Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural analysis of sterol distributions in the plasma membrane of living cells

Although plasma membrane (PM) cholesterol-rich and -poor domains have been isolated by subcellular fractionation, the real-time arrangement of cholesterol in such domains in living cells is still unclear. Therefore, dehydroergosterol (DHE), a naturally occurring fluorescent sterol, was incorporated into cultured L-cell fibroblasts. Two PM markers, the enhanced cyan fluorescent protein (ECFP-Mem) and 3'-dioctadecyloxacarbocyanine perchlorate [DiOC(18)(3)], were used to distinguish DHE localized at the PM of living cells. Spatial enrichment of DHE in the PM of living cells was visualized in real time by multiphoton laser scanning microscopy (MPLSM). Quantitative models and image-processing techniques were developed for statistical analysis of the distribution of DHE within the PM. The PM was resolved from the cytoplasm in a two-step process, and a smooth trajectory reference of the PM was refined by statistical regression and moments-based techniques. Thus, DHE intensities over the PM were measured following the major DHE intensity distributions. Spatial distributions of DHE within the PM were examined by a statistical inference technique, complete spatial randomness (CSR). For PM regions densely populated with DHE, the distributions of DHE exhibited statistical arrangements that were not spatial random (i.e., homogeneous Poisson process) or regular but, instead, exhibited strong cluster patterns. In effect, real-time MPLSM imaging data for the first time demonstrated that sterol enrichment occurred in clustered regions in the PM, consistent with the existence of cholesterol-rich domains in the plasma membrane of living cells.     

Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages

Accumulation of excess non-esterified free cholesterol (FC) in macrophages is a key factor in macrophage death during late stages of atheroslerosis. Raising FC content in macrophages has been shown to trigger Rac activation and actin polymerisation and to inhibit cell migration. Here, the plasma membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments. DHE's surface distribution matched exactly large ruffles and membrane protrusions which were pronounced in FC-loaded cells. Plasma membrane blebs, however, formed in FC-loaded J774 cells had a homogenous staining along the membrane bilayer at 20 degrees C. The results show that even in FC-loaded cells with increased membrane cholesterol content, sterols do not form a separate phase in the plasma membrane.     

Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells

Transport of the fluorescent cholesterol analog dehydroergosterol (DHE) from the plasma membrane was studied in J774 macrophages (Mphis) with normal and elevated cholesterol content. Cells were labeled with DHE bound to methyl-beta-cyclodextrin. In J774, Mphis with normal cholesterol, intracellular DHE became enriched in recycling endosomes, but was not highly concentrated in the trans-Golgi network or late endosomes and lysosomes. After raising cellular cholesterol by incubation with acetylated low-density lipoprotein (AcLDL), DHE was transported to lipid droplets, and less sterol was found in recycling endosomes. Transport of DHE to droplets was very rapid (t1/2 = 1.5 min after photobleaching) and did not require metabolic energy. In cholesterol-loaded J774 Mphis, the initial fraction of DHE in the plasma membrane was reduced, and rapid DHE efflux from the plasma membrane to intracellular organelles was observed. This rapid sterol transport was not related to plasma membrane vesiculation, as DHE did not become enriched in endocytic vesicles formed after sphingomyelinase C treatment of cells. When cells were incubated with DHE ester incorporated into AcLDL, fluorescence of the sterol was first found in punctate endosomes. After a chase, this DHE colocalized with transferrin in a distribution similar to cells labeled with DHE delivered by methyl-beta-cyclodextrin. Our results indicate that elevation of sterol levels in Mphis enhances transport of sterol from the plasma membrane by a non-vesicular pathway.     

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps – Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.     

Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells

We studied the transport of the fluorescent cholesterol analog dehydroergosterol (DHE) in polarized HepG2 human hepatoma cells. DHE delivered via methyl-beta-cyclodextrin was delivered to both the apical and basolateral membranes and became concentrated in the apical membrane within 1 min. Intracellular DHE was targeted mainly to vesicles of the subapical compartment or apical recycling compartment (SAC/ARC), where it colocalized with fluorescent transferrin and fluorescent analogs of phosphatidylcholine and sphingomyelin. In contrast, transport of DHE from the plasma membrane to the trans-Golgi network was found to be very low. Vesicles containing DHE traversed the cells in both directions, but vesicular export of DHE from the SAC/ARC to the plasma membrane domains was low. Disruption of the microtubule cytoskeleton disturbed vesicular transport of DHE but not its enrichment in the apical (canalicular) membrane. Transport of DHE to the canalicular membrane after photobleaching was very rapid (t(12) = 1.6 min) and was largely ATP-independent in contrast to enrichment of DHE in the SAC/ARC. Release of DHE from the canalicular membrane was also ATP-independent but slower than the enrichment of sterol in the biliary canaliculus (t(12) = 5.4 min). Canalicular DHE could completely redistribute to the basolateral plasma membrane but could not transfer from one cell to the other cell of an HepG2 couplet. We conclude that sterol shuttles rapidly among the plasma membrane domains and other membrane organelles and that this nonvesicular pathway includes fast transbilayer migration.     

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol‐auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE‐containing cells were exposed to membrane‐impermeant collisional quenchers (spin‐labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass‐bead lysis or repeated freeze‐thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.      

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehydroergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominant-negative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference of the sterol probe and causes some differential targeting of BCh2 and DHE in cells with high fat content.     

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence.

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Fluorescent sterols as tools in membrane biophysics and cell biology

Cholesterol is an important constituent of cellular membranes playing a fundamental role in many biological processes. This sterol affects membrane permeability, lateral lipid organization, signal transduction and membrane trafficking. Intracellular sterol transport modes and pathways as well as the regulation of sterol metabolism and disposition in various tissues are areas of intense research. Progress is intimately linked to development and use of appropriate analogs, which closely mimic the properties of cholesterol while allowing to be detected by spectroscopic or microscopic methods. This review provides an overview of various fluorescent sterols used in membrane biophysics and cell biology including analogs of cholesterol and cholesteryl esters. Attention is paid to the natural fluorescent sterol dehydroergosterol (DHE). A survey of the many applications of DHE in biological research is presented. Special emphasis is on recent developments in fluorescence microscopy instrumentation to visualize DHE as an intrinsically fluorescent analog of cholesterol in living cells.     

Distribution of sterol-specific complexes in a continually shearing region of a plasma membrane and at procaryotic-eucaryotic cell junctions

A narrow zone of plasma membrane between the head and body of a protozoan from termites undergoes continual in-plane shear because the head rotates continuously in the same direction relative to the cell body (Tamm, S.L., and S. Tamm, 1974, Proc. Natl. Acad. Sci. USA 71:4589- 4593). Using filipin and digitonin as cytochemical probes for cholesterol and related 3-beta-hydroxysterols, we found a high level of sterol-specific complexes, visible as membrane lesions in thin sections, in both shearing and nonshearing regions of the membrane, indicating no difference in sterol content. This confirmed previous observations that any region of the fluid membrane can undergo shear, but that this occurs only at certain locations due to cell geometry and proximity to rotating cytoskeletal structures. Filipin and digitonin did not disrupt the plasma membrane at the junctions with ectosymbiotic rod and fusiform bacteria (i.e., membrane pockets and ridges). However, pepsin degradation of dense material coating the junctional membranes resulted in a positive response of these regions to filipin. Fluorescence microscopy revealed a bright halo around each rod bacterium, due to filipin-sterol binding in the sides of the membrane pockets, but no fluorescence at the bottom of the pockets; the same fluorescence pattern was found in pepsin-treated cells despite the presence of sterols throughout the pocket membrane, as shown by electron microscopy. These findings indicate that (a) regional constraints may restrict the ability of filipin to interact with sterols or form visible membrane lesions, and (b) a negative response to filipin, assayed by either electron or fluorescence microscopy, is not sufficient to demonstrate low membrane sterol concentration, particularly in membrane domains characterized by closely associated proteins.     

Role of sterol superlattice in free radical-induced sterol oxidation in lipid membranes

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.     

Regulation of transbilayer distribution of a fluorescent sterol in tumor cell plasma membranes

The lipid composition and transbilayer distribution of plasma membrane isolated from primary tumor (L-929, LM, A-9 and C3H) and nine metastatic cell lines cultured under identical conditions was examined. Cultured primary tumor and metastatic cells differed two-fold in sterol/phospholipid molar ratios. There was a direct correlation between plasma membrane anionic phospholipid (phosphatidylinositol and phosphatidylserine) content and plasma membrane sterol/phospholipid ratio. This finding may bear on the possible link between oncogenes and inositol lipids. The fluorescent sterol, dehydroergosterol, was incorporated into primary tumor and metastatic cell lines. Selective quenching of outer monolayer fluorescence by covalently linked trinitrophenyl groups demonstrated an asymmetric transbilayer distribution of sterol in the plasma membranes. The inner monolayer of the plasma membranes from both cultured primary and metastatic tumor cells was enriched in sterol as compared with the outer monolayer. Consistent with this, the inner monolayer was distinctly more rigid as determined by the limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Dehydroergosterol fluorescence was temperature dependent and sensitive to lateral phase separations in phosphatidylcholine vesicles and in LM cell plasma membranes. Dehydroergosterol detected phase separations near 24 degrees C in the outer monolayer and at 21 degrees C and 37 degrees C in the inner monolayer of LM plasma membranes. Yet, no change in transbilayer sterol distribution was detected in ascending or descending temperature scans between 4 and 45 degrees C. Alterations in plasma membrane phospholipid polar head group composition by choline analogues (N,N-dimethylethanolamine, N-methylethanolamine, and ethanolamine) also did not perturb transbilayer sterol asymmetry. Treatment with phenobarbital or prilocaine, drugs that selectively fluidize the outer and inner monolayer of LM plasma membranes, respectively, did not change dehydroergosterol transbilayer distribution.     

Orthogonal arrays of particles in non-pigmented cells of rat ciliary epithelium: Relation to distribution of filipin- and digitonin-induced alterations of the basolateral membrane

Summary It has been suggested that orthogonal arrays of particles may increase the rigidity of plasma membrane, as does cholesterol. Therefore, using freeze-fractured non- pigmented ciliary epithelium, the distribution of such arrays was compared to the distribution of membrane deformations induced by the sterol-probes filipin and digitonin in different domains of the basolateral plasma membrane. The distribution of orthogonal arrays of particles was homogeneous between different regions of the basolateral membrane of the non-pigmented ciliary epithelium, while the number of filipin-induced alterations was nearly 4 times higher in the membrane domains not in contact with the basal lamina than in domains in contact with it. Contrary to the homogeneous distribution of arrays, digitonin-induced deformations also differed markedly in these two basolateral membrane domains. Considering that a marked positive response to sterol probes implies a high sterol content, we conclude that orthogonal arrays of particles can occur in plasma membrane regions well-provided with cholesterol and not in direct contact with the basal lamina. Other possible roles of these arrays are discussed.This paper was presented in part at the ARVO meeting, April– May 1986, Sarasota, USA     

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1(ts)) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.     

Structural changes in BHK cell plasma membrane caused by the binding of vesicular stomatitis virus

Spin label electron spin resonance techniques using a nitroxide derivative of stearic acid were used to detect changes in plasma membrane structure caused by the binding of vesicular stomatitis virus (VSV) to cell plasma membranes of intact BHK-21 cells. The results indicate that binding of VSV to cell surface receptors causes an increase in the observed rigidity of the plasma membrane lipid bilayer. This change in membrane structure, which appears to be caused by the cross-linking of receptors in the plane of the plasma membrane, could be prevented by treating the cells with colchicine before addition of virus and could be reversed by treating the cells with colchicine after addition of virus. Cells treated with a monovalent, water-soluble derivative of VSV G-protein (Gs) did not show an increase in plasma membrane bilayer rigidity. However, addition of anti-VSV G-protein immunoglobulin G to cells pretreated with G8 caused an increase in plasma membrane bilayer rigidity. This increased rigidity could also be reversed by the addition of colchicine. Fluorescence microscopy was used to determine the distribution of fluorescein-labeled VSV particles on the cell surface after addition of virus. Approximately 30 min after addition of virus, discrete areas on the cell surface showed fluorescent staining, which coalesced to apical regions of the cell after approximately 40 min.     

Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells

We analyzed the intracellular transport of HDL and its associated free sterol in polarized human hepatoma HepG2 cells. Using pulse-chase protocols, we demonstrated that HDL labeled with Alexa 488 at the apolipoprotein (Alexa 488-HDL) was internalized by a scavenger receptor class B type I (SR-BI)-dependent process at the basolateral membrane and became enriched in a subapical/apical recycling compartment. Most Alexa 488-HDL was rapidly recycled to the basolateral cell surface and released from cells. Within 30 min of chase at 37 degrees C, approximately 3% of the initial cell-associated Alexa 488-HDL accumulated in the biliary canaliculus (BC) formed at the apical pole of polarized HepG2 cells. Even less Alexa 488-HDL was transported to late endosomes or lysosomes. The fluorescent cholesterol analog dehydroergosterol (DHE) incorporated into Alexa 488-HDL was delivered to the BC within a few minutes, independent of the labeled apolipoprotein. This transport did not require metabolic energy and could be blocked by antibodies against SR-BI. The fraction of cell-associated DHE transported to the BC was comparable when cells were incubated with either Alexa 488-HDL containing DHE or with DHE bound to methyl-beta-cyclodextrin. We conclude that rapid, nonvesicular transport of sterol to the BC and efficient recycling of HDL particles underlies the selective sorting of sterol from HDLs in hepatocytes.     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Arv1 Regulates PM and ER Membrane Structure and Homeostasis But is Dispensable for Intracellular Sterol Transport

The pan‐eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild‐type and Arv1‐deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild‐type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C‐terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail‐anchored proteins involved in membrane homoeostasis .