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We report the results of a comparative study of the molecular order and dynamics of phosphatidylcholine (PC) bilayer membranes in the absence and presence of cholesterol, ergosterol and lanosterol, using deuterium (2H) nuclear magnetic resonance (NMR) of deuterated phospholipid molecules, in addition to solid state 13C and 31P-NMR. Using dimyristoylphosphatidylcholines (DMPCs) specifically labeled at positions 2', 3', 4', 6', 8', 10' and 12' of the sn-2 chain together with the perdeuterated 2-[2H27]DMPC derivative, the order profile for 9 of the 13 methylene groups of the sn-2 chain was established at 25 degrees C for DMPC, DMPC/cholesterol, DMPC/ergosterol and DMPC/lanosterol membranes, at a fixed sterol/phospholipid mol ratio of 30%, and in the presence of excess water. The overall ordering effects were found to be ergosterol > cholesterol >> lanosterol. Transverse relaxation (T2e) studies of these systems indicated that while for DMPC, DMPC/cholesterol and DMPC/ergosterol the relative relaxation rates were in qualitative agreement with models which assume cooperative motions of the bilayer molecules as the main relaxation mechanism, those in DMPC/lanosterol were anomalously high, suggesting alterations of lipid packing. Using dipalmitoylphosphatidylcholine (DPPC) deuterated at the trimethylammonium group of the choline moiety, we found that the differential ordering and motional effects induced by the sterols in the acyl chains were also reflected in the headgroup, both in the gel (L beta) and liquid-crystalline phases. 13C and 1H spin dynamics studies of these systems, including cross-polarization, rotating frame longitudinal relaxation and dipolar echo relaxation rates showed that the mobility of the different regions of the phospholipid molecules in the binary lipid systems were inversely correlated with the ordering effects induced by the sterols. A novel combination of C-D bond order parameters (obtained by 2H-NMR) and 13C-1H cross polarization rates confirmed these results. The effects of the same sterols at the same molar proportion on the unsaturated lipid 1-[2H31]palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (2H31-POPC) at 25 and 35 degrees C were different from those observed on DMPC and showed ordering effects which are largest for cholesterol, while ergosterol and lanosterol produced significantly smaller effects. Transverse relaxation studies indicate that while cholesterol does not perturb cooperative motions in POPC, both ergosterol and lanosterol do. Again, high-resolution solid state 13C-NMR studies support the conclusions of the 2H-NMR experiments.(ABSTRACT TRUNCATED AT 400 WORDS)
We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann-Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild-type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane-associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.
Physiologically relevant molecular species of plasmenylcholine and phosphatidylcholine were synthesized and their molecular dynamics and interactions with cholesterol were compared by determination of salient proton spin-lattice relaxation times and apparent activation energies for 1H-NMR observable motion. The molecular dynamics of PA PhosCho (1-hexadecanoyl-2-eicosatetra-5',8',11',14'-enoyl-sn-glycero-3-pho sphocholine) in multiple regions of the bilayer. Furthermore, the fluidity gradient of PA PhosCho was larger than that of PA PlasCho as ascertained by 1H spin-lattice relaxation time measurements. Introduction of cholesterol into each bilayer resulted in disparate effects on the dynamics of each subclass including: (1) increased motional freedom in the polar head group of PA PlasCho without substantial alterations in the dynamics of the polar head group of PA PhosCho; and (2) increased immobilization of the membrane interior in PA PlasCho in comparison to PA PhosCho. Analysis of Arrhenius plots of T1 relaxation times demonstrated that the apparent activation energies for vinyl and bisallylic methylene proton NMR observable motion in PA PhosCho were greater than that in PA PlasCho. Thus, comparisons of spin-lattice relaxation times and apparent activation energies demonstrate that vesicles comprised of PA PlasCho and PA PhosCho possess differential molecular dynamics and distinct interactions with cholesterol. Collectively, these results underscore the significance of the conjoint presence of the vinyl ether linkage and arachidonic acid as an important determinant of membrane dynamics in specialized mammalian membranes.
A variety of methods to incorporate cholesterol into lipid membrane systems have been applied with varying success. We tested an incorporation method based on cholesterol-loaded methyl-beta-cyclodextrins and compared it to a method that uses cholesterol-loaded liposomes. With methyl-beta-cyclodextrin, we increased the cholesterol content in microsomal membranes to almost the fourfold of the original content. With cholesterol-loaded liposomes instead, we achieved an elevation of 140%. Short incubation times and well-defined carrier properties favor the beta-cyclodextrin method. For direct detection of membrane cholesterol, we slightly modified a microenzymatic fluorescence assay originally developed for precise cholesterol detection in serum. Without the need to perform lipid extraction, this assay was reliable for cholesterol detection in liposomes and in microsomes. Additionally, we compared the sensitivity of the fluidity-sensitive fluorescent dyes pyrene, pyrene-methanol, bis-pyrene, 1-6-phenyl-1,3,5,-hexatrien, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5,-hexatrien in order to detect cholesterol indirectly by the dynamically relevant changes exerted on lipid matrices. These dyes differ not only in their membrane location but also in their dynamical behavior. We calibrated the dyes in liposomes of defined cholesterol content and used the most suited ones to follow and quantify the cholesterol incorporation into liposomal and microsomal membranes.
16Alpha-hydroxyestrone (16alpha-OHE(1)), an endogenous estrogen metabolite, is associated with increased bone density in postmenopausal women. This study was designed to evaluate the long-term activity of this metabolite on bone, uterus, and serum cholesterol in an animal model for postmenopausal bone loss. A preliminary dose-response study performed in weanling rats determined 2000 microg/kg/day to be the optimal dose of 16alpha-OHE(1) for studying estrogenic effect on bone. The long-term experiment was performed in 6-month-old animals that were either sham-operated or OVX. The OVX rats were implanted sc with 60-day continuous-release carrier, 17beta-estradiol (E(2)) (33 microg/kg/day) or 16alpha-OHE(1) pellets (2000 microg/kg/day). OVX decreased uterine weight, increased body weight, serum cholesterol, and all dynamic bone histomorphometric measurements in cortical and cancellous bone, and resulted in a 54% bone loss at the tibial metaphysis. E(2) completely prevented OVX-induced bone loss, suppressed bone turnover, and induced uterine hypertrophy and hypercholesterolemia. 16alpha-OHE(1) acted as an E(2) agonist on bone, suppressing bone formation and resorption. However, the estrogen metabolite lowered serum cholesterol and was only a partial E(2) agonist on uterine weight and epithelial cell height. These results suggest that 16alpha-OHE(1) is an estrogen agonist on bone and may be responsible, in part, for the cholesterol-lowering activity attributed to estrogen. As a consequence of its skeletal effects, older women who produce high levels of 16alpha-OHE(1) could have a lower risk for developing postmenopausal osteoporosis than women who produce less-active estrogen metabolites.
Japanese quail from the S16 generation of lines selected for high (HR) and low (LR) plasma cholesterol response to adrenocorticotropin (ACTH), and the randombred control line (C) from which the selected lines had been derived, were immunized with a heat-killed Salmonella pullorum antigen. Plasma from blood samples taken at 0,5,8, and 12 days postimmunization was titered for total agglutinin levels. A quadratic equation and its first derivative, calculated for each bird, produced mean values by line for maximum titers, mean day of maximum titer, and rate of titer decline. Results indicated that the LR line had significantly lower titers than the HR or C lines but that the HR line reached maximum titer later than the LR line. The rate of titer decline was fastest in the C line.
Cholesterol enrichment of rat liver mitochondria (CHM) impairs atractyloside-induced mitochondrial permeability transition (MPT) due to decreased membrane fluidity. In this study we addressed the effect of cholesterol enrichment on MPT induced by reactive oxygen species (ROS). Superoxide anion generated by xanthine plus xanthine oxidase triggered mitochondrial swelling and cytochrome c release in CHM, which was prevented by butylated hydroxytoluene, an anti-voltage-dependent anion channel antibody, or cyclosporin A. Furthermore, hydrogen peroxide generated by the combination of ganglioside GD3 and mitochondrial GSH depletion elicited mitochondrial swelling and release of cytochrome c, Smac/Diablo and apoptosis-inducing factor in control mitochondria and CHM. Thus, ROS induce MPT and apoptosome activation regardless of decreased mitochondrial membrane dynamics due to cholesterol enrichment.
Postparturient anoestry, spontaneously changing over to seasonal anoestry, represents an important reserve of full utilization of the reproductive potential of sheep. In spite of the fact that the length of gestation in sheep only amounts to 148 days (+/- 5 days), inability of most sheep to enter the cycle during the spring season acts as a factor limiting the number of lambings--and at the same time production of lambs--to one lambing per year. In order to obtain more detailed knowledge of hormonal conditions in sheep puerpery, the presented work was directed at the study of dynamic changes in concentrations of thyroxine (T4), triiodothyronine (T3), 17 beta-estradiol (E2), progesterone (P4) and cholesterol (Chol) in the blood serum of ewes and at their mutual correlative dependences between the 36th hour and the 51st postparturient day. The observation was carried out in nine nursing ewes of the Slovak Merino breed, with average weight 40-50 kg, lambed in January and February. Blood was sampled by means of jugular vein puncture 14 days (-14th day) before parturition (a. p.), up to 36 h after parturition (p. p.) and on day 4, 7, 14, 17, 21, 25, 34, 42 and 51 p. p. In the period from the 36th h to day 21 p. p., concentrations of T4 (Tab. I, Fig. 1) showed a decreasing tendency compared to the starting -14th day (69.55 +/- +/- 0.12 nmol.-1) with the exception of a temporary increase on the 7th day p. p. The lowest values were recorded on days 4 and 21 p. p. (45.66 +/- 21.61 nmol.l-1, P less than 0.05 and 54.89 +/- 11.06 nmol.l-1, P less than 0.05, resp.). An increase of these values to the starting level was observed between days 25 and 51 p. p. Compared to the values on -14th day (0.76 +/- 0.22 nmol.l-1), a significant increase of T3 concentrations (Tab. I, Fig. 2) was recorded at the 36th h and on days 4 and 7 p. p. with the highest values (1.48 +/- +/- 0.34 nmol.l-1, P less than 0.001) at 36 h p. p. After the temporary decrease between days 14 and 21 p. p. its concentrations showed a constantly rising tendency statistically significant on days 34, 42 and 51 p. p., compared to the -14th day (P less than 0.05). Concentrations of E2 (Tab. I, Fig. 3) reached the values of 0.44 +/- 0.41 nmol.l-1 on the -14th day.(ABSTRACT TRUNCATED AT 400 WORDS)
Natural abundance 13C solid-state nuclear magnetic resonance spectroscopy was used to investigate the effect of the incorporation of cholesterol on the dynamics of dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. In particular, the use of a combination of the cross-polarization and magic angle spinning techniques allows one to obtain very high resolution spectra from which can be distinguished several resonances attributed to the polar head group, the glycerol backbone, and the acyl chains of the lipid molecule. To examine both the fast and slow motions of the lipid bilayers, 1H spin-lattice relaxation times as well as proton and carbon spin-lattice relaxation times in the rotating frame were measured for each resolved resonance of DMPC. The use of the newly developed ramped-amplitude cross-polarization technique results in a significant increase in the stability of the cross-polarization conditions, especially for molecular groups undergoing rapid motions. The combination of T1 and T1 rho measurements indicates that the presence of cholesterol significantly decreases the rate and/or amplitude of both the high and low frequency motions in the DMPC bilayers. This effect is particularly important for the lipid acyl chains and the glycerol backbone region.
A mathematical equation of the shift of the dynamic balance of cholesterolemia to a new level is substantiated. A mathematical analysis of the results of experimental studies is presented. A correlation was established between the mechanism of the development of alimentary hypercholesterolemia in rabbits and the half-life period of cholesterol in the body (T1/2 per day). It is demonstrated that an equilibrium level of hypercholesterolemia (Xeqn,ch, mg%) is directly proportional to the speed of cholesterol accumulation in blood (Vch, mg%/day) and reciprocal to the constant rate of cholesterol decomposition in the body.
Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.
Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca(2+)-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.
NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca(2+)-dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.
We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin- Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.
A member of the annexin XIII sub-family, annexin XIIIb, has been implicated in the apical exocytosis of epithelial kidney cells. Annexins are phospholipid-binding proteins that have been suggested to be involved in membrane trafficking events although their actual physiological function remains open. Unlike the other annexins, annexin XIIIs are myristoylated. Here, we show by immunoelectron microscopy that annexin XIIIb is localized to the trans-Golgi network (TGN), vesicular carriers and the apical cell surface. Polarized apical sorting involves clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We now provide evidence for the raft association of annexin XIIIb. Using in vitro assays and either myristoylated or unmyristoylated recombinant annexin XIIIb, we demonstrate that annexin XIIIb in its native myristoylated form stimulates specifically apical transport whereas the unmyristoylated form inhibits this route. Moreover, we show that formation of apical carriers from the TGN is inhibited by an anti-annexin XIIIb antibody whereas it is stimulated by myristoylated recombinant annexin XIIIb. These results suggest that annexin XIIIb directly participates in apical delivery.
The dynamic interchange of cholesterol and the phase transition between nonmicellar and micellar aggregates in rat and model bile were characterized with gel-permeation chromatography, quasi-elastic light scattering, turbidity measurements, and by radiolabeling lipid aggregates in bile. Cholesterol partitioned into either the micellar or nonmicellar phases independent of the lipid aggregate structure. In model bile, increasing bile salt concentrations led to a decrease in the relative proportion of nonmicellar aggregates beginning at 5 mM taurocholate (TC), while the relative cholesterol content of the nonmicellar fraction increased from 1.0 to 2.7 +/- 2.0 (means +/- SD). In rats, creation of a biliary fistula resulted in a decrease of bile salts from 41 to 4 mM. Mixed micelles increased from 25 to 120 A in radius, while nonmicellar aggregates increased from 180 to 800 A in radius. Addition of TC to model bile (cholesterol:lecithin = 1:1) vesicles with total lipid concentrations less than 7 mM yielded a progressive shift of vesicles (450 A) to mixed micelles (30 A). For mixtures with higher total lipid concentrations, addition of TC promoted substantial vesicle aggregation and resulted in formation of a third phase containing lipid aggregates larger in size than the initial vesicles. These results suggest that rapid exchange of cholesterol occurs in bile and that significant remodeling of vesicles can occur. These alterations in vesicles include both enrichment in cholesterol content and formation of larger aggregates during increases in bile salt concentration.
Caveolae and rafts are specialized microdomains of the endothelial cell plasma membrane, which play an important role in signal transduction, transcellular transport, and cholesterol homeostasis. The dynamic protein composition of these subcellular lipid domains has been implicated in a variety of patho-physiological states of the vasculature, and is receiving increased attention. As a result of the membranous composition and abundance of insoluble intrinsic and membrane-associated proteins, determination of the raft/caveolae subproteome composition requires specially adapted methods. In this chapter, we present a straightforward protocol to obtain comprehensive and reliable peptide mixtures from this subproteome by subcellular fractionation and both one-dimensional and two-dimensional gel electrophoresis. These mixtures allow dynamic monitoring of composition and posttranslational modification of the raft/caveola subproteome using peptide mass fingerprinting and direct peptide sequencing tandem mass spectrometry.