The Use of DQ-BSA to Monitor the Turnover of Autophagy-Associated Cargo.

There is increasing evidence documenting the critical role played by autophagic and autophagy-associated processes in maintaining cell homeostasis and overall systemic health. Autophagy is considered a degradative as well as a recycling pathway that relies on encapsulated intracellular components trafficking to and fusing with degradative compartments, including lysosomes. In this chapter, we describe the use of DQ™-BSA to study autophagosome-lysosome fusion as well as a means by which to analyze hybrid autophagic pathways. Such noncanonical pathways include LC3-associated phagocytosis, better known as LAP. Both autophagosomes and LAPosomes (LC3-associated phagosomes) deliver cargo for degradation. The use of fluorescent DQ™-BSA in conjugation with autophagic makers and biomarkers of hybrid autophagy offers a reliable technique to monitor the formation of autolysosomes and LAPo-lysosomes in both fixed- and live-cell studies. This technique relies on cleavage of the self-quenched DQ™ Green- or DQ™ Red BSA protease substrates in an acidic compartment to generate a highly fluorescent product.

Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.

Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells.

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate.

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity and depletes lymphoid cells of PIP3. Hence we propose that Cdt toxicity results from depletion of this signaling lipid and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signaling. We have now focused on the relationship between cell susceptibility to CdtB and differences in the status of baseline PIP3 levels. Our studies demonstrate that the baseline level of PIP3, and likely the dependence of cells on steady-state activity of the PI-3K signaling pathway for growth and survival, influence cell susceptibility to the toxic effects of Cdt. Jurkat cells with known defects in both PIP3 degradative enzymes, PTEN and SHIP1, not only contain high baseline levels of PIP3, pAkt, and pGSK3ß, but also exhibit high sensitivity to Cdt. In contrast, HUT78 cells, with no known defects in this pathway, contain low levels of PIP3, pAkt, and pGSK3ß and likely minimal dependence on the PI-3K signaling pathway for growth and survival, and exhibit reduced susceptibility to Cdt. These differences in susceptibility to Cdt cannot be explained by differential toxin binding or internalization of the active subunit. Indeed, we now demonstrate that Jurkat and HUT78 cells bind toxin at comparable levels and internalize relatively equal amounts of CdtB. The relevance of these observations to the mode of action of Cdt and its potential role as a virulence factor is discussed.

Variants of Porphyromonas gingivalis lipopolysaccharide alter lipidation of autophagic protein, microtubule-associated protein 1 light chain 3, LC3.

Porphyromonas gingivalis often subverts host cell autophagic processes for its own survival. Our previous studies document the association of the cargo sorting protein, melanoregulin (MREG), with its binding partner, the autophagic protein, microtubule-associated protein 1 light chain 3 (LC3) in macrophages incubated with P. gingivalis (strain 33277). Differences in the lipid A moiety of lipopolysaccharide (LPS) affect the virulence of P. gingivalis; penta-acylated LPS1690 is a weak Toll-like receptor 4 agonist compared with Escherichia coli LPS, whereas tetra-acylated LPS1435/1449 acts as an LPS1690 antagonist. To determine how P. gingivalis LPS1690 affects autophagy we assessed LC3-dependent and MREG-dependent processes in green fluorescent protein (GFP)-LC3-expressing Saos-2 cells. LPS1690 stimulated the formation of very large LC3-positive vacuoles and MREG puncta. This LPS1690 -mediated LC3 lipidation decreased in the presence of LPS1435/1449 . When Saos-2 cells were incubated with P. gingivalis the bacteria internalized but did not traffic to GFP-LC3-positive structures. Nevertheless, increases in LC3 lipidation and MREG puncta were observed. Collectively, these results suggest that P. gingivalis internalization is not necessary for LC3 lipidation. Primary human gingival epithelial cells isolated from patients with periodontitis showed both LC3II and MREG puncta whereas cells from disease-free individuals exhibited little co-localization of these two proteins. These results suggest that the prevalence of a particular LPS moiety may modulate the degradative capacity of host cells, so influencing bacterial survival.

Pulse pressure variation as a predictor of fluid responsiveness in mechanically ventilated patients with spontaneous breathing activity: a pragmatic observational study.

INTRODUCTION: Pulse pressure variation predicts fluid responsiveness in mechanically ventilated patients passively adapted to the ventilator. Its usefulness in actively breathing ventilated patients was examined only by few studies with potential methodological shortcomings. This study sought to describe the performance of pulse pressure variation as a predictor of fluid responsiveness in hypotensive critically ill patients who trigger the ventilator. METHODS: We studied forty two hypotensive, mechanically ventilated patients with documented spontaneous breathing activity in whom a fluid challenge was deemed necessary by the attending physician. All patients were ventilated with a Maquet Servo-i Ventilator in different ventilatory modes with a flow-regulated inspiratory trigger set on position 4. Pulse pressure variation, mean and systolic arterial pressure were observed before and after the fluid challenge, which consisted in the intravenous administration of a 250 ml bolus of 6% hetastarch. Fluid responsiveness was defined as a more than 15% increase in arterial pressure after volume expansion. RESULTS: The area under the receiver operator characteristic curve for pulse pressure variation was 0.87 (95% CI 0.74 -0.99; p<0.0001) and the grey zone limits were 10% and 15%. Pulse pressure variation was correlated with increase in systolic arterial pressure (r2=0.32; p<0.001) and mean arterial pressure (r2=0.10; p=0.037). CONCLUSIONS: Pulse pressure variation predicts fluid responsiveness in patients who actively interact with a Servo-i ventilator with a flow-regulated inspiratory trigger set on position 4.

Membrane association and destabilization by Aggregatibacter actinomycetemcomitans leukotoxin requires changes in secondary structures.

Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains.

Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27-29%) and palmitolyl (C16:1 cis Δ9, 43-44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12-14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys(562) and Lys(687) are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coliα-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys(562) and Lys(687) with Arg blocks acylation, resulting in a >75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these post-translational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity.

Peripherin-2: an intracellular analogy to viral fusion proteins.

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.

Calcium-dependent association of calmodulin with the C-terminal domain of the tetraspanin protein peripherin/rds.

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.

A peptide analogue to a fusion domain within photoreceptor peripherin/rds promotes membrane adhesion and depolarization.

Photoreceptor peripherin/rds promotes membrane fusion, through a putative fusion domain located within the C-terminus (Boesze-Battaglia et al., Biochemistry 37 (1998) 9477-9487). A peptide analogue to this region, PP-5, competitively inhibits peripherin/rds mediated fusion in a cell free assay system. To characterize how this region is involved in the fusion process we investigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membrane depolarization was measured as the collapse of a valinomycin induced K(+) diffusion potential in model membranes, using a potential sensitive fluorescent probe, diS-C(2)-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic alpha-helix. Therefore, the requirement for an amphiphilic alpha-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the alpha-helical structure (PP-5HB). PP-5AB inhibited PP-5 induced depolarization when added in an equimolar ratio to PP-5. Neither mutant peptide alone or in combination with PP-5 had any effect on calcium dependent vesicle aggregation. Using non-denaturing gel electrophoresis and size exclusion chromatography techniques PP-5 was shown to form a tetrameric complex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetramers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data.

Collagen-stimulated unidirectional translocation of cholesterol in human platelet membranes.

When human platelets are stimulated with collagen or thrombin, the asymmetric distribution of membrane lipids is disrupted as phosphatidylserine and phosphatidylethanolamine translocate from the inner monolayer to the outer monolayer. Coincident with the stimulus-dependent rearrangement of membrane phospholipids is a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. This redistribution of cholesterol was observed when the stimulus was collagen or ADP. The data presented here show that epinephrine stimulation does not promote cholesterol translocation but does potentiate collagen-promoted movement of cholesterol. To investigate the process of cholesterol translocation, experiments were performed to determine whether collagen stimulated reverse cholesterol movement; i.e. from the inner to the outer monolayer. For this study, the fluorescent sterol cholestatrienol (C-3) was incorporated into platelet membranes by exchange from cholesterol-containing phosphatidylcholine small unilamellar vesicles. C-3 was then removed selectively from the outer monolayer by treatment of the platelets with bovine serum albumin (BSA). During the subsequent incubation of BSA-treated platelets, C-3 moved spontaneously into the outer from the inner monolayer. This translocation had an apparent half-time of approximately 25 min and was unaltered by the presence of collagen. These results suggest that collagen treatment of platelets selectively facilitates the inward movement of the sterol. We have hypothesized that cholesterol translocation may be thermodynamically driven as a result of an unfavorable entropy, resulting in cholesterol translocation out of an environment becoming enriched in phosphatidylethanolamine. The unidirectional nature of collagen-promoted cholesterol movement from the phosphatidylethanolamine-rich outer monolayer is consistent with this interpretation.

Differential rhodopsin regeneration in photoreceptor membranes is correlated with variations in membrane properties.

Rhodopsin, the major transmembrane protein in both the plasma membrane and the disk membranes of photoreceptor rod outer segments (ROS) forms the apo-protein opsin upon the absorption of light. In vivo the regeneration of rhodopsin is necessary for subsequent receptor activation and for adaptation, in vitro this regeneration can be followed after the addition of 11-cis retinal. In this study we investigated the ability of bleached rhodopsin to regenerate in the compositionally different membrane environments found in photoreceptor rod cells. When 11-cis retinal was added to bleached ROS plasma membrane preparations, rhodopsin did not regenerate within the same time course or to the same extent as bleached rhodopsin in disk membranes. Over 80% of the rhodopsin in newly formed disks regenerated within 90 minutes while only 40% regenerated in older disks. Since disk membrane cholesterol content increases as disks are displaced from the base to the apical tip of the outer segment, we looked at the affect of membrane cholesterol content on the regeneration process. Enrichment or depletion of disk membrane cholesterol did not alter the % rhodopsin that regenerated. Bulk membrane properties measured with a sterol analog, cholestatrienol and a fatty acid analog, cis parinaric acid, showed a more ordered, less "fluid", lipid environment within plasma membrane relative to the disks. Collectively these results show that the same membrane receptor, rhodopsin, functions differently as monitored by regeneration in the different lipid environments within photoreceptor rod cells. These differences may be due to the bulk properties of the various membranes.

Fusion between retinal rod outer segment membranes and model membranes: a role for photoreceptor peripherin/rds.

Peripherin/rds plays an essential role in the maintenance of photoreceptor rod cell disk membrane structure. The purification of this protein to homogeneity [Boesze-Battaglia, K., et al. (1997) Biochemistry 36, 6835-6846] has allowed us to characterize the functional role of peripherin/rds in the maintenance of rod outer segment (ROS) membrane fusion processes. Utilizing a cell-free fusion assay system, we report that the fusion of R18-labeled ROS plasma membrane (R18-PM) with disk membranes or peripherin/rds-enriched large unilammellar vesicles (LUVs) is inhibited upon trypsinolysis of peripherin/rds. To understand this phenomenon, we tested the ability of a series of overlapping synthetic C-terminal peripherin/rds peptides to mediate model membrane fusion. Within the 63 amino acid long region of the C-terminus, we identified a minimal 15 residue long amino acid sequence (PP-5), which is necessary to promote membrane fusion. PP-5 was able to inhibit R18-PM disk membrane fusion and promoted ANTS/DPX contents mixing in a pure vesicle system. This peptide (PP-5) promoted calcium-induced vesicle aggregation of phosphatidylethanolamine:phosphatidylserine LUVs. FTIR analysis confirmed the structural prediction of this peptide as alpha-helical. When modeled as an alpha-helix, this peptide is amphiphilic with a hydrophobicity index of 0.75 and a hydrophobic moment of 0.59. PP-5 has substantial biochemical and functional homology with other well-characterized membrane fusion proteins. These results demonstrate the necessity for peripherin/rds in ROS membrane fusion, specifically the requirement for an intact C-terminal region of this protein.

Cholesterol enhances the adhesion of human platelets to fibrinogen: studies using a novel fluorescence based assay.

This communication reports investigations on the effect of platelet cholesterol content on adhesion of platelets to a fibrinogen coated surface. The adhesion of platelets stimulated with thrombin or ADP was dramatically increased when the platelet cholesterol content was enriched by incubation with cholesterol containing phosphatidylcholine vesicles. In contrast, ADP failed to promote the adhesion of platelets to fibrinogen after they had been depleted of cholesterol, either by incubation with phosphatidylcholine vesicles or by brief exposure to cholesterol oxidase. By comparison, the adhesion of resting platelets to fibrinogen coated surface was unaltered following either enrichment or depletion of cholesterol. These data were obtained using a novel method of measuring the adhesion of platelets to a protein coated surface based upon the fluorescent detection of platelets containing the fluorescent probe octadecyl rhodamine (R(18)). R(18) was incorporated into platelet membranes using standard ethanol injection techniques at room temperature for 30 min. The platelets were introduced into fibrinogen coated wells of a 96-well microtiter plate in the presence of various cations and stimulatory or inhibitory ligands. The plate was then incubated at room temperature without agitation for various periods of time. Adhesion measured in this manner had characteristics similar to those reported using other methods. Thus the extent of adhesion ranged from 1-4% under basal conditions, and was increased in a dose-dependent manner by Mg(2+) and Ca(2+), increased further by ADP, collagen or thrombin and not affected by prostacyclin.

Purification and light-dependent phosphorylation of a candidate fusion protein, the photoreceptor cell peripherin/rds.

The proteins peripherin/rds and rom-1 form a protein complex in the rims of photoreceptor outer segment disk membranes. Peripherin/rds plays an essential role in the morphogenesis and maintenance of disk membrane structure, with peripherin/rds gene mutations resulting in photoreceptor cell degeneration. We report two different chromatographic procedures for the purification of native peripherin/rds from bovine photoreceptor cell outer segments and show that the protein is a phosphoprotein that promotes membrane fusion in vitro. During one procedure, peripherin/rds was copurified in association with rom-1 by hyroxylapatite and Mono Q FPLC. During the other, it was purified free from rom-1 by concanavalin-A affinity chromatography and chromatofocusing. Analysis of homogeneous peripherin/rds from the second procedure showed that exposure of photoreceptor outer segments to light resulted in the incorporation of nearly 2 mol of phosphate per mole of peripherin/rds and a concomitant shift in the isoelectric point of the protein. In addition, we found that recombination of purified peripherin/rds into lipid vesicles increased membrane fusion, with more rapid fusion detected with phosphorylated peripherin/rds. In conclusion, studies with purified peripherin/rds reveal that the protein undergoes light-dependent phosphorylation and that it may function in membrane fusion.

Fusion of intracellular rod outer segment disk membranes with the surrounding plasma membrane.

PURPOSE: The series of experiments described were undertaken to evaluate the use of a cell-free lysis model to measure membrane fusion events in photoreceptor rod outer segments (ROS). The experiments measure fusion initiated with the osmotic disruption of fluorescenty-labeled ROS and correlate these findings with previously described disc-plasma membrane fusion. The influence of calcium and disc membrane cholesterol content on fusion between ROS membrane species was evaluated. METHODS: Membrane fusion was followed by measuring the dilution of a membrane-associated fluorophore (R18) from labeled plasma membrane to an unlabeled membrane species; discs. Free calcium in the ROS preparations was measured using the calcium-sensitive fluorophore Quin-2. The affects of cholesterol content on fusion were investigated by increasing and decreasing disc membrane cholesterol content using well established lipid exchange techniques. RESULTS: There is an increase in R18 fluorescence on hypotonic lysis of ROS whose plasma membrane is labeled with R18. This increase in fluorescence is inhibited by EGTA and requires nanomolar levels of calcium. The initial rates of fusion between R18-labeled plasma membrane and discs were virtually identical in discs with increased and decreased levels of membrane cholesterol. CONCLUSIONS: The increase in R18 fluorescence on lysis of R18-labeled ROS is consistent with fusion between disc membranes and the surrounding plasma membrane. This fusion is dependent on nanomolar levels of calcium, is inhibited by EGTA, and is independent of the cholesterol content of these membranes.

Cell membrane lipid composition and distribution: implications for cell function and lessons learned from photoreceptors and platelets.

Photoreceptor rod cells and blood platelets are remarkably different, yet both illustrate a similar phenomenon. Both are strongly affected by membrane cholesterol, and the distribution of cholesterol in the membranes of both cell types is determined by the lipid composition within the membranes. In rod cells, cholesterol strongly inhibits rhodopsin activity. The relatively higher level of cholesterol in the plasma membrane serves to inhibit, and thereby conserve, the activity of rhodopsin, which becomes fully active in the low-cholesterol environment of the disk membranes of these same cells. This physiologically important partitioning of cholesterol between disk membranes and plasma membranes occurs because the disk membranes are enriched with phosphatidylethanolamine, thus providing a thermodynamically unfavorable environment for the sterol. Cholesterol enrichment of platelets renders these cells more responsive to stimuli of aggregation. Stimuli for platelet aggregation cause a rapid transbilayer movement of cholesterol from the outer monolayer. This stimulus-dependent redistribution of cholesterol appears to result from the concomitant movement of phosphatidylethanolamine into the outer monolayer. The attractive, yet still unproven, hypothesis is that cholesterol translocation plays an important role in the overall platelet response and is intimately related to the sensitizing actions of cholesterol on these cells.