Guanidine hydrochloride-induced shedding of a Dictyostelium discoideum plasma membrane fraction enriched in the cyclic adenosine 3',5'-monophosphate receptor

The cell surface cyclic AMP receptor of Dictyostelium discoideum is under study in a number of laboratories with respect to both its role in development of the organism and the physiology of excitation-response coupling. We report here that when starved amoebae are exposed to the chaotrope guanidine hydrochloride at 1.8 M, they shed a particulate cyclic AMP binding activity into the medium. This activity is due to membrane vesicles which originate from the cell surface. The vesicles are enriched up to 150-fold in cyclic AMP binding activity and up to 14-fold in phospholipid content when compared to the starting amoebae. The cyclic AMP binding activity of the membrane vesicles is identical to that of the cell surface receptor with respect to the following properties; (i) it is lacking in preparations from unstarved, vegetative amoebae; (ii) it is not inhibited by cyclic GMP and is stimulated by calcium ions; (iii) it has very rapid rates of association and dissociation of bound cyclic AMP; (iv) it has two classes of binding sites with dissociation constants similar to those of the surface receptors of whole amoebae. The binding activity of the isolated membranes is stable for several days at 4 degrees C and the lower affinity binding sites are stable up to several months when stored at -80 degrees C. Due to enrichment and stability of the receptor in this preparation, it should be highly suitable for many types of studies. The usefulness is enhanced by the fact that the preparation does not contain detectable cyclic AMP phosphodiesterase activity.     

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.     

Membrane cycling and macrovacuolation under the influence of cytochalasin: kinetic and morphometric studies

Fibroblasts exposed to higher doses of cytochalasin accumulate very big discrete endoplasmic vacuoles, the membrane of which is derived by internalization of plasmalemma. Morphometry confirms that the amount of surface interiorized is equal to the difference between the original cell surface area (before CD) and the reduced surface area measurable after CD-induced rounding. Correspondingly, there is a nearly two-fold increase in the activity of the ectoenzyme 5'-nucleotidase (a marker for plasma membrane) internally within the cytoplasm, after treatment with CD. Macrovacuolation increases cell volume by approximately 30%. Surface membrane is internalized as micropinocytotic vesicles at a rate measurable by the accumulation of HRP, a marker of fluid-phase pinocytosis. Uptake of HRP is shown to be enhanced at all times during exposure to CD, and is balanced by accelerated exocytic recycling of membrane except during a phase (approximately 4-8 hr) in which pinocytic uptake exceeds exocytosis. Vesicular membrane accumulated intracellularly in this period is retained in the endoplasm, and by successive fusions forms vacuoles in close approximation to microfilament aggregates. Once established, this new macrovacuolar membrane compartment is in dynamic equilibrium with the cell surface, and its membrane is cycled like the plasma membrane, in a mutual exchange of pinosomes between the several vacuoles and the cell surface. In drug-free medium vacuole membrane apparently reverts to the surface by pinocytotic recycling, and the cells recover normal characteristics 4-6 hr after withdrawal of cytochalasin.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

A description of seven Antarctic marine gymnamoebae including a new subspecies, two new species and a new genus: Neoparamoeba aestuarina antarctica n. subsp., Platyamoeba oblongata n. sp., Platyamoeba contorta n. sp. and Vermistella antarctica n. gen. n. sp

Seven marine gymnamoebae were isolated from different environments of seawater, slush (pack ice meltwater), and sediment in the Ross Sea area of Antarctica. All amoebae were isolated and maintained at temperatures below 4 degrees C. Growth, rate of locomotion, and general morphology were observed at an environmentally appropriate temperature (1 degrees C) and at room temperature (approximately 25 degrees C). Molecular (srDNA sequences) and microscopical techniques were used to identify the gymnamoebae and establish their phylogenetic affinities. Three isolates (S-131-2, SL-200, and W4-3) were assigned to a psychrophilic subspecies of Neoparamoeba aestuarina, N. aestuarina antarctica n. subsp., one isolate (S-205) was assigned to a new species of Platyamoeba, P. oblongata n. sp., two isolates (W51C#4 & W51C#5) were also assigned to a new species of Platyamoeba, P. contorta n. sp., and one isolate (S-241) was a novel psychrophilic gymnamoeba Vermistella antarctica n. gen. n. sp. Molecular and morphological results revealed that V. antarctica was not related to any described family of gymnamoebae. Strains S-205, W51C#4, and W51C#5 were capable of locomotion at room temperature, while strains SL-200, S-131-2, W4-3, and S-241 exhibited locomotion only below approximately 10 degrees C. Our results imply that the Antarctic environment is host both to cosmopolitan gymnamoebae that have acquired adaptations for existence at low environmental temperature and to apparently novel psychrophilic amoebae described here for the first time.     

Vacuolar pH is one factor that regulates hydrolase secretion

Lysosomal hydrolases are continually secreted by Acanthamoeba as a consequence of membrane cycling between the vacuolar compartment and the cell surface. In pinocytosing amoebae acid hydrolases can be separated into two groups on the basis of their secretion kinetics. We have previously shown that in Acanthamoeba acid hydrolases are almost exclusively restricted to a single compartment, digestive vacuoles, and that pH-dependent differential binding of hydrolases to vacuolar membrane can account for the different rates of hydrolase secretion from this compartment. In this report we show that the hydrolase secretion pattern changes and that all of the hydrolases are released with the same kinetics after phagocytosis of yeast or in growth media supplemented with ammonium acetate or chloroquine, but not after phagocytosis of polystyrene beads. The changes in the pattern of hydrolase secretion correlate with changes in vacuolar pH. The vacuolar pH of pinocytosing amoebae and amoebae saturated with beads is about 4.8. This value is increased to 6.8 by accumulation of weak bases and to about 6.1 when digestive vacuoles are saturated with yeast. These results indicate that vacuolar pH modulates hydrolase transport and secretion.     

The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: a modelling study

BACKGROUND AND AIMS: It has recently found that lowland rice grown hydroponically is exceptionally efficient in absorbing NO3-, raising the possibility that rice and other wetland plants growing in flooded soil may absorb significant amounts of NO3- formed by nitrification of NH4+ in the rhizosphere. This is important because (a) this NO3- is otherwise lost through denitrification in the soil bulk; and (b) plant growth and yield are generally improved when plants absorb their nitrogen as a mixture of NO3- and NH4+ compared with growth on either N source on its own. A mathematical model is developed here with which to assess the extent of NO3- absorption from the rhizosphere by wetland plants growing in flooded soil, considering the important plant and soil processes operating. METHODS: The model considers rates of O2 transport away from an individual root and simultaneous O2 consumption in microbial and non-microbial processes; transport of NH4+ towards the root and its consumption in nitrification and uptake at the root surface; and transport of NO3- formed from NH4+ towards the root and its consumption in denitrification and uptake by the root. The sensitivity of the model's predictions to its input parameters is tested over the range of conditions in which wetland plants grow. KEY RESULTS: The model calculations show that substantial quantities of NO3- can be produced in the rhizosphere of wetland plants through nitrification and taken up by the roots under field conditions. The rates of NO3- uptake can be comparable with those of NH4+. The model also shows that rates of denitrification and subsequent loss of N from the soil remain small even where NO3- production and uptake are considerable. CONCLUSIONS: Nitrate uptake by wetland plants may be far more important than thought hitherto. This has implications for managing wetland soils and water, as discussed in this paper.     

Disappearance of macrophage surface folds after antibody-dependent phagocytosis

We have employed the method of Burwen and Satir (J. Cell Biol., 1977, 74:690) to measure the disappearance of surface folds from resident guinea pig peritoneal macrophages after antibody-dependent phagocytosis. Unilamellar phospholipid vesicles containing dimyristoylphosphatidylcholine and 1 mol % dinitrophenyl-epsilon- aminocaproyl-phosphatidylethanolamine, a lipid that possesses a hapten headgroup, were prepared by an ether injection technique. These vesicles were taken up by macrophages in a time- and temperature- dependent fashion. Vesicles that contained ferritin trapped in the internal aqueous volume were identified within macrophages by transmission electron microscopy. Scanning electron microscopy has shown that macrophage surface folds decrease dramatically after phagocytosis. The surface fold length (micrometer) per unit smooth sphere surface area (micrometer2) decreases from 1.3 +/- 0.3 micrometer- 1 to 0.53 +/- 0.25 micrometer-1 when cells are incubated in the presence of specific anti-DNP antibody and vesicles at 37 degrees C. No significant effect was observed in the presence of antibody only or vesicles only. Our studies shown that phagocytosis is associated with a loss of cell surface folds and a loss of cell surface area, which is consonant with current views of the endocytic process. On the basis of our uptake data, we estimate that approximately 400 micrometer2 of vesicle surface membrane is internalized. The guinea pig macrophage plasma membrane has a total area of approximately 400 micrometer2 in control studies, whereas the cells have roughly 300 micrometer2 after phagocytosis. These estimates of surface areas include membrane ruffles and changes directly related to changes in cell volume. We suggest that during antibody-dependent phagocytosis a membrane reservoir is made available to the cell surface.     

Quantification of endocytosis-derived membrane traffic

The main data covered by this article have been summarized in Table I. A fairly uniform picture is obtained for endocytosis-derived membrane transfer and compartmentation. This may be due to the limited amount of information and the resulting low resolution. Data on mainly three cell types are presented: macrophages, fibroblasts and amoebae. The data vary as much for one cell type as between different cells. Therefore, no possible differences related to cell function emerge. More detailed data, for more cell types, may change the picture. The values for cell surface area, although significantly different in absolute terms (column S in Table I), are rather similar when related to cell diameter, all being about 3-fold in excess of the surface area of the smooth sphere of comparable volume (column xi in Table I). The rate of plasma membrane internalization for macrophages and amoebae both professional phagocytes, is about 2 cell surface area equivalents per h or more. This may be somewhat higher than for fibroblasts (column PM/h in Table I). The average residence time for membrane on the cell surface, therefore, is about 30 min. A most interesting finding seems to be the rather uniform values obtained for the average size (volume weighted) of primary pinosomes, being about 0.3 micron in diameter (column phi-Internalization in Table I). Due to their rapid increase in size as a result of fusion (cf. Fig. 2), it has not been feasible to directly measure the size of primary pinosomes by morphometric means. The values in Table I, give no information on the size distributions of primary pinosomes and on whether these consist of one or more size classes. The steady-state average diameter of pinosomes is noticeably larger than that of primary pinosomes (column phi-pinosomes in Table I; cf. Table II for Acanthamoebae). The corresponding decrease in surface-to-volume ratio can make about 50% of pinosomal membrane available for recycling directly from this membrane compartment. Membrane recycling from the pinosomal compartment occurs after an average residence time of about 3 min for macrophages and 4-6 min for fibroblasts (column tau-pinosomes in Table I). The relative pool size of intracellular membranes participating in shuttling to and from the cell surface is significantly different for animal cells and amoebae (column rho in Table I). For macrophages, fibroblasts, CHO cells, and mast cells, this intracellular membrane pool amounts to about 10-20% the plasma membrane area, compared to 150-200% in the case of amoebae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of DNA repair of a UV-sensitive mutant (radC) of Dictyostelium discoideum

Summary A radiation-sensitive mutant, TW8(radC), of Dictyostelium discoideum is more sensitive to ultraviolet light (UV) killing than the parental wild strain NC4(RAD ⁺), but is resistant to 4-nitroquinoline 1-oxide (4NQO) at almost the same level as NC4. In TW8 amoebae, single-strand breaks of DNA molecules were hardly detectable immediately after UV irradiation, and the removal of pyrimidine dimers was depressed during the postirradiation incubation when compared with that of NC4 amoebae. After treatment with 4NQO, however, single-strand breaks were detected in TW8 amoebae. The almost complete rejoining of these breaks was also detected after the removal of 4HAQO-adducts. The TW8 amoebae have an efficient repair capacity against DNA damage caused by 4NQO, MMS, MMC and MNNG but not UV.Abbreviations 4NQO 4-nitroquinoline 1-oxide - MMS methyl methanesulphonate - MMC mitomycin C - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Syntaxin 4 expression affects glucose transporter 8 translocation and embryo survival

Target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) are receptors that facilitate vesicle and target membrane fusion. Syntaxin 4 is the t-SNARE critical for insulin-stimulated glucose transporter 4 (GLUT4)-plasma membrane fusion in adipocytes. GLUT8 is a novel IGF-I/insulin-regulated glucose transporter expressed in the mouse blastocyst. Similar to GLUT4, GLUT8 translocates to the plasma membrane to increase glucose uptake at a stage in development when glucose serves as the main substrate. Any decrease in GLUT8 cell surface expression results in increased apoptosis and pregnancy loss. Previous studies have also shown that disruption of the syntaxin 4 (Stx4a) gene results in early embryonic lethality before embryonic d 7.5. We have now demonstrated that syntaxin 4 protein is localized predominantly to the apical plasma membrane of the murine blastocyst. Stx4a inheritance, as detected by protein expression, occurs with the expected Mendelian frequency up to embryonic d 4.5. In parallel, 22% of the blastocysts from Stx4a+/- matings had no significant insulin-stimulated translocation of GLUT8 whereas 77% displayed either partial or complete translocation to the apical plasma membrane. This difference in GLUT8 translocation directly correlated with one-third of blastocysts from Stx4a+/- mating having reduced rates of insulin-stimulated glucose uptake and 67% with wild-type rates. These data demonstrate that the lack of syntaxin 4 expression results in abnormal movement of GLUT8 in response to insulin, decreased insulin-stimulated glucose uptake, and increased apoptosis. Thus, syntaxin 4 functions as the necessary t-SNARE protein responsible for correct fusion of the GLUT8-containing vesicle with the plasma membrane in the mouse blastocyst.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Characterization of DNA from the cellular slime mould Dictyostelium discoideum after growth of the amoebae in different media

Amoebae of the cellular slime mould Dictyostelium discoideum Ax2 grown on Aerobacter aerogenes as food source have a DNA content (36.0 ± 0.9 × 10⁻¹⁴ g/cell) approximately twice that of the same amoebae grown axenically (16.8 ± 0.4 × 10⁻¹⁴ g/cell). Isolation and characterization of DNA from amoebae grown either axenically or on bacteria, by several methods (melting curve, density gradient centrifugation, DNA/DNA hybridization) suggests that not more than 16% of the DNA content of bacterially grown amoebae is of bacterial origin. Studies of the rate of reannealing of DNA samples isolated from amoebae grown either axenically or on bacteria and of the degree to which they hybridize with ribosomal RNA, suggests that the ‘extra’ DNA that bacterially grown cells contain is biologically similar to that contained in axenically grown cells. It is therefore concluded that amoebae growing exponentially on bacteria have, on average, 2.4 to 2.7 genome equivalents per cell and amoebae growing exponentially in axenic medium have 1.3 to 1.4 genome equivalents per cell. Since it is believed that amoebae of this strain growing on bacteria are haploid and since these differences in DNA content persist during their subsequent differentiation, it is concluded that axenically grown amoebae differentiate whilst in the G1 phase of the cell cycle and bacterially grown amoebae differentiate whilst in the G2 phase of the cell cycle.     

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.     

Limiting CO2 levels induce a blue light-dependent HCO3- uptake system in Monoraphidium braunii

The in situ photoactivation of an HCO3- uptake system in the green alga Monoraphidium braunii requires the irradiation of the cell suspensions with short wavelength radiation (blue, UVA and/or UVC). Plasma membrane ATPase inhibitors block the uptake of this monovalent anion at pH 9. M. braunii cells grown in high CO2 lack an HCO3- uptake system in their plasma membrane, but those grown in low CO2 can take up this anion at high rates. Cells grown in high CO2, transferred to CO2-limiting conditions in the light, start taking up HCO3- in 30 min, although they take 90 min to reach maximum rates of HCO3- transport. Therefore, this induction process seems to be triggered by low external CO2 concentration. In fact, increasing or decreasing the external HCO3- concentration does not induce the uptake system and only a decrease in CO2 concentration in the medium triggers the induction process. The appearance of the HCO3- transport activity is sensitive to cycloheximide, indicating that cytoplasmic protein biosynthesis is necessary for the induction of the uptake system. Photosynthetically active radiation, but not particularly blue light, is essential for induction of the uptake system to occur and the inhibition of photosynthesis by DCMU blocks it. From these results it can be inferred that when M. braunii cells detect a drop in CO2 concentration, they induce a blue light-dependent HCO3- uptake system.     

The Three-dimensional Structure of an Ionotropic Glutamate Receptor Reveals a Dimer-of-dimers Assembly

The ionotropic glutamate receptors (iGluRs) represent a major family of ion channels whose quaternary structure has not yet been defined. Here, we present the three-dimensional structure of a fully assembled iGluR, determined at approximately 20A resolution by electron microscopy. Analysis of negatively stained single-particle images reveals the presence of 2-fold, but not 4-fold, symmetry for these tetrameric channels, providing the first direct structural evidence for a dimer-of-dimers assembly. The receptor appears elongated, measuring approximately 170Ax140Ax110A, with the 2-fold symmetry centered on its longitudinal axis. The overall molecular shape and symmetry suggest an orientation relative to the membrane and permit the identification of a putative transmembrane domain. Internal cavities located along the longitudinal axis may represent components of the ion conduction pathway.     

Thermoelasticity of large lecithin bilayer vesicles.

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.     

Increased vesicle recycling in response to osmotic cell swelling. Cause and consequence of hypotonicity-provoked ATP release

Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1-43. In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2-3 min and lasting for approximately 10-15 min. The hypotonicity-induced increase in membrane surface area, like the cell swelling-induced release of ATP (Van der Wijk, T., De Jonge, H. R., and Tilly, B. C. (1999) Biochem. J. 343, 579-586), was diminished after 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester loading or cytochalasin B treatment. Uptake of TRITC-dextrans, however, was not affected. Treatment of the cells with the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-specific protease Clostridium botulinum toxin F not only nearly eliminated the hypotonicity-induced increase in membrane surface area but also strongly diminished the release of ATP, indicating the involvement of regulated exocytosis. Both the ATP hydrolase apyrase and the MEK inhibitor PD098059 diminished the osmotic swelling-induced increase in membrane surface area as well as the subsequent uptake of TRITC-dextrans. Taken together, the results indicate that extracellular ATP is required for the hypotonicity-induced vesicle recycling and suggest that a positive feedback loop, involving purinergic activation of the Erk-1/2 pathway, may contribute to the release of ATP from hypo-osmotically stimulated cells.     

Changes in the surface membrane during myoblast fusion.

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.     

A novel Na+/HCO3--codependent choline transporter in the syncytial epithelium of the cestode Hymenolepis diminuta

Absorption of exogenous choline by the cestode Hymenolepis diminuta was found to be both Na+- and HCO3--dependent and, at pH 6 to 7, accounted for up to 65% of the total choline uptake. Na+/HCO3- dependent choline uptake was activated at approximately 6 mM HCO3- (EC50 approximately 9 mM), and, above 100 mM Na+, the rate of uptake was directly proportional to the Na+ concentration. Atempts to uncouple Na+-dependent uptake from HCO3--dependent uptake were not successful: K+-depolarization was without effect on HCO3--dependent choline uptake, and use of valinoomycin to hyperpolarize the brush-border membrane resulted in inhibition of uptake. Na-/HCO3--dependent choline uptake was not associated with solvent drag. The Na+/HCO3--dependent choline uptake displayed a Q10 of 6.4 (27 degrees to 37 degrees) and a relatively high activation energy of 126 kJ x mol(-1). At pH 6.0 and 7.0, Na-/HCO3--dependent choline uptake rates were similar, but Na+/HCO3--dependent choline uptake was reduced at pH 5.0. The Na+/HCO3--dependent choline uptake, at pH 7.0, displayed a Kt of approximately 500 microM and a Vmax of 4.01 pmol x mg wet weight(-1) x min(-1). The Na+/HCO3--dependent choline uptake was hemicholinium-3 sensitive, but not significantly inhibited by 200 microM bumetanide, 100 microM amiloride, benzamil, or EIPA or by 1 mM 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) or 4-acetamido-4'-isothiocvanostilbene-2,2'-disulfonic acid (SITS). Although it remains to be shown that HCO3- uptake is coupled directly to both choline and Na+ uptake, the data suggest that choline up take occurs via choline/Na+/HCO3--co-trans porter.