood pressure). Optimization of the method for determination of cholesterol in erythrocyte membrane was based on the observation that primary cholesterol standards prepared by dissolving crystal cholesterol cannot be used due to the interference of the dissolved dry extract in organic solvents with the enzymatic reagent. Commercial standard solutions of cholesterol were used for calibration because they contain detergents for solubilisation of the dry extract in enzymatic reagent. The obtained mean value for cholesterol in erythrocyte membrane, as mmol/L erythrocyte is 4.44 ? 1.019; median 4.65 5-th percentile is 2.70, and 95-th percentile is 6.26. In the examined female group we tested cholesterol concentration in erythrocyte membrane according to age. Two groups were formed (females below and above 50 years) using nonparametric t-test no statistically significant difference was found between these two age groups (p&gt;0.05), while plasma lipid parameters of total cholesterol, triglycerides and LDL-cholesterol were different in the examined groups (p&lt;0.05). By Spearmen nonparametric correlation method we found no statistically significant correlation between cholesterol in erythrocyte membrane and other atherogenic risk factors.

The structural role of cholesterol in biological membranes.

The structural role of cholesterol in biological membranes.

Comparative behavior of sterols in phosphatidylcholine-sterol monolayer films

The ability of sterols other than cholesterol (CHOL) to support membrane functions in membranes that normally contain CHOL as the primary, if not sole, sterol may be due, in part, to how well such sterols can mimic CHOL’s behavior and physical properties in membranes. We compared the mixing properties of CHOL, 7-dehydrocholesterol (7DHC), and desmosterol (DES) in egg phosphatidylcholine-sterol monolayer films containing 10, 20, and 30 mol percent sterol, measuring pressure-area isotherms on a Langmuir–Blodgett trough with the aqueous, buffered subphase maintained at 37°C. Under the conditions employed, the pressure-area isotherms for all three sterols were similar, with 7DHC exhibiting slightly larger molecular areas on the water surface at all compositions. These results are discussed in the context of the ability of sterols such as 7DHC and DES to substitute structurally and functionally for CHOL in biological membranes.

Physical studies of cholesterol-phospholipid interactions

Our understanding of the role of cholesterol in biological membranes requires a detailed knowledge of the forces which mediate cholesterol-lipid interactions and of the lateral organization of cholesterol in natural and model membranes. The recent literature amply illustrates the strength of a multidisciplinary approach in determining the effects of cholesterol on the host bilayer lipids. To confidently apply our current knowledge of cholesterol-lipid interactions and cholesterol organization to natural membranes, however, a systematic analysis of the effects of cholesterol on a much broader range of host lipid bilayers will be required.

Sensitive assay for cholesterol in biological membranes reveals membrane‐specific differences in kinetics of cholesterol oxidase

Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membrane function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measure cholesterol, originally described for whole cells (Heider and Boyett [1978] J. Lipid Res., 19:514-518; Gamble et al. [1978] J. Lipid Res., 19:1068-1070) and serum (Huang et al. [1975] Clin Chem., 21:1605-1608). This method has a detection limit of 0.3 microgram cholesterol. As revealed by comparison with high-performance liquid chromatography, the fluorometric/enzymatic method with biological membranes is accurate (within 4% and 8% for intestinal and hepatic plasma membranes, respectively). The assay may be completed within 3 to 4 hours and requires neither lipid extraction nor chromatographic techniques. Kinetics of the cholesterol oxidase reaction are membrane-specific, and first-order rate constants (k) are positively correlated with membrane order.

Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs. Standardization of incubation conditions.

Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes. It binds to unesterified 3 beta-hydroxy-sterols forming 25 nm complexes which are readily recognized in freeze-fracture replicas. Since most investigations with filipin have been performed in isolated cells (tissue culture, cell suspensions etc.) we have investigated the conditions for reproducible labeling of cholesterol in membranes of parenchymatous organs. Vibratome sections of rat kidney fixed by glutaraldehyde perfusion were incubated in filipin and freeze-fracture replicas were prepared using standard techniques. The concentration of filipin, the thickness of vibratome sections and the incubation time and temperature were varied over a wide range. Optimal results were obtained with 50 micron thick tissue slices incubated in 400 micrograms/ml of filipin for 46 h at room temperature. Under these conditions lysosomes were consistently labeled while mitochondria and the endoplasmatic reticulum were negative. Peroxisomes showed a little or no labeling at all while the nuclear envelope was heavily labeled in some cells being negative in others. The method described here should be useful in investigation of the role of cholesterol in function of biological membranes in parenchymatous organs and compact tissues.

A detailed analysis of the motions of cholesterol in biological membranes by 2H-NMR relaxation

Spin-lattice relaxation, T 1z, measurements of [2,2,3,4,4,6- 2 H 6]cholesterol in model membranes of DMPC were performed as a function of temperature, Larmor frequency and position of labelling in the fused ring system. The results are interpreted according to a hierarchy of motions, such that motion i of correlation time τ i reduces the residual ordering set, characterizing motions i − 1, i − 2, etc.… by the amoount S i = d 00 (2)( β i ) , where β i is the angle between the axes of motional averaging of motions i and i − 1, respectively and d 00 (2) is the Wigner rotation matrix element. The appearance of minima in the temperature dependence of T 1 z for cholesterol, at 46.1 MHz and 30.7 MHz, and the scaling of these T 1 z (min) according to the orientation of each individual C 2H bond with respect to the axis of motional averaging of cholesterol, allows assignment of the sterol axial rotation to the second fastest motion, characterized by a correlation time of 3.2 × 10 −9 s at 25°C and an activation energy of 32 ± 5 kJ·mole −1. The fastest motion of cholesterol in DMPC could be a very rapid libration, ‘wobbling’, which does not contribute significantly to the T 1 z relaxation of cholesterol at physiological temperatures and Larmor frequencies smaller than 50 MHz, but does reduce the ordering of the cholesterol molecule in DMPC from S 0 = 1 to S 1 = 0.8, at 25°C.

Cholesterol oxidase as a structural probe of biological membranes: its application to brush-border membrane

Cholesterol present in intact brush-border membrane vesicles made from rabbit small intestine is a poor substrate for cholesterol oxidase (EC 1.1.3.6, from Nocardia sp. and Nocardia erythropolis). It becomes susceptible to oxidation by the enzyme only after the addition of detergent, e.g., Triton X-100, in quantities sufficient to disrupt the membrane. This is also true for cholesterol present in bilayers of small unilamellar phosphatidylcholine or phosphatidylserine vesicles made by ultrasonication. The data presented here on intestinal brush-border membrane are in good agreement with results reported on other biological membranes, e.g., from erythrocytes and vesicular stomatitis virus, but are somewhat different from those on rat intestinal brush-border membrane. Our results on phospholipid bilayers agree well with published work on model membranes. From the work presented we conclude that, with our present understanding, cholesterol oxidase can hardly be used to probe the distribution of cholesterol in biological membranes. A prerequisite for using the enzyme successfully as such a probe would be the understanding of the factors controlling the interaction of the enzyme with its substrate cholesterol. The question under which conditions cholesterol oxidase could be useful for probing the distribution and preferred location of cholesterol in biological membranes is discussed.

Membrane-associated proteins affect the formation of filipin-cholesterol complexes in viral membranes

The presence and distribution of cholesterol in biological membranes can be visualized by complex formation with the polyene antibiotic filipin after or during fixation with glutaraldehyde. In the envelopes of budding and immature retroviruses no filipin-cholesterol complexes are formed, but in the plasma membrane of host cells and in the envelopes of mature viruses filipin-cholesterol complexes are easily detected. However, after treatment of glutaraldehyde-fixed cells with pepsin, the presence of cholesterol in the envelopes of budding and immature viral particles could also be demonstrated. This indicates that in these structures the reaction of cholesterol with filipin is inhibited by proteins associated with the cholesterol-containing membrane. Treatment of fixed cells with trypsin, and of unfixed cells with cytochalasin B (CB) had no effect on detectability of cholesterol in these structures. On no occasion were cholesterol-filipin complexes formed in coated pits. The present findings call for caution when interpretating data on absence of filipincholesterol complexes in those membrane domains that are characterized by the presence of closely associated proteins.

Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus.

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP-sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.

Stabilizing effect of cholesterol on phosphatidylcholine vesicles observed by ultrasonic velocity measurement

The temperature dependence of the ultrasonic velocity was measured in sonicated vesicles of dipalmitoylphosphatidylcholine by varying the content of cholesterol. When cholesterol is incorporated, an anomalous dip of the ultrasonic velocity gradually smeared out. At the same time, the ultrasonic velocity of the membrane increased remarkably above 30 degrees C due to the increase of the bulk modulus by about 15%. On the other hand, the ultrasonic velocity and the bulk modulus decreased below 30 degrees C. Comparing the cholesterol-incorporated membrane with vesicles of bovine brain sphingomyelin and human erythrocyte membrane, we discuss the role of cholesterol in biological membranes in terms of the stability of the membrane as a barrier.

Studies on lecithin-cholesterol-water interactions by differential scanning calorimetry and X-ray diffraction

Addition of cholesterol to dipalmitoyl- l-lecithin in water lowers the transition temperature between the gel and liquid crystalline phase, and decreases the heat absorbed at the transition. No transition is observed with an equimolar ratio of the lecithin with cholesterol. This ratio corresponds to the maximum amount of cholesterol which can be introduced before cholesterol precipitation occurs. Unsaturated lecithins and the lipid extract of human erythrocyte ghosts exhibit similar behaviour. The cholesterol controls the fluidity of the hydrocarbon chains of the phospholipid by disruption of the crystalline chain lattice of the gel phase, and by inhibiting the flexing of chains in the dispersed liquid crystalline phase. The possible implications of this behaviour to the role of cholesterol in biological membranes are discussed.