Membrane Cholesterol Distribution Research Articles

Unexpected asymmetric distribution of cholesterol and phospholipids in equilibrium model membranes

Lipid compositional asymmetry across the leaflets of the plasma membrane is a ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly – taking many hours or days. However, a lipid like cholesterol – which is the most abundant lipid in the plasma membrane of animal cells – has been harder to pin-point in terms of its favored side. In the present work we show that when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long tail phospholipids organize asymmetrically across the membrane’s leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid – dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) – segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using Magic Angle Spinning (MAS) NMR in combination with Small Angle Neutron Scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol’s flip-flop half-time at 45°C is 10ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol’s flip-flop half-time at 37°C has an upper bound of 10ms.

Influence of Cholesterol Dysregulation and Cell Senescence on Hippocampal Neurovascular Integrity in Alzheimer’s Disease

The neurovascular unit (NVU) is an essential construct of cerebral blood flow control and contains neurons, astrocytes, microglia, smooth muscle, pericytes, and endothelial cells (ECs). This cellular ensemble, among other roles, balances cerebrovascular blood flow control with the metabolic demand of active neurons. Recent studies underscore the pivotal role of the NVU in Alzheimer’s disease (AD), particularly with molecular, cellular, and integrative evidence that vascular dysfunction precedes amyloid-beta (Aβ) plaque deposition. Cerebrovascular ECs are particularly vulnerable in the aging brain while central to regulation of the blood-brain barrier, hemodynamic flow, angiogenesis, and immunity. As a common characteristic of an array of cardiovascular and neurodegenerative diseases, interactions among dyslipidemia and EC health may establish cerebrovascular injury that precedes and accompanies AD. Furthermore, aberrant vascularization in the hippocampus coincides with development of mild cognitive impairment (MCI) and AD due to compromised oxygen and energy provisions in tandem with accumulation of metabolic waste and toxins. Thus, we hypothesize that interaction among cholesterol dysregulation and EC senescence signaling in the hippocampus contribute to progressive AD pathology. To test this hypothesis, we examine transgenic AD mice ( 3xTg-AD) demonstrating MCI (4-5 mo), presence of extracellular Aβ plaques (6-8 mo), and extracellular Aβ plaques with neurofibrillary tau tangles (≥12 mo) compared to young controls (1-2 mo). Our investigation employs an innovative tissue clearing method (iDISCO+), adapted for imaging on a confocal microscope, to probe three-dimensional vascular structure (CD31, Podocalyxin) and senescent cell aggregation (p16, β-Galactosidase) in the hippocampus. Second, Filipin III, selective blood vessel, and senescence markers are imaged in 25 μm coronal sections to examine broader spatial distribution of cholesterol around the hippocampus and cortex. Third, ex vivo blood vessel staining enables an in-depth analysis among membrane cholesterol inclusions and endothelial inward-rectifying K+ (KIR2.1) channels, an ion channel marker particularly sensitive to the distribution of membrane cholesterol while integral to cerebral blood flow control. As cholesterol dysregulation poses a significant risk to the integrity of the NVU including EC senescence and diminishment of KIR2.1 channel function, we anticipate that our results will ultimately highlight its potential role in the early to late stages of AD pathogenesis. This work was supported by the National Institutes of Health (R01AG073230). Imaging was performed in the LLUSM Advanced Imaging and Microscopy Core that is supported by NSF Grant No. MRI-DBI 0923559 and the Loma Linda University School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The asymmetric distribution of cholesterol in model membranes

The asymmetric distribution of cholesterol in model membranes

Distribution of cholesterol in asymmetric membranes driven by composition and differential stress.

Phospholipid asymmetry appears to be an evolutionarily conserved property of eukaryotic plasma membranes, suggesting it is crucial for their structure and function. It is fundamentally a non-equilibrium phenomenon since it can decay by lipids ‘‘flip-flopping’’ between leaflets. Cells must therefore maintain it by a variety of active mechanisms. This is easy for regular phospholipids, whose flip-flop takes hours/days but difficult for cholesterol, where these timescales are in the microsecond range. Cholesterol affects many properties of lipid membranes, from their elastic response to emergence of rafts in multi-component mixtures. It is therefore distressing that we know so little about its trans-membrane partitioning. We propose a theoretical model for the distribution of cholesterol in asymmetric membranes, focusing on the interplay of two dominant effects-difference in lipid composition, which leads to a partitioning bias in cholesterol, and lateral stress arising from crowding one leaflet with respect to the other. To test our theory, we employed coarse grained molecular dynamics simulations performed using the Cooke model. We find that our theory, which accounts for various elastic and thermodynamic drivers, explains the trends in our simulations. We show that the presence of cholesterol does not automatically result in tensionless membranes, even in the absence of preferential partitioning of this species. Additionally, we find that cholesterol's solvation bias can balance phospholipid number asymmetry, which results in strong correlations between cholesterol asymmetry and differential stress. Applying our theory to the most recent data on human RBC membranes suggests that close to 90% of all cholesterol resides in the exoplasmic leaflet, with a large remaining differential stress. This calls into question the notion of rafts being exoplasmic domains connected to liquid-ordered/liquid-disordered phase coexistence and forces us to rethink the common textbook picture of plasma membranes.

Distribution of cholesterol in asymmetric membranes driven by composition and differential stress

Many lipid membranes of eukaryotic cells are asymmetric, which means the two leaflets differ in at least one physical property, such as lipid composition or lateral stress. Maintaining this asymmetry is helped by the fact that ordinary phospholipids rarely transition between leaflets, but cholesterol is an exception: its flip-flop times are in the microsecond range, so that its distribution between leaflets is determined by a chemical equilibrium. In particular, preferential partitioning can draw cholesterol into a more saturated leaflet, and phospholipid number asymmetry can force it out of a compressed leaflet. Combining highly coarse-grained membrane simulations with theoretical modeling, we investigate how these two driving forces play against each other until cholesterol’s chemical potential is equilibrated. The theory includes two coupled elastic sheets and a Flory-Huggins mixing free energy with a χ parameter. We obtain a relationship between χ and the interaction strength between cholesterol and lipids in either of the two leaflets, and we find that it depends, albeit weakly, on lipid number asymmetry. The differential stress measurements under various asymmetry conditions agree with our theoretical predictions. Using the two kinds of asymmetries in combination, we find that it is possible to counteract the phospholipid number bias, and the resultant stress in the membrane, via the control of cholesterol mixing in the leaflets.

Global effects of pharmacologic inhibition of OSBP in human umbilical vein endothelial cells

Oxysterol-binding protein (OSBP) is a cholesterol/PI4P exchanger at contacts of the endoplasmic reticulum (ER) with trans-Golgi network (TGN) and endosomes. Several central endothelial cell (EC) functions depend on adequate cholesterol distribution in cellular membranes. Here we elucidated the effects of pharmacologic OSBP inhibition on the lipidome and transcriptome of human umbilical vein endothelial cells (HUVECs). OSBP was inhibited for 24 h with 25 nM Schweinfurthin G (SWG) or Orsaponin (OSW-1), followed by analyses of cellular cholesterol, 27-hydroxy-cholesterol, and triacylglycerol concentration, phosphatidylserine synthesis rate, the lipidome, as well as lipid droplet staining and western analysis of OSBP protein. Next-generation RNA sequencing of the SWG-treated and control HUVECs and angiogenesis assays were performed. Protein-normalized lipidomes of the inhibitor-treated cells revealed decreases in glycerophospholipids, the most pronounced effect being on phosphatidylserines and the rate of their synthesis, as well as increases in cholesteryl esters, triacylglycerols and lipid droplet number. Transcriptome analysis of SWG-treated cells suggested ER stress responses apparently caused by disturbed cholesterol exit from the ER, as indicated by suppression of cholesterol biosynthetic genes. OSBP was associated with the TGN in the absence of inhibitors and disappeared therefrom in inhibitor-treated cells in a time-dependent manner, coinciding with OSBP reduction on western blots. Prolonged treatment with SWG or OSW-1 inhibited angiogenesis in vitro. To conclude, inhibition of OSBP in primary endothelial cells induced multiple effects on the lipidome, transcriptome changes suggesting ER stress, and disruption of in vitro angiogenic capacity. Thus, OSBP is a crucial regulator of EC lipid homeostasis and angiogenic capacity.

Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction

Rationale for different formulations of omega-3 fatty acids leading to differences in residual cardiovascular risk reduction

Joint effect of differential stress and compositional asymmetry on cholesterol distribution in lipid membranes

Many lipid membranes of eukaryotic cells are asymmetric in nature, which means the two leaflets differ in at least one physical property, such as lipid composition or lateral stress. Maintaining this asymmetry is helped by the fact that ordinary phospholipids rarely transition between leaflets, but cholesterol is an exception: its flip-flop times are in the microsecond range, so that its distribution between leaflets is determined by a chemical equilibrium. In particular, preferential partitioning can draw cholesterol into a more saturated leaflet, and differential stress can force it out of a compressed leaflet.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. Statement of significanceA great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

SR‐BI & PCPE2: Friends or Foes in Adipocyte Cholesterol Transport?

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Specialized cells called adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity and organ failure. Since adipocytes have a reduced ability to synthesize their own cholesterol, they rely heavily on circulating lipoproteins for their cholesterol supply to support membrane expansion. Scavenger receptor BI (SR-BI) is believed to be important in the adipocyte cholesterol delivery process, as this integral membrane protein is the primary receptor for high-density lipoproteins (HDL) and regulates bi-directional cholesterol transport. Collaborative studies demonstrated that SR-BI-mediated HDL-cholesteryl ester (HDL-CE) uptake in the liver requires the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2). These published studies have prompted a novel hypothesis that PCPE2 facilitates SR-BI's cholesterol transport functions to regulate adipocyte cholesterol homeostasis. To test our hypothesis, we have replicated an adipocyte model system where ear mesenchymal stem cells (EMSCs) isolated from the outer ears of mice can be differentiated into adipocytes and further used to study cholesterol transport in vitro. We validated our adipocyte model system by Oil Red O staining, mRNA expression of adipogenesis markers (PPARγ, LPL, FABP4, cEBP2α), and adiponectin secretion over the 9-day post-differentiation period in wild-type (WT), SR-BI knock-out (SR-BI-/-), and PCPE2-/- adipocytes. All three genotypes showed increased Oil Red O staining, increased mRNA expression of adipogenesis markers, and increased adiponectin secretion over the post-differentiation period. Tandem mass spectrometry analysis showed that SR-BI-/- and PCPE2-/- adipocytes had half the amount of cholesteryl esters compared to WT adipocytes, indicating that there may be disrupted cholesterol transport in the SR-BI-/- and PCPE2-/- adipocytes. Early pilot data indicate no major changes in cholesterol efflux from [3H]-cholesterol-labeled cells to HDL between WT, SR-BI-/-, and PCPE2-/- adipocytes. Experiments to assess HDL binding and HDL-CE uptake using double radiolabeled HDL particles ([125I]-HDL-[3H]-CHE) are currently ongoing. Interestingly, using an assay that assesses cholesterol distribution in membranes, we were able to show that loss of PCPE2 causes changes in cholesterol distribution within the membrane, suggesting that it may play a role in lipid raft formation. All together, we are confident that we have developed a unique in vitro adipocyte model system that can help us assess how PCPE2 may impact SR-BI-mediated cholesterol delivery.

Developing a Purification System to Assess SR‐BI Structure and Function

High density lipoprotein (HDL) is considered anti-atherogenic due to its ability to remove cholesterol from the periphery and deliver it to the liver via its receptor, scavenger receptor BI (SR-BI). The interaction between HDL and SR-BI is the most important mechanism to remove bodily cholesterol. SR-BI is crucial for maintaining lipid homeostasis, as humans with SR-BI mutations display impaired cholesterol clearance and an elevated risk of cardiovascular disease (CVD). As there currently exists no full-length structure for any of the Class B scavenger receptors, the goal of this study is to resolve a high-resolution structure of functional full-length SR-BI. This structure would be a pivotal addition to the scientific toolbox employed to uncover questions of cholesterol transport in the context of cardiovascular disease. As first steps in the purification process, suspended sf9 cells were infected with baculovirus containing human SR-BI-encoding bacmid. After 96 hours post-infection, cells expressing SR-BI were centrifuged, lysed, and membrane proteins were solubilized into mixed micelles. SR-BI within micelles was then purified through a series of steps that included affinity column purification, desalting, and concentration. Upon successful purification, verifying the expression of functional SR-BI at the cellular surface was a critical step. To assess SR-BI function, sf9 cells were infected with baculovirus containing SR-BI encoding bacmid or empty vector and plated. Expression of human full-length SR-BI significantly increased HDL cellular association and uptake of HDL-cholesteryl esters compared to uninfected and empty vector infected cells. Additionally, free cholesterol efflux and membrane cholesterol distribution were increased with SR-BI expression. Lastly, SR-BI expressed in sf9 cells maintained its ability to form higher order oligomers, suggesting synthesized receptor displays its expected functions. This purification protocol and functional assays represent the first steps in resolving the first high-resolution structure of human full-length SR-BI and provide promise for delineating the ways in which the HDL/SR-BI relationship allows for efficient cholesterol clearance.

Platycodin D, a natural component of Platycodon grandiflorum, prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann–Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.

Distribution of Cholesterol in Asymmetric Membranes Driven by Composition and Differential Stress

Many biological membranes, in particular plasma membranes of eukaryotic cells, are asymmetric, which means that their two leaflets differ from one another. This asymmetry can arise in a number of ways, and we focus on the following two: a difference in lipid type, and a difference in lateral stress, arising from overfilling one leaflet with respect to the other. The two ways presuppose that lipids, once put in a given leaflet, stay in that leaflet. This is true for most phospholipids, but not for cholesterol, which transitions rapidly enough between leaflets so that its distribution is determined by chemical equilibrium. Given that compressive stress presumably excludes cholesterol from a leaflet, while preferential partitioning can draw it into a leaflet, this poses the question of how these two types of asymmetry cooperatively affect the distribution of cholesterol. Using a highly coarse-grained membrane model, we investigate how these two driving forces play against each other until cholesterol's chemical potential is equilibrated. We also model this system theoretically using two coupled elastic sheets whose composition degrees of freedom are described by a simple lattice gas model with a non-ideal mixing parameter χ. We find that the results from the simulations agree with the predictions if we allow for just one free parameter, which empirically determines how χ relates to the non-ideal interaction energy between cholesterol and lipids in either of the two leaflets. We anticipate that our simple model can help shed light on the longstanding puzzle of how cholesterol distributes in cellular plasma membranes, and whether the resulting equilibrium puts them under differential stress.

The Impact of TiO <sub>2</sub> Nanoparticle Exposure on Transmembrane Cholesterol Transport and Enhanced Bacterial Infectivity

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a new live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 12.5(2.2)-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 50.8(9.5) times higher, while the total cholesterol content increased only 20.5(2.4)%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity.

Proline residues in scavenger receptor-BI's C-terminal region support efficient cholesterol transport.

High-density lipoproteins (HDLs) facilitate reverse cholesterol transport, a process in which HDL removes cholesterol from circulation and carries it to the liver for biliary excretion. Reverse cholesterol transport is also facilitated by HDL's high-affinity receptor, scavenger receptor-BI (SR-BI), by mechanisms that are not fully understood. To improve our understanding of SR-BI function, we previously solved the NMR (nuclear magnetic resonance) structure of a peptide encompassing amino acids 405–475 of SR-BI. This segment of SR-BI, that includes the functionally critical C-terminal transmembrane domain and part of the extracellular domain, also contains four conserved proline (Pro) residues. We hypothesized that these proline residues support SR-BI in a conformation that allows for efficient cholesterol transport. To test this, we generated individual Pro-to-alanine mutations in full-length SR-BI and transiently expressed the mutant receptors in COS-7 cells to measure the effects on SR-BI-mediated cholesterol transport functions. Our findings reveal that HDL cell association and uptake of HDL-cholesteryl esters are impaired by mutation of Pro-412, Pro-438, or the transmembrane proline kink residue (Pro-459). In addition, SR-BI-mediated cholesterol efflux and membrane cholesterol distribution are impaired by mutation of Pro-412 or Pro-438, indicating that these residues are essential for a fully functional SR-BI receptor. Furthermore, we demonstrate that Pro-408 is necessary for proper SR-BI expression, but mutation of Pro-408 does not cause SR-BI to become misfolded or rapidly degraded by the proteasome or the lysosome. We conclude that key proline residues play an important role in SR-BI function by allowing for the efficient transport of cholesterol between cells and HDL.

Abstract 563: Working Out the Kinks of SR-BI-mediated Cholesterol Transport

High density lipoproteins (HDL) facilitate reverse cholesterol transport (RCT), the process by which cholesterol effluxed from peripheral tissues is delivered via selective uptake to the liver for excretion. These processes are mediated by HDL's high-affinity receptor, scavenger receptor BI (SR-BI). SR-BI is an 82 kDa glycoprotein with two transmembrane domains (TMD), two short cytoplasmic N/C-terminal domains, and a large extracellular domain. Our laboratory solved the NMR structure of the C-terminal TMD and nearby extracellular domain. This peptide contains four conserved proline residues, an amino acid that confers structural flexibility. In our structure, P408 and P412 are in an extracellular alpha-helix, P438 lies at a helix-loop junction, and P459 introduces a kink into the TMD helix. We hypothesized that these proline residues support SR-BI-mediated cholesterol transport. To test this hypothesis, we mutated each proline to alanine and transiently expressed wild-type (WT), P408A-, P412A-, P438A-, or P459A-SR-BI in COS-7 cells. Total lysate and cell surface expression of SR-BI was examined by immunoblot analysis and flow cytometry, respectively. All mutants expressed on the cell surface, except P408A-SR-BI, which was absent from whole cell lysates. P412A- and P438A-SR-BI were significantly impaired in their ability to efflux cell-associated [ 3 H]cholesterol to HDL, as compared to WT-SR-BI. Likewise, cellular binding of [ 125 I]HDL and selective uptake of [ 3 H]cholesteryl oleyl ether were reduced with P412A- and P438A-SR-BI. We assessed SR-BI’s ability to modulate membrane cholesterol pools by cholesterol oxidase treatment. SR-BI enhances the accessibility of cholesterol to the exogenous enzyme, and a proportional increase in cholestenone is detected by thin layer chromatography. Cholestenone was increased with WT-SR-BI but not P412A- or P438A-SR-BI, suggesting defective membrane cholesterol distribution. Taken together, key proline residues in the near-C-TMD extracellular domain support efficient HDL binding, selective uptake, cholesterol efflux, and membrane cholesterol organization. These critical residues appear to support SR-BI in a conformation that allows for efficient RCT.

Control of insulin granule formation and function by the ABC transporters ABCG1 and ABCA1 and by oxysterol binding protein OSBP.

In pancreatic β-cells, insulin granule membranes are enriched in cholesterol and are both recycled and newly generated. Cholesterol’s role in supporting granule membrane formation and function is poorly understood. ATP binding cassette transporters ABCG1 and ABCA1 regulate intracellular cholesterol and are important for insulin secretion. RNAi inter­ference–induced depletion in cultured pancreatic β-cells shows that ABCG1 is needed to stabilize newly made insulin granules against lysosomal degradation; ABCA1 is also involved but to a lesser extent. Both transporters are also required for optimum glucose-stimulated insulin secretion, likely via complementary roles. Exogenous cholesterol addition rescues knockdown-induced granule loss (ABCG1) and reduced secretion (both transporters). Another cholesterol transport protein, oxysterol binding protein (OSBP), appears to act proximally as a source of endogenous cholesterol for granule formation. Its knockdown caused similar defective stability of young granules and glucose-stimulated insulin secretion, neither of which were rescued with exogenous cholesterol. Dual knockdowns of OSBP and ABC transporters support their serial function in supplying and concentrating cholesterol for granule formation. OSBP knockdown also decreased proinsulin synthesis consistent with a proximal endoplasmic reticulum defect. Thus, membrane cholesterol distribution contributes to insulin homeostasis at production, packaging, and export levels through the actions of OSBP and ABCs G1 and A1.

Investigating the Transbilayer Distribution of Cholesterol in Asymmetric Unilamellar Vesicles using Small-Angle Scattering

Cholesterol is the most abundant component of eukaryotic plasma membranes (PMs), accounting for 30-50% of the total lipid. In addition to modulating the structural and mechanical properties of lipid bilayers, cholesterol promotes the formation of ordered+disordered fluid phase separation in model membranes, and may similarly contribute to microdomain or “raft” formation in the PM. A major unresolved question is how PM properties and functions are influenced by the asymmetric transbilayer distribution of lipids, including cholesterol. While the distribution of most phospholipids and sphingolipids is known, determining the average transbilayer distribution of cholesterol has been challenging due to its rapid translocation between the PM leaflets. Previous attempts to probe the cholesterol distribution in cell membranes using fluorescence-based methods have yielded conflicting results. Moreover, studies using model vesicles have been limited due to the difficulty of preparing asymmetric liposomes. Making use of recent advancements enabling the production of asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange, we prepared cholesterol-containing aLUVs in which one leaflet was enriched in egg sphingomyelin (eSM), and the other in phosphatidylethanolamine (POPE). We then used small-angle neutron and x-ray scattering combined with isotopic labeling strategies to probe the average cholesterol location within these bilayers. An analysis of scattering data, together with complementary Molecular Dynamics simulations, provides insight into the transbilayer distribution of cholesterol in asymmetric membranes.

Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces.

Cholesterol Rich Domains Identified in Unilamellar Supported Biomimetic Membranes via Nano-Viscosity Measurements

Understanding the distribution of cholesterol in phospholipid membranes is of key importance in membrane biophysics, primarily since cholesterol enriched regions, rafts, are known to play a special role in protein function. In this work, quartz crystal microbalance with dissipation (QCM)-based viscosity measurements were used to study cholesterol-induced domain formation in partially suspended single bilayer membranes. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its mixtures with different amounts of cholesterol were studied. QCM temperature ramping experiments identified domains of different phase transition temperatures in the mixed membranes. The phase transition of DMPC shifted from 23.4 °C toward lower temperatures with increasing cholesterol content. A second, continuous but much broader, transition peak has been observed for the DMPC: cholesterol mixtures suggest that a separate cholesterol rich domain coexists with the DMPC rich domain. Importantly, the sharp DMC phase transition peak gradually diminished and eventually disappeared over 15% cholesterol content, suggesting that the cholesterol rich domain has a definite stoichiometry and once this cholesterol concentration is reached the DMPC-rich domain disappears. DSC control experiments do not show the second domain, suggesting that the phase separation only happens in nontensioned (flat) membranes.