Movement Of Lipids Research Articles

Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane.

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulfonic acid, suggesting the existence of protein-mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.

Cholesterol transport function of pancreatic cholesterol esterase: directed sterol uptake and esterification in enterocytes.

We have recently hypothesized that neutral lipids can, in part, move across biological membranes via a mechanism involving enzymes anchored to membrane proteoglycans such as those found in the brush border of the enterocyte [Bosner, M. S., Gulick, T., Riley, D. J. S., Spilburg, C. A., & Lange, L. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7438-7442]. Present results now show a subsequent, essential protein-mediated sorting of neutral lipids for further intracellular metabolism. Thus, in the absence of enzyme, 0.002 pmol of cellular ester appeared after 2 h, and its level increased only 3.5-fold after 12 h. However, in the presence of cholesterol esterase, the level of cholesterol ester increased 39-fold in the same time period, indicating that the enzyme-mediated uptake accounts for 10-fold greater ester synthesis than that from basal absorption. Kinetic analysis reveals that both enzyme-mediated and background absorption depend on taurocholate concentration and are second-order reactions more likely dependent on collision than diffusion. Other lipid-recognizing proteins such as pancreatic triglyceride lipase and the intestinal fatty acid binding protein are not stimulatory to intracellular cholesterol processing. Taken together, these data suggest that pancreatic cholesterol esterase and possibly other proteoglycan-binding extracellular enzymes of neutral lipid metabolism may facilitate movement of neutral lipids into the plasma membrane and direct them into functional intracellular sites.

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane.

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.

Lipid sorting and trafficking in cells: Current Opinion in Structural Biology 1993, 3:495–498

Recent advances in the field of lipid sorting include the report of a cell-free system that exhibits ATP-dependent transport of phosphatidylserine to mitochondria. Other investigations describe co-transport of glycosphinogo-lipids with proteins, intra-Golgi movement of gangliosides and mobilization of cholesterol from lysosomes. In addition, the role of transfer proteins in intracellular lipid movement has again been challenged.

Effects of beta-(N-methylamino)-L-alanine on cytoskeletal proteins of erythrocyte membranes.

The interaction of the amino acid beta-(N-methylamino)-L-alanine, a neurotoxin found in the seed of the false sago palm, with erythrocyte membranes has been monitored by electron paramagnetic resonance techniques of spin labeling. This neurotoxin did not alter the motion or order of bilayer lipids, but a highly-significant and dose-dependent alteration in the physical state of cytoskeletal proteins was observed. These results are discussed in reference to potential mechanisms involved in the neurotoxicity produced by beta-(N-methylamino)-L-alanine and in reference to the unusual neurological disorders among the Chamorro population of Guam and other Marianas Islands.

Chicken Sterol Carrier Protein m2/Sterol Carrier Protein x: cDNA Cloning Reveals Evolutionary Conservation of Structure and Regulated Expression

The chicken is a useful model for studies of lipid biochemistry because of the profound changes in lipid metabolism during development and the marked effects of estrogen on its lipids. Here we report the cloning of a cDNA encoding chicken sterol carrier protein 2 (SCP2)/sterol carrier protein x (SCPx), proteins that are believed to play key roles in intracellular lipid movement and metabolism. The 2.4-kb clone, isolated from a liver cDNA library by homology screening with a rat SCP2 cDNA, encodes a 58-kDa polypeptide, SCPx, which encompasses the sequence for SCP2 at its C-terminus. Comparison of the deduced amino acid sequence of the chicken protein with those of mammals revealed conservation of structure from an evolutionary standpoint. Like the other vertebrate SCPx proteins, the chicken protein contains a conserved Arg-Gly-Asp sequence and a cysteine residue in the N-terminus that aligns with the active site cysteine of Escherichia coli 3-ketoacyl-CoA thiolase, a protein that was previously shown to be homologous to vertebrate SCPx. Northern blot analysis of poly(A)+ RNA from different chicken tissues revealed two mRNA species, one of 2.5 kb, encoding SCPx, and one of 1.4 kb, presumably encoding SCP2. High levels of the 2.5 and 1.4-kb mRNAs were found in liver, intestine, and ovarian granulosa cells, consistent with a role for these proteins in lipid metabolism. There was no change in the 2.5-kb mRNA in chicken liver with development (Day 20 embryo to 4 weeks posthatch), but there was a 10-fold in-crease in the 1.4-kb mRNA by 1 week posthatch. Treatment of roosters with a single injection of estradiol (25 mg/kg) caused a twofold increase in the 2.5-kb mRNA in liver at 6 h after estradiol administration, reaching a maximum fourfold increase at 48 h, while the 1.4-kb mRNA increased twofold at 48 h. The two SCP2/SCPx mRNAs were most abundant in granulosa cells from small follicles. A reduction in SCP2/SCPx gene expression was observed with follicular maturation, correlating with falling estrogen production. In summary, we have documented (i) marked conservation of SCP2/SCPx structure across species and (ii) developmental and hormonal regulation of the mRNAs which encode proteins thought to be involved in lipid metabolism.

Interaction of avidin with spin-labelled N-biotinyl phosphatidylethanolamine in a lipid membrane

N-Biotinyl phosphatidylethanolamine spin labelled at the C-14 position of the sn-2 chain has been incorporated at a level of 1 mol% in bilayers of dimyristoyl phosphatidylcholine, and the effects on the chain mobility of binding avidin to the biotin lipid headgroup have been studied by electron spin resonance spectroscopy. In the fluid phase, avidin causes a large and selective restriction in the chain motion of the biotin lipids to which it is attached, without perturbing appreciably the mobility of the bulk lipid chains. This specific type of lipid-protein interaction is different in kind from that observed both with integral and peripheral membrane proteins and may be involved in transmembrane communication on ligand binding to lipid headgroups, as well as lateral communication (at high packing densities) between proteins with covalent lipid anchors.

Signalling via plasmodesmata—the neglected pathway

This review considers recent studies on the role of plasmodesmata in the conduction of small solutes and signalling molecules between plant cells. The substructure of plasmodesmata is described in relation to the potential pathways available for symplastic signalling between cells. At least two discrete pathways are available for transport through plasmodesmata, the cytoplasmic sleeve between the desmotubule and the plasmalemma, and the endoplasmic reticulum which connects contiguous cells via the central desmotubule. This latter pathway has been shown recently to function as a dynamic continuum for the movement of lipids and lipid-signalling molecules between plant cells. The role of plasmodesmata in the conduction of hormones and electrical signals is also considered, as is the potential for movement of macromolecular signalling molecules via the symplast. The factors which regulate plasmodesmatal conductance and the significance of symplast 'domains' are discussed in relation to the control of movement of signalling molecules in the symplast.

Deuterium NMR relaxation studies of peptide-lipid interactions.

A unique model membrane system composed of a synthetic amphiphilic peptide (Lys2-Gly-Leu16-Lys2-Ala-amide) and a specifically labeled phospholipid (1,2-[7,7-2H2]dipalmitoyl-sn-glycero-3-phosphocholine) has been studied by 2H NMR, using inversion recovery, quadrupolar echo, and modified Jeener-Broekaert sequences, from 213 to 333 K, at molar peptide concentrations of 0, 2, 4, and 6%. Analysis of the experiments, employing a density matrix treatment based on the stochastic Liouville equation, revealed information about the dynamic organization of the lipid in the model membrane system, whose phase behavior has been determined previously [Huschilt et al. (1985) Biochemistry 24, 1377-1386]. The dynamic organization is described in terms of segmental and molecular order parameters and in terms of correlation times corresponding to both internal and overall lipid motions. In the liquid crystalline phase, the molecular order parameter, SZZ, was observed to decrease slightly upon addition of peptide while the conformational order parameter corresponding to the seventh segment, SZ'Z', did not change for any concentration of peptide. In general, the gauche-trans isomerization rate in the middle of the chain was not observed to change upon peptide addition, whereas the whole body reorientational correlation times (tau R parallel and tau R perpendicular) increased by nearly an order of magnitude. The anisotropy ratio (tau R perpendicular/tau R parallel) decreased with peptide added. An additional motion which involves a jump about the axis of the sn-2 chain is also observed to be slowed down significantly in the presence of peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

A functional barrier to movement of lipids in polarized neurons.

In polarized neurons, axons and dendrites perform different functions, which are reflected in their different molecular organization. Studies on the sorting of viral and endogenous glycoproteins in epithelial cells and hippocampal neurons suggest that there may be similarities in the mechanism of sorting in these two cell types. The mechanisms that maintain the distinct composition of the two plasma membrane domains in these two cell types must, however, be different. We have proposed the existence of a functional barrier at the axonal hillock/initial segment which prevents the intermixing of membrane constituents. Here we test this hypothesis by fusing liposomes containing fluorescent phospholipids into the plasma membrane of polarized hippocampal cells in culture. Fusion was induced by lowering the pH and mediated by influenza virus haemagglutinin expressed on the axonal surface of neurons infected with fowl plague virus. Labelling was found exclusively on axons after fusion. Although the fused lipids were mobile on the axonal membrane, no labelling was detected on the cell body and dendritic surfaces. These results suggest that there is a diffusion barrier at the axonal hillock/initial segment which maintains the compositional differences between the axonal and somatodendritic domains.

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.

Effects of thermal acclimation on seasonal accumulation of an apolipoprotein A-I-like molecule in a cold-adapted lizard

Using two-dimensional electrophoresis on total extracts of ovary, liver, and muscle of a cold-adapted lizard, Lacerta vivipara, we investigated the effects of cold at the protein level during autumn and winter. The major phenomenon observed was an accumulation in tissue of an apolipoprotein A-I-like molecule which displayed the same electrophoretic pattern as that previously described in human serum and peripheral tissues (isoform number, charge, apparent molecular mass). However, we detected the isoforms I4, I5, and I6 in all the lizard tissues examined, and two additional more acidic forms (I7, I8), which might come from proteolysis of the basic components. In this study, we report that the accumulation and distribution of the apolipoprotein A-I-like isoforms differ according to thermal acclimation, season, and the particular organ. These findings strongly suggest that significant movements of lipids are involved in the ectothermic hibernation process.

Sarcoplasmic reticulum from rabbit and winter flounder: Temperature-dependence of protein conformation and lipid motion

A comparative study of lipids and proteins in sarcoplasmic reticulum (SR) from rabbit and flounder has been undertaken. The protein/phospholipid ratio (w/w) was 3:1 in flounder SR (FSR) and 2.2:1 in rabbit SR (RSR). Both membranes had similar contents of PC (70%) and PI (6%). PE constituted 15% in RSR and 21% in FSR. PS and sphingomyelin were minor components of both SR (< 4%). There were differences in the unsaturated chains of the total lipid extracts, PC, PE, and PI between FSR and RSR. RSR was high in linoleate and arachidonate while FSR contained substantial amounts of eicosapentaenoate and docosahexaenoate. FTIR spectroscopy revealed that the lipids of both membranes did not undergo a phase transition between 0 and 50°C. The lipids were in the liquid-crystalline state at physiological temperatures and underwent monotonic increases in conformational disorder as the temperature was raised. CD spectra indicated higher content of α-helical structure of proteins in RSR than in FSR. Increasing temperature caused diminution of α-helix content. Relatively large decreases in ellipticity were observed between 20°C and 40°C for FSR and 30°C and 60°C for RSR. Measurements of intrinsic tryptophan fluorescence as a function of temperature gave similar results for membrane proteins in both FSR and RSR. The rate of change of tryptophan fluorescence and fluorescence lifetimes was constant over the temperature ranges studied, and no abrupt shifts in fluorescence occurred in the temperature regions where ellipticity decreased rapidly.

Rates of spontaneous exchange of synthetic radiolabeled sterols between lipid vesicles

14C-labeled sterols with structural variation in the polar function [3 alpha-OH, 3-O(CH2)2O-(CH2)2O(CH2)2OH, 3 alpha-NH2, 3 beta-NH2, and 3-OC(O)CHN = N] and at the 7 position (7-oxo, 7 alpha-OH, and 7 beta-OH) were synthesized and incorporated into unilamellar vesicles for studies of the rates of transfer to an excess of acceptor vesicles. Cholesterol, cholestanol, and epicholesterol underwent full exchange in a single kinetic pool, and 90% of the 3 alpha-triethoxycholesterol was exchangeable in one pool. Biphasic kinetics with full exchangeability were observed for cholesterylamines, which bear a positive charge at the 3 position; the slow phase reflects the high activation energy for inner-to-outer leaflet movement of the charged lipid. Biphasic kinetics were also found for cholesteryl diazoacetate, indicating that this photoaffinity probe and cholesterol have different mechanisms of transfer. Sterols that are more hydrophilic than cholesterol as estimated by reversed-phase high-performance chromatography (elution with acetonitrile-2-propanol, 4:1 v/v, with varying proportions of water) gave faster exchange rates than cholesterol, whereas sterols that are more hydrophobic gave slower exchange rates. However, the rates of [14C]sterol desorption from the lipid-water interface are not correlated with the relative sterol hydrophobicity as estimated by the logarithm of the capacity factors using acetonitrile-2-propanol-water as the mobile phase. These studies suggest that the interaction of sterols with phospholipids provides the principal physical-chemical basis for determining the rates of spontaneous exchange of sterols between bilayers.

Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.

Organelle biogenesis and intracellular lipid transport in eukaryotes

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.

CDNAs Encoding Members of a Family of Proteins Related to Human Sterol Carrier Protein 2 and Assignment of the Gene to Human Chromosome 1 p21→pter

Sterol carrier protein 2 (SCP2) is believed to play a key role in intracellular lipid movement. Here we report the cloning and nucleotide sequences of cDNAs encoding SCP2-related proteins of 58.85 kD and 30.8 kD and the assignment of the SCP2 gene to human chromosome 1 p21-pter. The SCP2-related proteins share common deduced carboxyl amino acid sequences with SCP2 and the cDNAs have a common 3' untranslated nucleotide sequence. The mRNAs encoding these proteins increased in a coordinate fashion as human placental cytotrophoblasts differentiated into syncytiotrophoblasts in culture. Our observations document the existence of a family of related proteins encoded by the human SCP2 gene.

Direct observation of brownian motion of lipids in a membrane.

Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 10(-8) cm2/sec, and paucivalent probes had an average D of 0.73 x 10(-8) cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 x 10(-8) cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 x 10(-8) cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results. Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes.

Collective lipid motions in bilayer membranes studied by transverse deuteron spin relaxation

The anisotropy and pulse frequency dispersion of the spin–spin relaxation time TCP2E from Carr–Purcell–Meiboom–Gill pulse sequences is employed to evaluate the major contribution to transverse 2H spin relaxation in bilayer membranes. Analysis of the experiments is achieved in terms of a density operator formalism, employing the stochastic Liouville approach. From a comparison of the observed angular and frequency dependences of TCP2E with those predicted for order director fluctuations, we conclude that collective lipid motions constitute the dominant transverse relaxation process. Computer simulations provide the viscoelastic parameters of the lipid membranes. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers at T=318 K an average elastic constant of K=2×10−11 N and an effective viscosity of η=0.1 P have been determined. Using the experimentally accessible value for the long wavelength cutoff of the elastic modes, one obtains the mean square amplitude of the director fluctuations 〈θ20〉=0.04. This corresponds to an order parameter of SOF=0.94. Apparently, the contributions of the collective motions to the measured order parameters are marginal.

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.