Can hydration forces induce lateral phase separations in lamellar phases?

Large repulsive forces measured between membranes of lamellar lipid phases at low hydration are attributed to hydration interactions which vary widely among lipid species. We include this interaction in a model of lamellar phases of two membrane components (two lipids or lipid and protein). The surface polarization of a mixture is taken as a linear combination of those of the components. The model predicts phase separation at low hydration. This may have important consequences for living cells which are dehydrated either by the osmotic effects of tissue freezing, or by desiccation in unsaturated atmospheres.Abbreviations used ACC cold acclimated protoplasts - NA non cold acclimated protoplasts - DLPC dilauralphosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DPPE dipalmitoylphosphatidylethanolamine - PC phosphatidylcholine - PE phosphatidylethanolamine - L fluid lamellar phase - H_ii inverse hexagonal phase     

Primary Invagination of the Vegetal Plate During Sea Urchin Gastrulation

The initial phase of echinoid gastrulation, primary invagination,involves an inpocketing of a monolayered epithelium. To gaininformation about the nature of the mechanical forces that areresponsible for primary invagination, several experimental approacheshave been taken, using the transparent embryos of the sea urchin,Lytechinus pictus, as the principal material. Vegetal platesisolated microsurgically well before the onset of gastrulationwill invaginate normally, demonstrating that the forces responsiblefor primary invagination are generated by the cells in the vegetal to of the embryo. As shown by serial reconstructions of L.pictus embryos, relatively few cells (about 100) take part inprimary invagination. Both the number of cells and the totalvolume of tissue in the wall of the archenteron increase withtime. Even so, it can be shown that very little movement ofcells over the lip of the blastopore takes place during primaryinvagination, and this process is best viewed as a simple inpocketingof the vegetal epithelium. The cells in the wall of the archenteronhave a distinctive shape; they are elongated along their apico-basalaxes and frequently have enlarged, rounded, basal ends. However,they do not undergo any dramatic changes in shape during primaryinvagination. In particular, there is only a slight decreasein the height of the cells (length along the apico-basal axis),a result that is inconsistent with the hypothesis that invaginationis due to cell rounding (Gustafson and Wolpert, 1967). Examinationof L. pictus and Strongylocentrotus purpuratus gastrulae bytransmission electron microscopy reveals that cells in the wallof the archenteron continue to be joined by typical junctionalcomplexes during primary invagination. In addition, the morphologyof the junctional complex at the gastrula stage is more elaboratethan previously described. Sparse bands of micronlaments areassociated with the plasma membrane at the level of the junctionalcomplexes in both endodermal and ectodermal cells. These andother relevant data on early echinoid gastrulation are discussedin relation to several possible mechanisms of epithelial morphogenesis.     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

Plasma membrane of younger and outer cells is the primary specific site for aluminium toxicity in roots

Experiments were carried out to identify the primary site for aluminium (Al) toxicity in roots. Al accumulated in large amounts in the younger and outer cells in roots of pea and was retarded when the ionic strength of the Al solution was high. Cell destruction was extensive in the regions with high Al accumulation. The accumulation of Al in, and potassium (K) leakage from, the root tip were in the order pea>maize>rice, the same order as their sensitivity to Al.The protoplasts from the root tip portion of pea incubated with Al showed a wrinkled and uneven surface. The protoplasts progressively shrank and eventually collapsed. Viability decreased in this process. In the control protoplasts of maize, -glucan formation was uniform on the spherical surfaces, whereas it was spotty in the Al-treated protoplasts; the cell wall material of the latter contained partly 1, 3--glucan which is known to be synthesised by 1, 3--glucan synthase embedded in the plasma membrane. These results suggest that the specific site for Al toxicity is the plasma membrane of younger and outer cells in roots and that Al tolerance depends largely on the integrity of the plasma membrane.     

Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. I. Protein-carbohydrate bond.

We previously described the use of a counter-rotating cone and plate rheoscope to measure the time and force dependence of break-up of doublets of sphered, swollen, and fixed red cells (SSRC) cross-linked by monoclonal IgM antibody. It has been shown that doublet break-up can occur by extraction of receptors from the membrane, rather than by antibody-antigen bond break-up, and is a stochastic process. We therefore prepared 4.62-microns carboxyl modified latex spheres with a covalently coupled synthetic blood group B antigen trisaccharide. Using a two-step carbodiimide process, ethylene diamine was covalently linked to the carboxyl modified latex spheres, and the trisaccharide, having an eight carbon spacer modified to bear a terminal carboxyl group, was linked to the ethylene diamine. Using these antigen spheres we carried out studies in Couette flow, in a transparent cone and plate rheoscope, of the shear-induced break-up of doublets cross-linked by monoclonal IgM anti-B antibody in 19% and 15% Dextran 40. As previously found with SSRC, over a range of normal force from 55 to 175 pN, there was a distribution in times to break-up. However, the fraction of antigen sphere doublets broken up, which increased from 0.08 to 0.43 at 75 pM IgM, and from 0.06 to 0.20 at 150 pM IgM, was significantly lower than that for the SSRC, where the fraction broken up at 150 pM IgM increased from 0.10 to 0.47. Thus, significantly higher forces were required to achieve the same degree of break-up for doublets of antigen-linked spheres than for SSRC. Computer simulation using a stochastic model of break-up showed that the differences between antigen sphere and SSRC doublet break-up were due to a change in bond character (the range and depth of the bond energy minimum) rather than to an increase in the number of bonds linking antigen-sphere doublets. This supports the notion that antibody-antigen bonds are ruptured in the case of antigen spheres, whereas antigen is able to be extracted from the membrane of SSRC, although changes of receptor substrate from cell to latex and the possibility of latex strand extraction from the microspheres are potential complicating factors.     

Integrin-like protein at the invaginated plasma membrane of epidermal cells in mature leaves of the marine angiosperm<Emphasis Type="Italic"> Zostera marina</Emphasis> L.

By immunoblotting with anti-human integrin polyclonal antibodies (1, 3 or 5), a single distinct band of about 60 kDa was detected in total protein extracts from mature leaves of the seagrass Zostera marina L., but no band was detected in total protein extracts from immature seagrass leaves, freshwater grass leaves or Arabidopsis thaliana (L.) Heynh. leaves. This integrin-like protein was detected by indirect immunofluorescence microscopy on the surface of non-spherical protoplasts of epidermal cells isolated from mature seagrass leaves using an anti-integrin 3 polyclonal antibody. Electron-microscopic analyses with the same antibody indicated that this integrin-like protein was localized specifically in the invaginated plasma membrane of epidermal cells in mature seagrass leaves. Therefore, this integrin-like protein of about 60 kDa may be involved in the developmentally regulated invagination of the plasma membrane in epidermal cells of the seagrass Z. marina.     

Pinocytosis and locomotion of amoebae: XII. Dynamics and motive force generation during induced pinocytosis in A. proteus

Summary The mechanism of induced pinocytosis was investigated in Amoeba proteus by light and electron microscopy. The application of nine different inducing substances revealed that pinocytotic channel formation, elongation, vesiculation, shortening and disappearance are the result of the successive or simultaneous action of both traction and pressure forces, which are produced by the contractile activity of a plasma membrane-associated layer of filaments ranging from a few hundred nm to several in thickness. The initial phase of channel formation is caused by traction forces according to the membrane flow concept, whereas channel elongation and vesiculation mainly result from pressure forces in conjunction with the extrusion of small hyaline pseudopodia. Shortening and disappearance of the pinocytotic channels are brought about by local contractions of the cortical filament layer in the basal region of the hyaline pseudopodia. Experiments using latex beads as marker particles together with inducing substances show that a rapid membrane turnover during pinocytosis can be excluded, and that the plasma membrane slides as an entire structure over the underlying cytoplasm.The authors are most grateful to Mrs. J. Ruch for technical assistance     

Membrane mobility and the Concanavalin A binding system of the plasmalemma of higher plant protoplasts

The binding of a colloidal gold-Concanavalin A (ConA) complex to the plasmalemma of tobacco leaf protoplasts has been investigated using scanning electron microscopy. At 5° C the particles of gold-ConA appear to be randomly distributed over the surface of the protoplast. If the temperature is raised, the particles associate into clusters. Saturation of the membrane with particles can only occur when the weight of ConA in solution exceeds 1 g/10⁴ protoplasts in suspension, and when its concentration exceeds 15 g/ml. These results are discussed in terms of the properties of the ConA binding site and the mobility of such sites within the membrane surface.Abbreviations ConA Concanavalin A - AuConA Colloidal gold-Concanavalin A complex     

A simple technique for estimating operative environmental temperature

• 1.1.|Operative environmental temperature (T_c) is commonly measured using a taxidermic mount consisting of a hollow copper cast of an animal's body covered by the animal's integument. We compare estimates of T_c made using such mounts with those derived from use of painted metal sphere thermometers, which are easier to construct and more rugged than taxidermic mounts.
• 2.2.|Comparison of data for 4 bird species indicates that metal spheres may be acceptable T_c-thermometers for analyses involving multiple measurements over moderately long time-scales (e.g. several hours).
• 3.3.|In this case, positive and negative differences between operative temperature estimated from use of taxidermic mounts and painted spheres tend to compensate and the average difference is usually less than 2°C. This difference is similar to that resulting from postural variation of taxidermic mounts or variation among individual mounts in identical postures.
• 4.4.|Sensitivity analysis indicates that use of painted spheres is unlikely to be an important source of error in estimates of total daily energy expenditure.
• 5.5.|In contrast, use of painted sphere thermometers in analyses involving fewer measurements over shorter-time scales can produce unacceptable discrepancies from values obtained from taxidermic mounts (i.e. up to 6.3°C).

     

Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability

By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 s/1.5 kV*cm^–1 and 200 s/0.5 kV*cm^–1, hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths.     

Movement of surface markers along the pinocytotic pseudopodia ofAmoeba proteus

Summary The movement of latex beads over pinocytotic pseudopodia produced byAmoeba proteus was recorded in the presence of 117.65 mM EGTA as an inducer of pinocytosis. The results show that all particles flow in the direction of pseudopodial growth, with a slightly higher velocity than the advancing frontal edge. This means that markers are removed from the base of a pinocytotic pseudopodium and gradually approach the pseudopodium tip. Two particles on the surface of the same pseudopodium can move at the same rate or differ slightly in the velocity of their forward flow. A bead can move even if another blocks the channel orifice. Retrograde particle movement has never been observed. Whether all latex spheres bound to pinocytotic pseudopodia flow with the laterally mobile plasma membrane fraction, which slides over submembranous contractile layer, or whether the whole cortical complex, the actin network and the plasma membrane, move together towards the invagination site is discussed.     

Effect of urea on the plasma membrane particles in yeast cells and protoplasts

Summary Urea in 4 to 8 M concentrations causes aggregation of the plasma membrane particles in yeast cells. Particle counting and the direction of the thread-like deformed particles indicates the translational movement of the particles along the supporting membrane. The aggregation of the particles was prevented by fixation of the cells with glutaraldehyde. Urea changed also the splitting characteristics of the membranes: faces of the plasma membrane covered with the particles were exposed less often. Yeast protoplasts were mostly desintegrated by urea solutions. In resistant protoplasts the particles were normally dispersed. Due to a strong hypertony of the urea solutions profound changes in plasma membrane invaginations were induced in yeast cells.     

Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts

A functional assay for gibberellin (GA) receptors is described based on the induction of -amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA₄ immobilised to Sepharose beads. A 17-thiol derivative of GA₄, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA₄-17-Sepharose induces high levels of -amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA₄. In order to eliminate the possibility that GA₄ may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA₄, GA₄-17-Sepharose and control Sepharose estimated by determining the relative amounts of -amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA₄17-Sepharose induces -amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.Abbreviation GA(n) gibberellin A(n) We thank Professor Jake MacMillan and Drs. Peter M. Chandler (CSIRO, Division of Plant Industry, Canberra, Australia), Peter Hedden and Johnathan Weir (Unilever, Port Sunlight, UK) for helpful discussions and suggestions. Computer graphics were performed by the University of Bristol Molecular Recognition Centre.     

High frequency fusion of plant protoplasts by electric fields

Mesophyll cell protoplasts of Vicia faba were collected by dielectrophoresis in a highly inhomogeneous alternating electric field (sine wave, 5 to 10 V peak-to-peak value, 500 kHz, electrode distance 200 m). Under these conditions, the cells formed aggregates of two or three on the electrodes or bridges consisting of 4 to 6 protoplasts between the electrodes. This pearl chain arrangement of the cells was only stable for the duration of the applied field. By the additional application of a high single field pulse (square wave, 15 V, 50 s), it was possible to induce cell fusion within the aggregates or bridges. This electrically stimulated fusion of cells proceeded at room temperature and under physiological pH-conditions, without the use of chemical reagents, and gave a high yield. Smaller fused aggregates formed spheres within a few minutes. During the dielectrophoretically induced adhesion of the protoplasts to one another, the field strength must be chosen such that dielectric breakdown of the membrane is avoided, but at the same time, the strength of the subsequently applied single field pulse must be high enough to induce dielectric breakdown at the sites of contact between the protoplast membranes. From these results, one can conclude that in addition to close contact between membranes, the prerequisite for electrically stimulated cell fusion is dielectric breakdown which leads to changes in the membrane conductance, permeability, and probably fluidity.Presented at II Congress FESPP, Santiago de Compostela, Spain, 27.7.–1.8.1980, and Gordon Research Conference of Bioelectrochemistry, Tilton, New Hampshire, USA, 4.8.–8.8.1980     

RGD-mediated membrane-matrix adhesion triggers agarose-induced embryoid formation in sunflower protoplasts

Summary Agarose embedding of sunflower (Helianthus annuus L.) hypocotyl protoplasts induces an asymmetric division pattern and subsequent polarized development leading to embryoid formation. We cultured protoplasts in media with different mannitol concentrations. Induction of plasmolysis of agarose-embedded protoplasts by increasing the mannitol concentration lowered the proportion of embryoids formed. This indicates that adhesion sites between the plasma membrane and the agarose matrix are involved in embryoid formation. The involvement of such adhesion sites was tested by incubating embedded protoplasts with RGD peptide. 1 M RGD heptapeptide reduced embryoid formation by 50% as compared to the control DGR peptide. We also showed that RGD heptapeptide acts on the cytoskeleton by disrupting cortical microtubules. The results are discussed in terms of a model in which the anchorage of the protoplast membrane to the agarose matrix is mediated by RGD-binding proteins connected with microtubules, determining asymmetric division of the cell and polarized development.Abbreviations ECM extracellular matrix - TBS Tris-buffered saline     

Surface charge and calcium binding in sarcoplasmic reticulum membranes

Vesicles of fragmented sarcoplasmic reticulum membranes have been adsorbed on to 2.68 latex spheres. Observation of these vesicle containing spheres in the presence of an electric field allows a calculation of the electrophoretic mobility of the vesicles Following this determination, the net membrane surface charge has been estimated. The mobility of sarcoplasmic reticulum membranes exhibited a dependency on pH. At an ionic strength of 0.10 a mobility (pH=7.0) of –0.67±0.10/sec/volt/cm was observed. At pH=7.0 and /2=0.150 the net excess negative charge density was 2.0×10^–2 coul/m². This is equivalent to one charge per 10³ A² (assuming a uniform charge distribution). With an average vesicle volume of 2.8×10⁸ A³ and a surface area of 2×10⁶ A² the surface of one vesicle would contain a net of approximately 2×10³ negative charges. While the mobility did not change during uptake of calcium by the vesicles, both glutaraldehyde fixation and lecithin extraction by phospholipase C greatly altered the mobility of the vesicle membrane. Calcium binding and uptake both exhibited a dependence on pH.     

An investigation into the feasibility of using azide-insensitive ATPase and ConA as yeast plasma membrane markers

Cytochemical localization of Concanavalin A binding sites in protoplasts of Candida tropicalis, investigated with glycosylated-ferritin and electron microscopy, showed that the lectin was specifically bound to the external protoplast surface. Thus, the plasma membranes have been labelled with ¹²⁵I-Concanavalin A and followed through the isolation procedure. Relative distribution of ¹²⁵I-radioactivity and azide-insensitive ATPase activity in the obtained fractions, suggested that this enzyme was an equivocal plasma membrane marker. Despite the presence of internal Concanavalin A binding sites, Concanavalin A could be used unambiguously as an exogenous plasma membrane marker of intact protoplasts.Abbreviations ConA Concanavalin A - MM -Methyl-D-Mannoside     

Translational control of cyclins

Background

An oocyte undergoes two rounds of asymmetric division to generate a haploid gamete and two small polar bodies designed for apoptosis. Chromosomes play important roles in specifying the asymmetric meiotic divisions in the oocytes but the underlying mechanism is poorly understood.

Results

Chromosomes independently induce spindle formation and cortical actomyosin assembly into special cap and ring structures in the cortex of the oocyte. The spindle and the cortical cap/ring interact to generate mechanical forces, leading to polar body extrusion. Two distinct force-driven membrane changes were observed during 2^nd polar body extrusion: a protrusion of the cortical cap and a membrane invagination induced by an anaphase spindle midzone. The cortical cap protrusion and invagination help rotate the spindle perpendicularly so that the spindle midzone can induce bilateral furrows at the shoulder of the protruding cap, leading to an abscission of the polar body. It is interesting to note that while the mitotic spindle midzone induces bilateral furrowing, leading to efficient symmetric division in the zygote, the meiotic spindle midzone induced cytokinetic furrowing only locally.

Conclusions

Distinct forces driving cortical cap protrusion and membrane invagination are involved in spindle rotation and polar body extrusion during meiosis II in mouse oocytes.     

Virus removal from bioproducts using ultrafiltration membranes modified with latex particle pretreatment

Ultrafiltration is an attractive process for virusremoval from bioproducts owing to its high throughputas well as the fact that the operation is carried outunder ambient conditions (damage to proteins is highlylimited). The principal concern regarding the adoptionof conventional ultrafiltration membranes for virusremoval is the possibility of the virus passingthrough abnormally large pores or surfaceimperfections on the membrane surface. The chiefprinciple behind the present work is to pretreat themembrane by blocking the abnormally large pores usinglatex particles. Experimental work was conducted tovalidate this pretreatment using the bacteriophagex174 as a model virus.The results attained were highly encouraging.Different sizes of latex particles were tested bytreating a 100 KD molecular weight cut-off membrane,and the transmission of phage (suspended in buffer)through this membrane assessed. In the absence of anyparticle pretreatment, a virus clearance of 4.78 logreduction value was observed for this membrane. Thetransmission of phage through the membrane could bereduced by an order of magnitude using 0.11 mlatex particles, or two orders of magnitude using acombination of 0.11 and 0.50 m particles.The application of latex particles did nothinder the transport of protein through the 100 KDmembrane. Protein sieving coefficients obtained usingthis membrane were 91%, 16% and 2%, for lysozyme,HSA and IgG, respectively.     

β-1-4-and β-1-3-glucan synthases are associated with the plasma membrane of the fungus Saprolegnia

Cytoplasmic membranes from mycelium or protoplasts of Saprolegnia monoica (a cellulosic cell-wall fungus) were separated by continuous sucrose-density-gradient centrifugation. Glucan synthases assayed at low (micromolar uridine 5-diphosphate (UDP) glucose for -1-4-glucan synthase) and high (millimolar UDP glucose for -1-3-glucan synthase) substrate concentrations were associated with membranes exhibiting vanadate-sensitive, oligomycin-insensitive ATPase and equilibrating at density 1.16 g cm^-3. Synthase activities were also bound to membranes of lower density (1.10 and 1.145 g cm^-3). Plasma membranes were stabilized by coating protoplasts with concanavalin A. After lysis of the protoplasts, plasma membranes recovered by low centrifugal forces were isolated in continuous isopycinic gradients. Both synthase activities peaked with [³H]concanavalin A and Na-vanadate ATPase indicating that the synthetases are located at the plasma membrane. Treatments of intact protoplasts with cold glutaraldehyde or proteases before disruption lead to a diminution of glucan-synthase activities indicating that at least part of the enzymes of plasma membrane face the outside of the cell.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - GSI -1,4-glucan synthase - GSH -1,3-glucan synthase - UDP uridine 5-diphosphate