Primary cell wall inspired micro containers as a step towards a synthetic plant cell.

The structural integrity of living plant cells heavily relies on the plant cell wall containing a nanofibrous cellulose skeleton. Hence, if synthetic plant cells consist of such a cell wall, they would allow for manipulation into more complex synthetic plant structures. Herein, we have overcome the fundamental difficulties associated with assembling lipid vesicles with cellulosic nanofibers (CNFs). We prepare plantosomes with an outer shell of CNF and pectin, and beneath this, a thin layer of lipids (oleic acid and phospholipids) that surrounds a water core. By exploiting the phase behavior of the lipids, regulated by pH and Mg2+ ions, we form vesicle-crowded interiors that change the outer dimension of the plantosomes, mimicking the expansion in real plant cells during, e.g., growth. The internal pressure enables growth of lipid tubules through the plantosome cell wall, which paves the way to the development of hierarchical plant structures and advanced synthetic plant cell mimics.

Phospholipid flipping involves a central cavity in P4 ATPases.

P4 ATPase flippases translocate phospholipids across biomembranes, thus contributing to the establishment of transmembrane lipid asymmetry, a feature important for multiple cellular processes. The mechanism by which such phospholipid flipping occurs remains elusive as P4 ATPases transport a giant substrate very different from that of other P-type ATPases such as Na+/K+- and Ca2+-ATPases. Based on available crystal structures of cation-transporting P-type ATPases, we generated a structural model of the broad-specificity flippase ALA10. In this model, a cavity delimited by transmembrane segments TM3, TM4, and TM5 is present in the transmembrane domain at a similar position as the cation-binding region in related P-type ATPases. Docking of a phosphatidylcholine headgroup in silico showed that the cavity can accommodate a phospholipid headgroup, likely leaving the fatty acid tails in contact with the hydrophobic portion of the lipid bilayer. Mutagenesis data support this interpretation and suggests that two residues in TM4 (Y374 and F375) are important for coordination of the phospholipid headgroup. Our results point to a general mechanism of lipid translocation by P4 ATPases, which closely resembles that of cation-transporting pumps, through coordination of the hydrophilic portion of the substrate in a central membrane cavity.

Lipid flippases and their biological functions.

The typically distinct phospholipid composition of the two leaflets of a membrane bilayer is generated and maintained by bi-directional transport (flip-flop) of lipids between the leaflets. Specific membrane proteins, termed lipid flippases, play an essential role in this transport process. Energy-independent flippases allow common phospholipids to equilibrate rapidly between the two monolayers and also play a role in the biosynthesis of a variety of glycoconjugates such as glycosphingolipids, N-glycoproteins, and glycosylphosphatidylinositol (GPI)-anchored proteins. ATP-dependent flippases, including members of a conserved subfamily of P-type ATPases and ATP-binding cassette transporters, mediate the net transfer of specific phospholipids to one leaflet of a membrane and are involved in the creation and maintenance of transbilayer lipid asymmetry of membranes such as the plasma membrane of eukaryotes. Energy-dependent flippases also play a role in the biosynthesis of glycoconjugates such as bacterial lipopolysaccharide. This review summarizes recent progress on the identification and characterization of the various flippases and the demonstration of their biological functions.

The organizing potential of sphingolipids in intracellular membrane transport.

Eukaryotes are characterized by endomembranes that are connected by vesicular transport along secretory and endocytic pathways. The compositional differences between the various cellular membranes are maintained by sorting events, and it has long been believed that sorting is based solely on protein-protein interactions. However, the central sorting station along the secretory pathway is the Golgi apparatus, and this is the site of synthesis of the sphingolipids. Sphingolipids are essential for eukaryotic life, and this review ascribes the sorting power of the Golgi to its capability to act as a distillation apparatus for sphingolipids and cholesterol. As Golgi cisternae mature, ongoing sphingolipid synthesis attracts endoplasmic reticulum-derived cholesterol and drives a fluid-fluid lipid phase separation that segregates sphingolipids and sterols from unsaturated glycerolipids into lateral domains. While sphingolipid domains move forward, unsaturated glycerolipids are retrieved by recycling vesicles budding from the sphingolipid-poor environment. We hypothesize that by this mechanism, the composition of the sphingolipid domains, and the surrounding membrane changes along the cis-trans axis. At the same time the membrane thickens. These features are recognized by a number of membrane proteins that as a consequence of partitioning between domain and environment follow the domains but can enter recycling vesicles at any stage of the pathway. The interplay between protein- and lipid-mediated sorting is discussed.

Lipid distribution and transport across cellular membranes.

In eukaryotic cells, the membranes of different intracellular organelles have different lipid composition, and various biomembranes show an asymmetric distribution of lipid types across the membrane bilayer. Membrane lipid organization reflects a dynamic equilibrium of lipids moving across the bilayer in both directions. In this review, we summarize data supporting the role of specific membrane proteins in catalyzing transbilayer lipid movement, thereby controlling and regulating the distribution of lipids over the leaflets of biomembranes.

Helicobacter pylori-induced prostaglandin E(2) synthesis involves activation of cytosolic phospholipase A(2) in epithelial cells.

Helicobacter pylori initiates an inflammatory response and gastric diseases, which are more common in patients infected with H. pylori strains carrying the pathogenicity island, by colonizing the gastric epithelium. In the present study we investigated the mechanism of prostaglandin E(2) (PGE(2)) synthesis in response to H. pylori infection. We demonstrate that H. pylori induces the synthesis of PGE(2) via release of arachidonic acid predominately from phosphatidylinositol. In contrast to H. pylori wild type, an isogenic H. pylori strain with a mutation in the pathogenicity island exerts only weak arachidonic acid and PGE(2) synthesis. The H. pylori-induced arachidonic acid release was abolished by phospholipase A(2) (PLA(2)) inhibitors and by pertussis toxin (affects the activity of G alpha(i)/G alpha(o)). The role of phospholipase C, diacylglycerol lipase, or phospholipase D was excluded by using specific inhibitors. An inhibitor of the stress-activated p38 kinase (SB202190), but neither inhibitors of protein kinase C nor an inhibitor of the extracellular-regulated kinase pathway (PD98059), decreased the H. pylori-induced arachidonic acid release. H. pylori-induced phosphorylation of p38 kinase and cytosolic PLA(2) was blocked by SB202190. These results indicate that H. pylori induces the release of PGE(2) from epithelial cells by cytosolic PLA(2) activation via G alpha(i)/G alpha(o) proteins and the p38 kinase pathway.

Transmembrane movement of diether phospholipids in human erythrocytes and human fibroblasts.

We have synthesized spin-labeled (SL) and fluorescently labeled diacyl, 1-alkyl-2-acyl-, and di-alkyl glycerophospholipids. The sn-2 chain was a short chain with either a nitroxide group or a 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD). After incorporation in the exoplasmic leaflet of human erythrocytes, we found that SL-phosphatidylcholine (PC) redistributed very slowly across the plasma membrane, less than 20% reaching the cytoplasmic leaflet in 3 h at 37 degrees C. In contrast, SL-phosphatidylserine (PS) accumulated on the cytoplasmic leaflet with the same plateau corresponding to 90% of the probes inside. The characteristic times for inward redistribution were different for the three PS analogues: at 37 degrees C, the t(1/2) for the diacyl, alkyl-acyl, and dialkyl compounds were 2.3, 3.5, and 41 min, respectively. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid translocation of the PS derivatives. The diether PS bearing an NBD group translocated very slowly in human erythrocytes and no acceleration by ATP could be measured. On the other hand, in human fibroblasts, the diether NBD-PS and SL-PS were both transported from the exoplasmic to the cytoplasmic monolayer of the plasma membrane as it is the case for the transport of the respective diester PS analogues. These results prove that the ether bonds do not prevent completely PS binding and translocation by the aminophospholipid translocase despite a probable hindrance due to the ether linkage on the sn-2 chain. Because of the high stability of the ether linkage, SL and NBD diether analogues should be useful to investigate lipid traffic in cultured cells.

Lipid traffic: the ABC of transbilayer movement.

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs.

Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.

Lipid domains in the exoplasmic and cytoplasmic leaflet of the human erythrocyte membrane: a spin label approach.

The existence of different lipid domains in the monolayers of the human erythrocyte membrane was investigated at 4 degrees C by employing spin-labelled phospholipid analogues. Spectra of analogues located exclusively either in the exoplasmic or in the cytoplasmic leaflet of erythrocyte membranes were recorded. Spectra were simulated by variation of order parameter describing the average amplitude of motion of the long molecular axis of the nitrogen 2 p pi orbital of the spin label and of the respective correlation times. For both leaflets at least three components were required to fit the experimental spectra, differing mainly in the order parameter. While the parameters of each component are not very different between both membrane halves, the relative contribution of each component to the spectrum is different between the exoplasmic and cytoplasmic leaflet. The order parameter of the most fluid component, presumably resembling the lipid bulk phase, is smaller in the cytoplasmic leaflet in comparison to the exoplasmic one. The lateral coexistence of different lipid domains in the human red blood cell membrane is concluded. The molecular nature of those domains is discussed.

Internalization of phospholipids from the plasma membrane of human osteoblasts depends on the lipid head group.

The redistribution of spin- or fluorescence-labeled phospholipid analogs across the plasma membrane of human osteoblast cells, either in suspension or grown as monolayers, was investigated. After incorporation into the outer membrane leaflet, analogs of the aminophospholipids phosphatidylserine and phosphatidylethanolamine moved rapidly to the inner monolayer, whereas the choline-containing analogs of phosphatidylcholine and sphingomyelin disappeared more slowly from the outer leaflet. The fast inward movement of the aminophospholipids became reduced after lowering the intracellular ATP, suggesting the presence of an aminophospholipid translocase activity in the plasma membrane of these cells. From these data, a transverse phospholipid asymmetry in osteoblasts can be inferred with the aminophospholipids mainly concentrated in the inner monolayer and the choline-containing phospholipids in the outer leaflet. A similar pattern of phospholipid internalization was inferred for osteoblasts from human osteoporotic bones and for a human osteosarcoma cell line. The relevance of the enrichment of phosphatidylserine in the cytoplasmic membrane leaflet for calcification in skeletal tissues is emphasized.

Protein-mediated inward translocation of phospholipids occurs in both the apical and basolateral plasma membrane domains of epithelial cells.

The translocation of spin-labeled analogues of phosphatidylcholine (4-doxylpentanoyl-PC, SL-PC), phosphatidylethanolamine (SL-PE), phosphatidylserine (SL-PS), and sphingomyelin (SL-SM) from the outer to the inner leaflet of the plasma membrane bilayer was investigated in dog kidney MDCK II and human colon Caco-2 cells. Disappearance from the outer leaflet was assayed using back-exchange to serum albumin. Experiments with cells in suspension as well as with polarized cells on filters were performed at reduced temperatures (10 and 20 degreesC) to suppress endocytosis and hydrolysis of spin-labeled lipids. For both epithelial cell lines, a fast ATP-dependent inward movement of the aminophospholipids SL-PS and SL-PE was found, while SL-SM was only slowly internalized without any effect of ATP depletion. The kinetics of redistribution of SL-PC were clearly different between the two cell lines. In MDCK II cells, SL-PC was rapidly internalized in an ATP-dependent and N-ethylmaleimide-sensitive manner and at a rate similar to that of the aminophospholipids. In contrast, in Caco-2 cells the inward movement of SL-PC was much slower than that of the aminophospholipids, did not depend on ATP, and was not N-ethylmaleimide-sensitive. Inhibitor studies indicated that the outward-translocating multidrug resistance P-glycoprotein present in these cells did not affect the kinetics of inward translocation. Internalization was always similar on the apical and basolateral cell surface, suggesting the presence of the same phospholipid translocator(s) on both surface domains of epithelial cells. We propose that Caco-2 cells contain the well-known aminophospholipid translocase, while MDCK II cells contain either two translocases, namely, the aminophospholipid translocase and a phosphatidylcholine-specific translocase, or one translocase of a new type, translocating aminophospholipids as well as phosphatidylcholine.

Stability of transbilayer phospholipid asymmetry in viable ram sperm cells after cryotreatment.

The transbilayer dynamics of lipids in the plasma membrane of mammalian sperm cells is crucial for the fertilization process. Here, the transbilayer movement and distribution of phospholipids in the plasma membrane of fresh, ejaculated and cryopreserved ram spermatozoa was studied by labeling cells with fluorescent analogues of phosphatidylserine and phosphatidylcholine. By co-labeling cells with the DNA-binding dye propidiumiodide as well as by employing fluorescence microscopy and flow cytometry we were able to determine the transbilayer redistribution of fluorescent phospholipid analogues in intact (propidiumiodide-negative) and in impaired (propidiumiodide-positive) spermatozoa. The transbilayer distribution of the fluorescent phosphatidylserine and phosphatidylcholine analogues was not perturbed in intact sperm cells after cryopreservation. In those cells, the phosphatidylserine analogue became rapidly enriched on the cytoplasmic leaflet by the activity of a putative aminophospholipid translocase similar to intact cells of fresh, ejaculated samples. However, upon cryopreservation the activity of the putative aminophospholipid translocase was significantly reduced in intact cells. Employing annexin V-FITC, we found that even after cryopreservation the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells. The phosphatidylcholine analogue redistributed very slowly remaining essentially confined to the exoplasmic leaflet of the plasma membrane of intact cells from both fresh, ejaculated and cryopreserved samples. The physiological consequences of a perturbed transbilayer asymmetry in sperm plasma membranes is discussed.

Release of phospholipids from erythrocyte membranes by taurocholate is determined by their transbilayer orientation and hydrophobic backbone.

Bile salts mediate a specific release of phosphatidylcholine (PC) from the canalicular membrane into the bile fluid. We utilized human red blood cells (RBC) as a model system to study the release of endogenous phospholipids as well as phospholipid analogues from plasma membranes in the presence of the bile salt taurocholate (TC). Short- and long-chain fluorescent as well as spin-labeled analogues with various headgroups were chosen. RBC were labeled either on the exoplasmic or on the cytoplasmic leaflet with the analogues and incubated with various concentrations of TC. Analogues on the exoplasmic layer could be readily released by TC. Release was most efficient above the critical micellar concentration (CMC) of TC. Release was independent of the headgroup, but depended on the fatty acid chain length of the analogues; i.e., it was lower for long-chain than for short-chain labeled phospholipids. Analogues on the cytoplasmic leaflet were efficiently shielded from TC-mediated release. The preferential release of endogenous PC and sphingomyelin (SM) from the erythrocyte membrane above the CMC supports the conclusion that TC-mediated release of phospholipids occurs preferentially from the exoplasmic leaflet independent of their headgroup. However, the extent of release of endogenous phospholipids was significantly lower in comparison to that of analogues, endorsing the relevance of the hydrophobic backbone for bile salt mediated release of phospholipids. Implications for the mechanism of the release of PC from the canalicular membrane into the bile fluid are discussed.

Influence of pH on phospholipid redistribution in human erythrocyte membrane.

The influence of the suspension pH (pHo) on the transmembrane mobility of spin-labeled phospholipid analogues in the human red blood cell was investigated. The passive transverse diffusion of spin-labeled phospholipid analogues was independent of pHo in the investigated range (5.8 to 8.5). However, upon acidification to pHo 5.8, a significant decrease of the rapid adenosine triphosphate (ATP)-dependent inward movement of aminophospholipids was found at physiologic ionic concentration, whereas a change of pH from 7.4 to 8.5 did not affect this transport. Evidence is given that the intracellular pH affects the active transport of aminophospholipids but not the extracellular pH. Suppression of the ATP-dependent outside-inside redistribution of aminophospholipid analogues by low pH was reversible because original transport activity was re-established upon reneutralization. pH dependence of the active phospholipid transport was not caused by the spin-labeled reporter group or by depletion of intracellular ATP. Because the same influence of pH on aminophospholipid movement could be observed for resealed ghosts, constituents of the red blood cell cytoplasm do not mediate the influence of pH on the ATP-dependent inward movement of aminophospholipids.

Transverse movement of spin-labeled phospholipids in the plasma membrane of a hepatocytic cell line (HepG2): implications for biliary lipid secretion.

The redistribution of spin-labeled phospholipid analogs across the plasma membrane of HepG2 cells, either in suspension or grown as monolayers, was investigated. After incorporation into the outer membrane leaflet spin-labeled aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) moved rapidly to the inner monolayer, whereas the analog of phosphatidylcholine (PC) disappeared more slowly from the outer leaflet. The fast, inward movement of the aminophospholipids was abolished after adenosine triphosphate (ATP)-depletion of cells, suggesting the presence of an aminophospholipid translocase in the plasma membrane of these cells. Compared with human red blood cells, the activity of the aminophospholipid translocase is two orders of magnitude higher in HepG2 cells. From these data, a transverse phospholipid asymmetry can be inferred with the aminophospholipids mainly concentrated on the inner monolayer and the choline-containing phospholipids on the outer leaflet. The relevance of the enrichment of PC in the outer membrane leaflet for the formation and composition of the bile is discussed.

The influence of external surface potential and transmembrane potential on the passive transbilayer movement of phospholipids in the red blood cell membrane.

The passive transbilayer movement of spin-labelled analogues of phosphatidyl-choline (PC), phosphatidyl-ethanolamine (PE), and phosphatidyl-serine (PS) in red blood cell membranes was investigated at physiological and low ionic strength of the extracellular solution. Passive transbilayer movement of aminophospholipids PS and PE was measured in ATP-depleted cells. To discriminate between a possible surface potential and a transmembrane potential effect, NaCl in physiological ionic strength solution was replaced either by sucrose or by Na-tartrate (constant osmolarity). Neither in sucrose (low ionic strength) nor in Na-tartrate media a significant change of the translocation rate of the phospholipids was observed. From these results in can be concluded that changes of the external surface potential as well as of the transmembrane potential do not affect the passive transbilayer movement of phospholipids in human red blood cells.

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach.

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

ATP-dependent redistribution of phosphatidylethanolamine in the plasma membrane of an epithelial and a hepatocytic cell line.

The redistribution of spin-labeled phospholipid analogues of sphingomyelin (SM) and phosphatidylethanolamine (PE) from the outer to the inner leaflet of the plasma membrane of an epithelial (CaCo-2) and a hepatocytic cell line (HepG2) was investigated. The amount of analogues in the outer leaflet was determined by their back-exchange to bovine serum albumin (BSA). For both cell lines a fast ATP-dependent inward movement of spinlabeled PE (SL-PE) was found while SL-SM redistributed only slowly by a passive mechanism. After depletion of intracellular ATP transverse diffusion of SL-PE was similar to that of SL-SM. The data are compatible with the presence of an aminophospholipid translocase in both cell lines.