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.

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.

An improved assay for measuring the transverse redistribution of fluorescent phospholipids in plasma membranes.

The internalization of fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phospholipids from the plasma membrane can be assessed by the irreversible quenching of analogues in the outer leaflet by dithionite. Here we have utilized this assay to follow the redistribution of short-chain C6-NBD-sphingomyelin and C6-NBD-phosphatidylserine from the cell membrane of human gingiva fibroblasts. The significant uptake of dithionite across the plasma membrane and the subsequent reduction of NBD-analogues exposed to the cytoplasmic lumen does not allow an accurate measurement of the amount of internalized lipid probes even at low temperature. We could show that a precise determination can be achieved by extraction of analogues remaining in the exoplasmic half by a short pretreatment with bovine serum albumin prior to addition of dithionite. The fluorescence of those analogues localized to the cytoplasmic lumen was slowly destroyed by permeating dithionite. The fluorescence of those NBD-probes which are localized in the inner layer of intracellular vesicles remained almost unaffected in the time course of the assay. Thus, this approach allows to distinguish between different routes of internalization of NBD-phospholipids from the plasma membrane.

On the lateral distribution of spicula on echinocytes.

Human erythrocytes were transformed to advanced stages of echinocytes by means of an increase of the pH, by addition of 2,4-dinitrophenol or by an increase in temperature. Scanning electron microscopy pictures were taken and the lateral distribution of the spicula was analyzed. Regardless of the method of the production of the echinocytes, no correlation of the spatial distribution of the spicula was detected. Except for the exclusion due to the finite size of the spicula basis, the distribution was random. The conclusion was drawn that the generation of spicula is a local process. No long-range ordering interaction between the spicula could be detected.

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.

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.

Incorporation of phospholipid analogues into the plasma membrane affects ATP-induced vesiculation of human erythrocyte ghosts.

The influence of various spin-labelled phospholipid analogues on the ATP-dependent vesiculation of human erythrocyte ghosts at 37 degrees C was investigated by monitoring the acetylcholinesterase activity. After incorporation of analogues into the outer leaflet a decline of endocytic vesiculation was observed. However, suppression was arrested after about 20 min when ghosts have been labelled with a phosphatidylserine analogue known to redistribute rapidly to the inner leaflet. These data suggest that vesiculation of erythrocyte ghosts is affected by differential expansion of the inner and outer leaflet.

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.

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.

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.

Protein-dependent translocation of aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells.

We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the exoplasmic monolayer. Variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated into the membrane suggests a saturability of the transbilayer movement of aminophospholipids. ATP depletion or pretreatment with N-ethylmaleimide of ram sperm cells reduced the fast inward motion of PS and PE, indicating a protein-mediated aminophospholipid translocation. The results suggest for the plasma membrane of ram sperm cells the presence of an aminophospholipid translocase and an asymmetric transversal lipid distribution with aminophospholipids preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet. The relevance of the transversal segregation of phospholipids for membrane fusion processes occurring during fertilization is discussed.

Rapid determination of the transbilayer distribution of NBD-phospholipids in erythrocyte membranes with dithionite.

The assessment of the transverse distribution and mobility of NBD-labelled phospholipid analogues in biological membranes by selective chemical destruction of fluorescent label in the outer monolayer with dithionite has been investigated using resealed erythrocyte ghosts as a model system. The distribution of those analogues can be determined in < 30 s directly in the cell suspension provided the permeation of dithionite across the membrane is suppressed. The results were compared with data on translocation of either NBD- or spin-labelled phospholipid analogues obtained with the technique of back exchange to BSA. It is shown that the passage of dithionite can be mediated by anion-transport systems such as band 3 which is inhibited by DIDS. Appropriate conditions for the applicability of the assay were elucidated also using resealed ghosts having fluorescent NBD-taurine in the intracellular lumen. The application of the assay to measure fast translocation processes, e.g. those mediated by the aminophospholipid translocase, is described.

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.

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.

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 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.