Changes In Membrane Architecture Research Articles

A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria

Aging is one of the most fundamental, yet least understood biological processes that affect all forms of eukaryotic life. Mitochondria are intimately involved in aging, but the underlying molecular mechanisms are largely unknown. Electron cryotomography of whole mitochondria from the aging model organism Podospora anserina revealed profound age-dependent changes in membrane architecture. With increasing age, the typical cristae disappear and the inner membrane vesiculates. The ATP synthase dimers that form rows at the cristae tips dissociate into monomers in inner-membrane vesicles, and the membrane curvature at the ATP synthase inverts. Dissociation of the ATP synthase dimer may involve the peptidyl prolyl isomerase cyclophilin D. Finally, the outer membrane ruptures near large contact-site complexes, releasing apoptogens into the cytoplasm. Inner-membrane vesiculation and dissociation of ATP synthase dimers would impair the ability of mitochondria to supply the cell with sufficient ATP to maintain essential cellular functions.

Mechanisms of ESCRT‐mediated cargo sorting and degradation

Vesicle‐mediated cargo transport within the endomembrane system requires precise coordination between adaptor molecules, which recognize sorting signals on substrates, and factors that promote changes in membrane architecture. At endosomal compartments, a set of protein complexes collectively known as the ESCRT machinery sequesters transmembrane cargoes that harbor a ubiquitin modification and packages them into vesicles that bud into the endosome lumen. Using atomic force microscopy, we define the architecture of each ESCRT complex in the presence of physiologically relevant lipid bilayers. This analysis indicates that ESCRT‐0, ESCRT‐I, and ESCRT‐II function as heterotetrameric complexes that collectively bind and sequester ubiquitin‐modified cargoes. Additionally, our analysis reveals a mechanism by which ESCRT‐III membrane scission activity is both spatially and temporally regulated during multivesicular endosome biogenesis. A revised model describing ESCRT function during protein sorting in the endocytic pathway will be presented.

The lens barrier: ultrastructural evidence

Abstract Purpose Studies on cortical 1 and nuclear 2 cataracts suggest that the human lens consists of two compartments differentially susceptible to ageing. Biochemical studies support this view and show that the lens can be divided into a metabolically active cortex and a metabolically inert nucleus. We investigated the presence of gap junctions (GJ), square arrays (SA) and the cholesterol content of the fiber membranes in the axial poles of clear human lenses. Methods Small pieces with known localization were prepared for freeze fracture. Filipin was used as cholesterol probe. Results We find that the outermost posterior fibres are thin (<1 µm), the amount of cholesterol in their membranes high but with a normal membrane architecture with IMPs and GJs. This aspect changes abruptly at a depth of approx. 0.2 mm. There the amount of cholesterol in the membranes increases steeply, the amount of IMPs drops and the GJs become degenerate. On 1 or 2 fibres, at the border of this change in membrane architecture, SAs are found. SAs are absent everywhere else. Anterior in the lens the same phenomena are observed, however, deeper (0.5mm). This is probably related to thicker fibres (>4 µm). Conclusion A high cholesterol content limits water permeability of membranes and it inhibits the function of carriers and transporters. Degenerate GJs will reduce solute flux from superficial fibres to deeper fibres. So it is concluded that the centre of the lens (ca 65% of the total lens volume) is effectively sealed. This supports the existence of a physiological barrier in the lens as proposed by Truscott2 . 1)Vrensen GFJM 2009 A Ophthalmol 87:602‐610. 2)Truscott RJW 2000 Ophthalmic Res 32: 185–194.

Beta-chemokine receptor CCR5 in human spermatozoa and its relationship with seminal parameters

Chemokine receptor CCR5, the main HIV-1 coreceptor, is present in the human spermatozoa. This study aimed to investigate (i) whether the percentage of CCR5-positive spermatozoa varies under conditions associated with changes in the membrane architecture, such as capacitation and fixation/permeabilization procedures; (ii) whether there is any relationship between individual variability in sperm CCR5 expression and semen parameters. In cytometric analysis, the percentage of CCR5-positive unfixed spermatozoa varied from approximately 10 to approximately 60%, and it significantly decreased after 5 h capacitation. The percentage of CCR5-positive spermatozoa was increased to more than 90% following fixation and permeabilization, suggesting the existence of large intracellular pools of the receptor. Immunocytochemistry showed positive staining in the anterior region of the sperm head. In ejaculates from male partner of 102 infertile couples, the CCR5 expression rate significantly correlated with sperm count, total sperm number and forward motility, but not with sperm morphology. In stepwise analysis, only forward motility entered into the model; however, this explained only approximately 8% of the variability in CCR5 expression. Interquartile analysis showed significant differences between the first and fourth quartiles of CCR5 expression for all semen parameters, except morphology. The percentage of CCR5-positive spermatozoa may vary under conditions associated with changes in membrane architecture and spermatozoa showed large intracellular pools of CCR5. A lower expression of CCR5 in asthenozoospermia seems to be suggested; however, it would only partially contribute to the inter-individual variability in the CCR5 expression. A genetic basis can be hypothesized to explain the variability.

Strboh A homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers

During 30-months of storage at 4 degrees C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18-24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca(+2) declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25-30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.

Annexins: putative linkers in dynamic membrane–cytoskeleton interactions in plant cells

The plasma membrane, the most external cellular structure, is at the forefront between the plant cell and its environment. Hence, it is naturally adapted to function in detection of external signals, their transduction throughout the cell, and finally, in cell reactions. Membrane lipids and the cytoskeleton, once regarded as simple and static structures, have recently been recognized as significant players in signal transduction. Proteins involved in signal detection and transduction are organised in specific domains at the plasma membrane. Their aggregation allows to bring together and orient the downstream and upstream members of signalling pathways. The cortical cytoskeleton provides a structural framework for rapid signal transduction from the cell periphery into the nucleus. It leads to intracellular reorganisation and wide-scale modulation of cellular metabolism which results in accumulation of newly synthesised proteins and/or secondary metabolites which, in turn, have to be distributed to the appropriate cell compartments. And again, in plant cells, the secretory vesicles that govern polar cellular transport are delivered to their target membranes by interaction with actin microfilaments. In search for factors that could govern subsequent steps of the cell response delineated above we focused on an evolutionary conserved protein family, the annexins, that bind in a calcium-dependent manner to membrane phospholipids. Annexins were proposed to regulate dynamic changes in membrane architecture and to organise the interface between secretory vesicles and the membrane. Certain proteins from this family were also identified as actin binding, making them ideal mediators in cell membrane and cytoskeleton interactions.

Expression and functional characterization of isoforms 4 of the plasma membrane calcium pump.

PMCA isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII had a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but had a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone was almost 3 times higher than in PMCA4CI and was increased by La3+ less than in the PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) had no Ca2+-dependent ATPase activity, but was still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform was retained in the endoplasmic reticulum; changes in membrane architecture apparently occurred during its expression; the C-terminal portion of the isoform was located in the cytosol, indicating that the deletion of the 10th transmembrane domain resulted in the loss of at least another transmembrane domain.

Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells.

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.

Effects of sterol biosynthesis-inhibiting fungicides and plant growth regulators on the sterol composition of barley plants

A number of sterol biosynthesis-inhibiting (SBI) fungicides and plant growth regulator analogs were applied as root drenches to barley seedlings and their effect on the total sterol composition of the roots and shoots was measured by gas-liquid chromatography. Prochloraz was found to be inactive in this system, probably because of poor uptake, while the other compounds could be divided into two groups according to their mode of action as assessed by sterol profiling. The morpholines tridemorph and fenpropimorph inhibited the enzyme cycloeucalenol—obtusifoliol isomerase whereas triadimenol, nuarimol, paclobutrazol, and triapenthenol (RSW 0411) inhibited the enzyme responsible for the removal of the C-14 methyl group. Effects of individual diastereo-isomers and enantiomers of some compounds on sterol profiles were compared with their known fungicidal and anti-gibberellin properties. Shoot growth was reduced by all the compounds tested, paclobutrazol, nuarimol, and triapenthenol being the most effective. As well as inducing accumulation of abnormal sterols, SBI fungicide treatment changed the ratio of campesterol to stigmasterol and sitosterol. It is hypothesized that this may reflect changes in membrane architecture and may offer an explanation for the increased frost hardiness sometimes observed with SBI fungicide-treated plants.

Studies on the selective toxicity of guanazole in mice

Guanazole ( 3,5-diamino- 1,2,4-triazole (GZ), an antileukemic agent effective against acute myelocytic leukemia, seems to exert its action through inhibition of ribonucleoside diphosphate reductase. Using recently developed technique for drug infusion in unrestricted mice, 5–30 mg/ml guanazole was infused at a rate of 0.2–0.3 ml/hr in normal mice and mice bearing leukemia L 1210 or a subline resistant to guanazole (L1210/GZ). Incorporation of 14C -uridine (UR) into DNA was considerably reduced in spleen, thymus and bone marrow; incorporation of 3H -thymidine was reduced to a much greater extent in these tissues. The inhibition of precursor incorporation was dependent on the concentration of guanazole used and was somewhat correlated to the intracellular drug concentration in different tissues. This inhibition was in the following order: L 1210 spleen > normal spleen > L1210/GZ spleen. Analysis of free intracellular ribonucleotide pools indicated the presence of a high IMP and a low ATP pool in normal spleen, and a moderate IMP and a high ATP pool in spleen infiltrated by cells from either leukemia; this profile resembled that observed in intraperitoneally growing L 1210 cells. Guanazole infusion resulted in the reversion of pools profile from that characteristic of L 1210 spleen to that of normal spleens. The morphological form and microscopic characteristics of L 1210 spleen from mice treated with GZ also resemble those of spleen from non-leukemic mice. Study of in vitro uptake of guanazole in leukemia L 1210, indicated that the transport occurred by a passive diffusion process. In the subline, L1210/GZ, the initial rate of uptake was 1/3rd to 1/4th of that in the parent line. This suggested that a generalized change in membrane architecture is likely to have accurred in L1210/GZ. The results of this study are consistent with the idea that in different tissues in mice, the mechanism of action of guanazole is dependent upon inhibition of ribonucleotide reductase, whereas its selectivity of action is dependent upon differences in uptake.

Action of pure phospholipase A 2 and phospholipase C on human erythrocytes and ghosts

1. 1.|Pancreatic phospholipase A 2 (phosphatide acyl-hydrolase, EC 3.1.1.4) and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus appeared not to be lytic for human erythrocytes, either before or after treatment of the cells with trypsin, pronase or neuraminidase. No significant breakdown of phospholipids could be observed. 2. 2.|Both phospholipases were found to evoke hemolysis in the presence of sublytic concentrations of sodium deoxycholate, whereas sublytic concentrations of Triton X-100 were effective only in combination with phospholipase C. 3. 3.|Treatment of human red cell ghosts with either phospholipase A 2 or phospholipase C resulted in a complete hydrolysis of lecithin, phosphatidylethanolamine and phosphatidylserine, whereas sphingomyelin was not attacked. Similar results were obtained with liposomes derived from human erythrocytes, indicating that the degree of hydrolysis depends only upon the chemical nature of the phospholipids involved. 4. 4.|In the native human erythrocyte membrane the fatty acid-ester linkage at C 2 and the phosphoryl-glycerol linkage at C 3 of the phosphoglyceride molecules apparently are not accessible to phospholipase A and C attack. Removal of the sialic acid residues from the membrane surface does not promote the action of these lipolytic enzymes. Changes in membrane architecture occurring during membrane isolation or as induced by nonlytic concentrations of detergents lead to exposure of membrane phosphoglycerides to phospholipases A and C.