Isolation and properties of the plasmalemma in yeast

Summary A method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg⁺⁺-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

Quantitative characteristics and reconstruction of the surface topography of histochemical markers on nerve cell plasmalemma

An electron-microscopic study of the topography of carbohydrate residues on the surface of the cell body membrane of cultured spinal neurons was carried out using lectins from wheat (WGA) and snail (HPL), labeled with colloidal gold, as specific molecular probes. Mathematical methods of analysis suggested a set of surface markers, from the distribution of particles observed in electron micrographs of random sections, corresponding to two random functions. Analysis of these functions allows the required quantitative characteristics to be obtained. The Monte Carlo reconstructing model is described, and results of its use (based on the aforementioned experimental data) are demonstrated in the form of "averaged" surface topography of the studied markers in a limited section of the membrane. The results obtained are discussed in connection with cooperative properties of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 595–603, September–October, 1991.     

Electrophysiological properties of macrophages

Electrophysiological studies indicate that the macrophage can display at least two different K conductances, a Ca-mediated K conductance and an inward rectifying K conductance, as well as an electrogenic Na+-K+ pump. Spontaneous hyperpolarizations associated with a Ca-mediated K permeability have been noted in all types of macrophages studied. Similar membrane hyperpolarizations can be elicited by a variety of stimuli that presumably increase intracellular calcium. These include mechanical and electrical stimulation as well as exposure to endotoxin-activated serum, chemotactic peptides, and the Ca ionophore A23187. Recent patch clamp studies on macrophages demonstrated channel activity that probably corresponds to currents through the inward rectifying K conductance previously described with current clamp techniques. With the advent of the patch clamp, this and other conductances can be effectively examined by using both whole-cell voltage clamp and patch recordings in a variety of different macrophages, including small freshly isolated cells.     

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Architectonics of the surface of alveolar macrophages

The architectonics of the alveolar macrophage surface has been investigated in the raster electron microscope. The material is obtained by means of washing from the lungs of intact noninbred white rats and also 24 h after a single intragastric administration of a cancerogenic agent--nitrosodimethylamine (NDMA)--in a toxic dose (30 mg/kg). The alveolar macrophages are studied both as a suspension and also after 30 min of cultivation. The preparations are dried in the air and by the critical point method. When the latter method is used, the architectonics of the alveolar macrophage surface is much richer. Nevertheless, the former method also gives enough information. NDMA administration produces a damaging effect on the surface architectonics and on the character of the macrophages spreading over the glass. The morphological characteristics of the changes in the surface architectonics of the alveolar macrophages can be used to estimate the cytotoxic effect of different harmful factors of the environment.     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Cell surface fibronectin of mouse peritoneal macrophages

Fibronectin (FN) was detected on thioglycollate-induced mouse peritoneal macrophages by binding the 125I-labeled F(ab')2 fragment of rabbit anti-human plasma fibronectin. The cell surface fibronectin (sFN) was removed from the surface of the macrophage monolayer by limited trypsinization. After trypsinization, binding of 125I-labeled plasma fibronectin (125I-pFN) to the macrophage monolayer was increased, suggesting that the FN receptor covered with sFN was exposed by trypsinization without destroying the receptor activity. The amounts of saturation binding of 125I-pFN to the macrophage monolayers before and after trypsinization were about 2.4 and 6.3 micrograms per 10(6) cells, respectively, indicating that the macrophage monolayer has the capacity of binding 6.3 micrograms FN per 10(6) cells, and the FN receptor equivalent to about 4 micrograms pFN per 10(6) cells is covered with sFN.     

Calcium fluxes at plasmalemma and tonoplast

Abstract. An account is given of the characterization of calcium fluxes across plasmalemma and tonoplast membranes of root cortical cells, using compartmental analysis. Some of the assumptions associated with the method are discussed. Recent evidence regarding the concentration of free Ca²⁺ in plant cells, and the mechanisms driving active calcium transport across cell membranes, is reviewed. It is proposed that the evidence from whole cell studies and work at the molecular level is mutually supportive, and some speculation is ventured about the general pattern of calcium transport in higher plant cells.     

Effect of volume and pH on surface receptor number in macrophages

Incubation of rabbit alveolar macrophages in hypo-osmotic solutions transiently increases cell volume and inhibits membrane internalization, resulting in an increase in surface receptor number. Since recent reports suggest that hypo-osmotic treatment decreases intracellular pH, and that reduced pH inhibits receptor internalization, pH was measured in hypo-osmotically treated macrophages. We found that cells incubated in iso-osmotic solutions of pH less than 7.2 exhibited a decrease in intracellular pH upon exposure to hypo-osmotic solutions, while cells in iso-osmotic solutions of pH greater than 7.2 had an increase in pH upon exposure to hypo-osmotic solutions. The relative increase in surface receptor number was unaffected by the initial pH or by the direction of change in pH. Incubation of cells in high K+/low Na+ hypotonic buffers induced a persistent increase in cell volume and surface receptor number. Cell volume and surface receptor number fell to baseline values after restoration of isotonicity by the addition of hypertonic sucrose. These manipulations had little effect on intracellular pH. We conclude that the inhibition of membrane internalization observed in cells exposed to hypo-osmotic solutions is independent of changes in intracellular pH. The inhibition of internalization observed in this system may be due directly to forces produced as a consequence of cell swelling.     

Membrane surface of Mycobacterium microti-infected macrophages antigenically differs from that of uninfected macrophages

Identification of the antigenic changes in mycobacteria-infected macrophage may be important in understanding the mechanisms responsible for the intracellular survival of the bacteria. In the present study, Mycobacterium microti-infected macrophages were utilized to investigate the possibility of differentiating the infected cells from normal cells, based on the antigenic changes occurring in the membranes. Antisera were generated against bacterial extract, heat-killed bacteria and crude preparation of M. microti-infected homologous macrophage membrane. The reactivity of these antisera, towards in vitro infected macrophages, was compared by flow cytometry. Unlike anti-bacterial extract antiserum or anti-heat-killed bacterial antiserum, anti-infected macrophage membrane antiserum reacted with infected macrophage surface. This reactivity increased with the increase in post-infection time. However, it was not observed with uninfected macrophages, PMA- or lipopolysaccharide-activated macrophages and those harboring Mycobacterium tuberculosis H37Ra, heat-killed M. microti and Leishmania donovani. Interestingly, anti-infected macrophage membrane antiserum identified a 63-kDa antigen in M. microti-infected macrophage membranes which was not present in the membranes of normal macrophages, activated macrophages and of those infected with M. tuberculosis H37Ra, heat-killed M. microti and L. donovani. Thus, membranes of M. microti-infected macrophages differ antigenically from those of the normal macrophages and infected homologous macrophage membrane antiserum provides a useful tool in studying such changes.     

Fine structure of the plasmalemma surface of poplar parenchyma cells observed by the freeze etching technique

The plasmalemma surface of poplar parenchyma cells observed by the freeze etching technique is characterized by 11 nm particles, primary pit fields, lomasome-like structures and fibrillar structures. The most remarkable feature is the occurrence of fibrillar structures, which are considered to be the imprint of underlying microtubules on the plasmalemma surface. The previously reported observation on the possible appearance of microtubules at the cytoplasmic surface inside the plasmalemma is questionable. Although the fibrillar structures run almost perpendicular to the main cell axis, longitudinally oriented ones are also found, the occurrence of which is discussed in relation to the orientation of the cellulose microfibrils.     

Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534/mum2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300/MUM2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation, we analyzed synthetic events taking place at this time, but no correlation with 25SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibroblasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma.     

Significance of plasmalemma disruption in bovine and equine spermatozoa

We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their incidence in both fresh and frozen-thawed material from both animals. The vesicles were formed by the separation and expansion of the plasmalemma away from the underlying structure but were not caused by the freeze-thaw process. We suggest that these structures form part of the normal ultrastructure of spermatozoa and are damaged during preparation of the samples for transmission electron microscopy, resulting in a structure previously and incorrectly identified as damaged by the freezing and thawing process.     

Cellulose microfibril deposition at the plasmalemma surface of regenerating tobacco mesophyll protoplasts: A deep-etch study

Summary The reappearance of cellulose microfibrils at the naked surface of protoplasts enzymatically isolated from tobacco (Nicotiana tabacum L. var. Xanthi) mesophyll tissue has been closely studied using the techniques of thin-sectoining and the deep-etch modification of the freeze fracture procedure.A 16 h lag period was recorded between the time of isolation and the sudden appearance of considerable lengths of cellulose microfibril at the outer protoplast surface. The microfibrils were not associated with any structured particles or apparently differentiated regions of the plasmalemma. Terminal regions of the microfibrils appeared to have tapering ends, or else be sinking into the membrane substance. There was no evidence to suggest transport of intact microfibrils in vesicles through the cytoplasm to the plasmalemma.The reported observations have been discussed with respect to the various working hypotheses which have been proposed for the in vivo construction of cellulose microfibrils.     

Identification of albumin-binding proteins of thymocyte plasmalemma

In the present work we examined whether the interaction between albumin molecules and thymocytes involves albumin-binding proteins (ABP). Two plasmalemma-rich fractions obtained by differential centrifugation from rat thymus lymphocytes were characterized biochemically and morphologically. These fractions were examined by ligand-blotting and ligand affinity chromatography techniques. Plasmalemma proteins separated by SDS-PAGE were electrotransferred onto nitrocellulose membranes and incubated with¹²⁵I-albumin, in the presence or absence of excess native albumin. The autoradiogram revealed specific binding to two sets of polypeptides of 16–18 and 29–31 kDa, which could be blocked by native albumin. To elucidate whether albumin-binding proteins are exposed on the cell surface, intact lymphocytes were surface radioiodinated and membrane fractions prepared from them were subjected to affinity chromatography on albumin-agarose beads. The proteins thus purified had, like ABP, M_r of 16 and 31. These data indicate that ABP (i) are components of thymocyte plasma membrane, (ii) have apparent molecular mass of 16–18 and 29–31 kDa, and (iii) are exposed on the outer membrane surface.Abbreviations ABP albumin-binding proteins - Alb bovine serum albumin - Au gold - DAPI 4,6-diamidino-2-phenylindol - EM electron microscopy - NC nitrocellulose - PAGE polyacrylamidegel electrophoresis - PBS phosphate buffered saline - PEG polyethylene glycol - PMSF phenylmethylsulfonyl fluoride - WGA Wheat germ agglutinin