Behaviour of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells

During plasmolysis of onion epidermal cells, the contracting protoplast remains connected to the cell wall by an intricate, branched system of plasma membrane (PM) ‘Hechtian strands’ which stain strongly with the fluorescent probe DiOC₆. In addition, extensive regions of the cortical endoplasmic reticulum (ER) network remain anchored to the cell wall during plasmolysis and do not become incorporated into the contracting protoplast with the other cell organelles. These ER profiles become tightly encased by the PM as the latter contracts towards the centre of the cell. Thus, although the cortical ER is left outside the main protoplast body, it is nonetheless still bound by the PM of the cell. As well as being anchored to the wall, the cortical ER remains intimately linked with plasmodesmata and retains continuity between cells via the central desmotubules which become distended during plasmolysis. The PM also remains in close contact with the plasmodesmatal pore following plasmolysis. It is suggested that plasmodesmata, although sealed, may not be broken during plasmolysis, their substructure being preserved by continuity of both ER and PM through the plasmodesmatal pore. A structural model is presented which links the behaviour of PM, ER and plasmodesmata during plasmolysis.     

Zones of membrane adhesion in the cryofixed envelope of Escherichia coli

The envelopes of Escherichia coli B and E. coli K29 were examined using cryofixation and freeze substitution. Emphasis was directed toward the question whether membrane adhesion zones (which connect inner membrane (IM) and outer membrane (OM) after plasmolysis in 10-20% sucrose) can be visualized with the use of cryotechniques. Plasmolysis in 10-20% sucrose was observed to have no effect on cell viability. We found that simple plunge-freezing methods preserve adhesion sites, whereas these sites were not observed after impact-freezing. Also, plasmolysis "bays," visible in light microscopic preparations of living cells, were seen to be maintained intact after plunge-freezing. Employment of photocrosslinking with UV-flashes before or after plasmolysis showed a significant increase in the number of adhesion areas compared to noncrosslinked specimens. To control the contact speed of the specimen during immersion into the cryogen, a hollow rotor was constructed in which the cryogenic liquid is moving at desired high speeds. Adhesion sites presented themselves in the plasmolyzed cell as sites of close contact of the outer and inner membrane, an arrangement that would leave very limited space for peptidoglycan layers at the contact site of the two membranes. Adhesion sites may occur either as single, isolated sites or within stretches of IM/OM apposition where they appear to function as "spot welds" between the two membranes. Exposure of cells to sucrose concentrations of 35% caused rupture of adhesions with cytoplasmic fragments remaining attached to the envelope. The cryofixation procedures described here do not presently yield the number of membrane adhesions obtainable with conventional aldehyde fixation. However, since the combination of millisecond photocrosslinking and cryofixation of plasmolyzed cells resulted in a higher membrane stabilization and in an increase of the number of adhesion sites, this combination appears to be a useful tool for the analysis of sensitive membrane structures.     

Wall-to-membrane linkers in onion epidermis: some hypotheses

Wall-to-wall linkage may help maintain cell integrity and polarity, and focus mechanical stress from wall to mech-anotransductive ion channels within the plasm a lemma. When cells of onion bulb scale epidermis shrink during plasmolysis with CaCl₂, the plasmalemma remains attached to the cell wall by Hechtian strands which we hypothesize might possibly be drawn out from linkages fulfilling the above functions. We show that at least many of the attachment loci are independent of the plasmodesmata. A priori, wall glycoproteins seem good candidates for the wall-to-membrane linkers; therefore, we investigated the distribution in wall and plasmalemma of antigen recognized by antibody to hydroxyproline-rich glycoprotein (HRGP). Using fluorescent secondary antibodies, we showed that polyclonal antibodies prepared against wall HRGP from soybean bind to the onion walls (following mild depectination), but also bind to the plasmalemma after the wall is enzymatically digested. The distribution of the antibodies is punctate. On the plasmalemma, the points tend to be scattered more or less uniformly, but can cluster at termini of large streaming strands (which rarely form in wall-constrained cells.) These streaming strands can be seen to exert tension on the membrane. We hypothesize that (1) the antigen on the surface of the protoplast may correspond to the antigen in the walls, (2) such antigen may be responsible for adhesion of membrane to wall at the linkage sites visualized by CaCl₂ plasmolysis, and (3) the linkage sites may be transmembrane proteins to which cytoskeleton can attach at the inner surface.     

A plasmolytic cycle: The fate of cytoskeletal elements

Summary In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.Abbreviations DIC differential interference contrast - DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

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     

The ''Bayer bridges'' confronted with results from improved electron microscopy methods

In electron micrographs of conventionally prepared thin sections of Escherichia coli one observes (i) a wavy appearance of the two membranes showing frequent appositions (named adhesion sites) and (ii) intermembrane bridges after plasmolysis which, it is claimed, occur at the adhesion sites and are related to intermembrane protein transport (transmigration). When chemical fixation is replaced by cryofixation, the observations are very different. (a) The two membranes are equally spaced and no contacts, adhesions or other sorts of connections are visible. (b) After plasmolysis the protoplast is shrunken, but the typical bridges are no longer produced. (c) In addition, when peptidoglycan is stained on conventionally prepared sections, it is revealed as a 7-nm-thick sacculus which is not interrupted at the sites of apposition. In view of the new observations, the structural concepts derived from conventionally prepared material must be revised. It is proposed that the intermembrane space is entirely filled by a gel, the outer part of which is the 7 nm thick, very stable, chemically resistant peptidoglycan (or murein). The inner part is much less stable and is proposed to undergo rapid autolytic changes upon cell death. The large 'Bayer bridges' might then tentatively be explained as an artificial post-mortem enhancement of either a stream of proteins transmigrating across the periplasm or of a pre-existing, but not yet resolved, structure. This enhancement probably occurs during the 7-10 min between plasmolysis and fixation that are prescribed for the procedure necessary for revealing 'Bayer bridges'.     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm.

The chaperone DnaK can be released (up to 40%) by osmotic shock, a procedure which is known to release the periplasmic proteins and a select group of cytoplasmic proteins (including thioredoxin and elongation factor Tu) possibly associated with the inner face of the inner membrane. As distinct from periplasmic proteins, DnaK is retained within spheroplasts prepared with lysozyme and EDTA. The ability to isolate DnaK with a membrane fraction prepared under gentle lysis conditions supports a peripheral association between DnaK and the cytoplasmic membrane. Furthermore, heat shock transiently increases the localization of DnaK in the osmotic-shock-sensitive compartment of the cytoplasm. We conclude that DnaK belongs to the select group of cytoplasmic proteins released by osmotic shock, which are possibly located at Bayer adhesion sites, where the inner and outer membranes are contiguous.     

Bacterial plasmolysis as a physical indicator of viability.

Bacterial plasmolytic response to osmotic stress was evaluated as a physical indicator of membrane integrity and hence cellular viability. Digital image analysis and either low-magnification dark-field, high-magnification phase-contrast, or confocal laser microscopy, in conjunction with pulse application of a 1.5 M NaCl solution, were used as a rapid, growth-independent method for quantifying the viability of attached biofilm bacteria. Bacteria were considered viable if they were capable of plasmolysis, as quantified by changes in cell area or light scattering. When viable Salmonella enteritidis biofilm cells were exposed to 1.5 M NaCl, an approximately 50% reduction in cell protoplast area (as determined by high-magnification phase-contrast microscopy) was observed. In contrast, heat- and formalin-killed S. enteritidis cells were unresponsive to NaCl treatment. Furthermore, the mean dark-field cell area of a viable, sessile population of Pseudomonas fluorescens cells (approximately 1,100 cells) increased by 50% as a result of salt stress, from 1,035 +/- 162 to 1,588 +/- 284 microns2, because of increased light scattering of the condensed, plasmolyzed cell protoplast. Light scattering of ethanol-killed control biofilm cells underwent little change following salt stress. When the results obtained with scanning confocal laser microscopy and a fluorescent viability probe were compared with the accuracy of plasmolysis as a viability indicator, it was found that the two methods were in close agreement. Used alone or in conjunction with fluorochemical probes, physical indicators of membrane integrity provided a rapid, direct, growth-independent method for determining the viability of biofilm bacteria known to undergo plasmolysis, and this method may have value during efficacy testing of biocides and other antimicrobial agents when nondestructive time course analyses are required.     

Plasmolysis and Cell Shape Depend on Solute Outer-Membrane Permeability during Hyperosmotic Shock in E. coli

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.     

Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.     

Compartmentalization of the periplasmic space at division sites in gram-negative bacteria.

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in lkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983). When cell division was blocked by growing divC(Ts) cells at elevated temperatures, the localized regions of plasmolysis were clustered along the aseptate filaments at positions that corresponded to sites where septum formation occurred when cell division was permitted to resume by a shift back to the permissive temperature. Taken together the results are consistent with a model in which extended zones of adhesion define localized compartments within the periplasmic space, predominantly located at future sites of cell division.     

Specific localization of the lysis protein of bacteriophage MS2 in membrane adhesion sites of Escherichia coli.

Specific localization of the lysis (L) protein of bacteriophage MS2 in the cell wall of Escherichia coli was determined by immunoelectron microscopy. After induction of the cloned lysis gene, the cells were plasmolyzed, fixed, and embedded in either Epon or Lowicryl K4M. Polyclonal L-protein-specific antiserum was purified by preabsorption to membranes from cells harboring a control plasmid. Protein A-gold was used to label the protein-antibody complexes. Between 42.8% (Lowicryl) and 33.8% (Epon) of the label was found in inner and outer membranes, but 30.3% (Lowicryl) and 32.8% (Epon) was present mostly in clusters in the adhesion sites visible after plasmolysis. The remaining label (26.9 and 33.4%, respectively) appeared to be present in the periplasmic space but may also have been part of membrane junctions not visible because of poor contrast of the specimen. In contrast, a quite different distribution of the L protein was found in cells grown under conditions of penicillin tolerance, i.e., at pH 5, a condition that had previously been shown to protect cells from L-protein-induced lysis. At tolerant conditions, only 21.0% of the L protein was in the adhesion sites; most of the protein (68.2%) was found in inner and outer membranes. It is concluded that lysis of the host, E. coli, was a result of the formation of specific L-protein-mediated membrane adhesion sites.     

Plasmolysis bays in Escherichia coli: are they related to development and positioning of division sites?

Plasmolysis bays, induced in Escherichia coli by hypertonic treatment, are flanked by zones of adhesion between the plasma membrane and the cell wall. To test the proposition of Cook et al. (W. R. Cook, F. Joseleau-Petit, T. J. MacAlister, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 84:7144-7148, 1987) that these zones, called periseptal annuli, play a role in determining the division site, we analyzed the positions of these zones by phase-contrast and electron microscopy. In situ treatment of cells grown in agar showed that the youngest cell pole was the most susceptible to plasmolysis, whereas the constriction site was resistant. Lateral bays occurred only at some distance from a polar bay or a resistant constriction site. Orienting cells with their most prominently plasmolyzed polar bay in one direction showed that the lateral bays were always displaced away from the polar bay at about half the distance to the other cell pole. If no poles were plasmolyzed, lateral bays occurred either in the centers of nonconstricting cells or at the 1/4 or 3/4 position of cell length in constricting cells. The asymmetric positions of lateral plasmolysis bays, caused by their abrupt displacement in the presence of polar bays or constriction sites, does not confirm the periseptal annulus model (Cook et al.), which predicts a gradual and symmetric change in the position of lateral bays with increasing cell length. Our analysis indicates that plasmolysis bays have no relation to the development and positioning of the future division site.     

Plasmolysis of Pteridium protoplasts: A study using light and scanning-electron microscopy

A study was undertaken using gametophytes of the fern Pteridium aquilinum to examine the effects of plasmolysis on the topography of protoplasts. Methods are described whereby the surfaces of non-isolated protoplasts can be observed in the plasmolysed condition using scanning electron microscopy. Plasmolysed gametophytes were also examined in the light microscope using differential interference contrast and ultra-violet fluorescence microscopy after staining with fluorescein diacetate. With scanning electron microscopy, plasmolysed protoplast surfaces appeared smooth with no evidence of wrinkling or infolding of excess membrane. The formation of irregular-shaped protoplasts, protoplasmic threads, subprotoplasts, and protoplasmic networks covering internal wall surfaces all provided evidence for strong wall adhesion of the protoplasm. The availability of membrane for uptake into folds or vesicles is therefore thought to be minimal. Transmission electron microscopy showed some protoplasmic threads to be plasmodesmata, the remainder being cell-wall contact points. Remnants of these threads were occasionally observed on isolated protoplasts in both the light and electron microscopes.     

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall

The heptapeptide Tyr-Gly- Arg-Gly-Asp- Ser-Pro containing the sequence Arg-Gly-Asp (RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K_d1 1 nM, and K_d2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.     

Contesting the ‘model minority’: racialization,youth culture and ‘British Chinese’/‘Oriental’ nights

While racialized youth are often central in debates on citizenship, multiculturalism and belonging, those ascribed as ‘British Chinese’ are constructed as model minorities, lacking a hybridized culture but insulated from racism, and thus invisible in these discussions. This article argues, however, that the model minority discourse is itself a specific form of contemporary racialization that revives ‘yellow peril’ discourses on the capacities of particular ‘Oriental’ bodies. Drawing on ethnographic fieldwork, it examines how young people challenge these constructions, by drawing on popular culture to organize and participate in what they call ‘British Chinese’ and, more provocatively, ‘Oriental’ nightlife spaces. It analyses how through these spaces participants forge a sense of identity that allows them to reimagine themselves as racialized subjects. It demonstrates how these spaces constitute transient sites of experimental belonging, facilitating new cultural politics and social identifications that at once contest reified conceptions of British Chineseness yet also create new exclusions.     

Characterization of adhesion zones in E. coli cells

After plasmolysis of Escherichia coli cells, the adhesion zones were characterized using the cytochemical PTA and SP procedures which stain peptidoglycan and lipopolysaccharides (LPS) respectively. A PTA-stained layer was detected at the adhesion sites. This layer was visualized irrespective of the electron microscopy procedure used. Also, using SP staining an outer membrane in which LPS molecules were asymmetrically distributed, was observed.     

Membrane–wall attachments in plasmolysed plant cells

Summary. Field emission scanning electron microscopy of plasmolysed Tradescantia virginiana leaf epidermal cells gave novel insights into the three-dimensional architecture of Hechtian strands, Hechtian reticulum, and the inner surface of the cell wall without the need for extraction. At high magnification, we observed fibres that pin the plasma membrane to the cell wall after plasmolysis. Treatment with cellulase caused these connecting fibres to be lost and the pinned out plasma membrane of the Hechtian reticulum to disintegrate into vesicles with diameters of 100–250nm. This suggests that the fibres may be cellulose. After 4h of plasmolysis, a fibrous meshwork that labelled with anti-callose antibodies was observed within the space between the plasmolysed protoplast and the cell wall by field emission scanning electron microscopy. Interestingly, macerase-pectinase treatment resulted in the loss of this meshwork, suggesting that it was stabilised by pectins. We suggest that cellulose microfibrils extending from strands of the Hechtian reticulum and entwining into the cell wall matrix act as anchors for the plasma membrane as it moves away from the wall during plasmolysis.Correspondence and reprints: Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.     

Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus

A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single-step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.