Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

Background and Aims

Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium.

Methods

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

Key Results

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

Conclusions

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.     

Mechanical properties of external confinement modulate the rounding dynamics of cells

Many studies have demonstrated that mitotic cells can round up against external impediments. However, how the stiffness of external confinement affects the dynamics of rounding force/pressure and cell volume remains largely unknown. Here, we develop a theoretical framework to study the rounding of adherent cells confined between a substrate and a cantilever. We show that the rounding force and pressure increase exclusively with the effective confinement on the cell, which is related to the cantilever stiffness and the separation between cantilever and substrate. Remarkably, an increase of cantilever stiffness from 0.001 to 1 N/m can lead to a 100-fold change in rounding force. This model also predicts an active role of confinement stiffness in regulating the dynamics of cell volume and hydrostatic pressure. We find that the dynamic changes of cellular volume and hydrostatic pressure after osmotic shocks are opposite if the cantilever is soft, whereas the dynamic changes of cellular volume and pressure are the same if the cantilever is stiff. Taken together, this work demonstrates that confinement stiffness appears as a critical regulator in regulating the dynamics of rounding force and pressure. Our findings also indicate that the difference in cantilever stiffness need to be considered when comparing the measured rounding force and pressure from various experiments.     

A micromanipulation method to measure the mechanical properties of single tomato suspension cells

A micromanipulation method has been developed to measure the force required to burst single tomato cells (Lycopersicon esculentum vf36) taken from suspension cultures. The method works by compressing a cell between parallel surfaces whilst measuring the force being imposed on the cell, and its deformation. The mean bursting force for two-week-old cells was 3.6 mN (standard error 0.1 mN), at a compression speed of 23 m s^–1. Usually force-deformation curves showed a single bursting event, but sometimes multiple bursts were observed, implying cells could reseal after failure. If cells were deformed without bursting, and then held, they showed a relaxation of the force. This was attributed to water loss, although wall relaxation was also a possibility. The half time of this relaxation was between 1–10 s. Tests on protoplasts gave bursting forces 1000 fold lower than intact cells, and cells treated with Triton to disrupt the membranes and destroy turgor collapsed with no bursting. As expected, both turgor and the presence of a wall were essential to maintaining cell strength.     

A role for caveola-forming proteins caveolin-1 and CAVIN1 in the pro-invasive response of glioblastoma to osmotic and hydrostatic pressure

In solid tumours, elevated interstitial fluid pressure (osmotic and hydrostatic pressure) is a barrier to drug delivery and correlates with poor prognosis. Glioblastoma (GBM) further experience compressive force when growing within a space limited by the skull. Caveolae are proposed to play mechanosensing roles, and caveola-forming proteins are overexpressed in GBM. We asked whether caveolae mediate the GBM response to osmotic pressure. We evaluated in vitro the influence of spontaneous or experimental down-regulation of caveola-forming proteins (caveolin-1, CAVIN1) on the proteolytic profile and invasiveness of GBM cells in response to osmotic pressure. In response to osmotic pressure, GBM cell lines expressing caveola-forming proteins up-regulated plasminogen activator (uPA) and/or matrix metalloproteinases (MMPs), some EMT markers and increased their in vitro invasion potential. Down-regulation of caveola-forming proteins impaired this response and prevented hyperosmolarity-induced mRNA expression of the water channel aquaporin 1. CRISPR ablation of caveola-forming proteins further lowered expression of matrix proteases and EMT markers in response to hydrostatic pressure, as a model of mechanical force. GBM respond to pressure by increasing matrix-degrading enzyme production, mesenchymal phenotype and invasion. Caveola-forming proteins mediate, at least in part, the pro-invasive response of GBM to pressure. This may represent a novel target in GBM treatment.     

The freezing point depression of mammalian tissues in relation to the question of osmotic activity of cell fluid

The freezing point depression of freshly excised frozen tissues, pulverized in a hydraulic press or in a mortar, is greater than that of plasma. Even at 0°C. the freezing point depression of such homogenates increases significantly with time. Dilution data indicate that such freezing point data are valid. The presence of intact cells has been shown in smears of tissues pulverized in a mortar, but not in smears of those crushed in a hydraulic press. The osmolarity of various diluent solutions affects the calculated osmotic activity of tissue homogenates presumably because of delayed diffusion between the diluent and cell fluid. With a hypertonic NaCl diluent, spuriously low values of tissue osmotic activity are found from calculations assuming instantaneous mixing between homogenates and diluents. The limitations of data from cryoscopic experiments and from tissue-swelling experiments are discussed in relation to the basic question of whether or not cell fluid is isotonic to extracellular fluid.     

Effect of inositol hexaphosphate on the transient behavior of red cells following a DMSO-induced osmotic pulse

An osmotic pulse can be used to incorporate inositol hexaphosphate (IHP) into red cells. The pulse is induced by equilibrating a red cell suspension with DMSO and then rapidly diluting with an isotonic IHP solution. Since IHP binds to hemoglobin and lowers the affinity for oxygen, this method may find application in the preparation of low-affinity cells for experimental and clinical use. The experiments reported here examined the dynamic changes of several red cell variables immediately following the osmotic pulse. The effect of IHP, which has been shown to dissociate red cell cytoskeletons, was evaluated by comparison with a matched phosphate-buffered saline (PBS) diluent. Red cell morphology, volume, and hemoglobin permeability were studied by fixing the cells at times ranging from 0.06 to 300 sec after dilution. Mechanical fragility was measured by subjecting the cells to a short period of shear stress at the same times after dilution. With both diluents, the cells underwent a rapid increase in volume followed by a return towards normal volume with a maximum at less than 250 msec. With IHP diluent, the period of hemoglobin permeability immediately followed the size peak and was completed by about 1 sec after dilution. PBS also induced a second leakage at longer times (10-120 sec), which resulted in a morphological dichotomy with ghosts and intact cells. The choice of diluent also affected sensitivity to shear stress. The IHP-treated cells had a mechanical fragility maximum at about 1 sec. The PBS-treated cells exhibited no enhanced mechanical fragility. An unexpected result was the inhibition of the second phase of lysis in PBS-treated cells by a properly timed shear stress.     

Cell Wall Architecture Prerequisite for the Cell Division in the Protoplasts of White Poplar, Populus alba L.

White poplar (Populus alba L.) protoplasts were investigatedat 0, 3,10, 20 and 30 d after regeneration to visualize thecell wall architecture prerequisite for cell division. The 10day-old cells just before cell division developed a thin walllayer with uneven deposition of cell wall materials and weresaved from bursting by suspension in low osmotic medium. Thethree dimensional architecture of the cell wall, as revealedby rapid-freezing and deep-etching electron microscopy, in 10day-old cells, constituted thin innermost lamellae on the plasmamembrane along with highly extended micronbrillar networks.These results suggest that the deposition of thin lamellae isimportant not only for cells to withstand bursting but alsoto induce cell division. The present investigations give thefirst account of the visualization of the three-dimensionalarchitecture of regenerated cell wall right before cell division. (Received July 31, 1997; Accepted April 6, 1998)     

An osmotic model of the growing pollen tube

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip.     

A mathematical model of the dynamics of odontogenic cyst growth

OBJECTIVE: To formulate a mathematical model of odontogenic cyst growth and establish the dynamics of cyst enlargement and role of osmotic pressure forces throughout its growth. STUDY DESIGN: The model assumed a spherical cyst with a semipermeable lining of living cells and a core consisting of degraded cellular material, including generic osmotic material, fed by the continuous death of epithelial cells in the lining. The lining cells were assumed to have both elastic and viscous properties, reflecting the action of physical stresses by the surrounding cyst capsule, composed of fibroblasts and collagen fibers. The model couples the cyst radius and osmotic pressure differences resulting in a system of 2 nonlinear ordinary differential equations. RESULTS: The model predicts that in all parameter regimens the long-time behavior of the cyst is the same and that linear radial expansion results. CONCLUSION: In the early and intermediate stages of cystic growth, osmotic pressure differences play an important role; however, in very large cysts, this role becomes negligible, and cell birth in the lining dominates growth.     

不同稀释液对冻干A、C群脑膜炎球菌结合疫苗质量的影响

为选择合适的的冻干A、C群脑膜炎球菌结合疫苗的稀释液,以注射用水(H2O)、生理盐水(NS)、磷酸盐缓冲液(PBSpH6.8～7.2)作为候选稀释液,进行了相关溶解度、pH、渗透压、异常毒性、免疫原性的检测比较。溶解度、pH、免疫原性结果显示三种稀释液无差异;H2O作为稀释液的渗透压不符合人用制剂渗透压的要求,另两者则符合要求;三种稀释液溶解制品后的pH检测数据均符合要求,但PBS的缓冲能力更强。故认为PBS更适合作为冻干A、C群脑膜炎球菌结合疫苗的稀释液。     

Thermodynamic analysis of the interaction of the xylem water and phloem sugar solution and its significance for the cohesion theory

The cohesion theory explains water transport in trees by the evaporation of water in the leaves (transpiration), which in turn generates the tension required for sap ascent, i.e. the flow of pure water from the soil through the root system and the non-living cells of the tree (xylem tracheids) up to the leaves. Only a small part of this water flow entering the leaves is used in photosynthesis to produce sugar solution, which is transported from the leaves through the living cells (phloem) to everywhere in the tree where it is needed and used. The phloem sieves are connected to the xylem tracheids by water transparent membranes, which means that the upflow of pure water and downflow of sugar solution interact with each other, causing the osmotic pressure in the sugar solution (Münch model). In this paper we analyse this interaction with a thermodynamic approach and we show that some open questions in the cohesion theory can then perhaps be better understood. For example, why under a quite high tension the water can flow in the xylem mostly without any notable cavitation, and how the suction force itself depends on the cavitation. Minimizing Gibbs energy of the system of xylem and phloem, we derive extended vapor pressure and osmotic pressure equations, which include gas bubbles in the xylem conduits as well as the cellulose-air-water interface term. With the aid of the vapor pressure equation derived here, we estimate the suction force that the cavitation controlled by the phloem sugar solution can generate at high moisture contents. We also estimate the suction force that the transpiration can generate by moisture gradient at low moisture contents. From the general osmotic pressure equation we derive an equation for calculating the degree of cavitation with different sugar solution concentrations and we show the conditions under which the cavitation in the xylem is totally avoided. Using recent field measurement results for a Scotch pine, the theory is demonstrated by showing its predictions for possible amounts of cavitation or embolism from morning hours to late afternoon.     

Mechanical and failure properties of single attached cells under compression

Eukaryotic cells are continuously subjected to mechanical forces under normal physiological conditions. These forces and associated cellular deformations induce a variety of biological processes. The degree of deformation depends on the mechanical properties of the cell. As most cells are anchorage dependent for normal functioning, it is important to study the mechanical properties of cells in their attached configuration. The goal of the present study was to obtain the mechanical and failure properties of attached cells. Individual, attached C2C12 mouse myoblasts were subjected to unconfined compression experiments using a recently developed loading device. The device allows global compression of the cell until cell rupture and simultaneously measures the associated forces. Cell bursting was characterized by a typical reduction in the force, referred to as the bursting force. Mean bursting forces were calculated as 8.7+/-2.5 microN at an axial strain of 72+/-4%. Visualization of the cell using confocal microscopy revealed that cell bursting was preceded by the formation of bulges at the cell membrane, which eventually led to rupturing of the cell membrane. Finite element calculations were performed to simulate the obtained force-deformation curves. A finite element mesh was built for each cell to account for its specific geometrical features. Using an axisymmetric approximation of the cell geometry, and a Neo-Hookean constitutive model, excellent agreement between predicted and measured force-deformation curves was obtained, yielding an average Young's modulus of 1.14+/-0.32 kPa.     

From Plateau to Pseudo-Plateau Bursting: Making the Transition

Bursting electrical activity is ubiquitous in excitable cells such as neurons and many endocrine cells. The technique of fast/slow analysis, which takes advantage of time scale differences, is typically used to analyze the dynamics of bursting in mathematical models. Two classes of bursting oscillations that have been identified with this technique, plateau and pseudo-plateau bursting, are often observed in neurons and endocrine cells, respectively. These two types of bursting have very different properties and likely serve different functions. This latter point is supported by the divergent expression of the bursting patterns into different cell types, and raises the question of whether it is even possible for a model for one type of cell to produce bursting of the type seen in the other type without large changes to the model. Using fast/slow analysis, we show here that this is possible, and we provide a procedure for achieving this transition. This suggests that the design principles for bursting in endocrine cells are just quantitative variations of those for bursting in neurons.     

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.     

Insights into protective effects of medium additives on animal cells under fluid stresses: the hydrophobic interactions

Animal cells in suspension culture can suffer severe mechanical damage from bursting gas bubbles or other hydrodynamic force sources. Certain chemical additives in the culture media, particularly some surface-active chemicals, can effectively protect animal cells against such damage. Previously we proposed that the protective effect is associated with the adsorption of the additives in the cell membrane through hydrophobic binding of the surface-active molecules to the membrane. Adsorption of the additives to the cell membrane may lead to decreased hydrophobicity of the cell surface, thus eliminating cell adhesion to bubbles and reducing cell damage from bursting bubbles. In this study, we measured the hydrophobicity of two insect cell lines based on cell adhesion to hydrocarbon phase and its influence by surface-active chemicals, Pluronic F68, a methylcellulose and a polyethylene glycol. The experimental results showed strong support for the aforecited cell protection mechanism.     

Investigation of ampicillin effect on morphological and mechanical properties of Escherichia coli and Bacillus cereus cells with atomic force microscopy

The effect of subbacteriostatic concentrations of ampicillin on morphological and mechanical properties of gramnegative and grampositive cells of Escherichia coli K12 TG1 and Bacillus cereus IP 5832 respectively was studied with atomic force microscopy. Significant heterogeneity of the bacterial populations was shown by the character of the response to the antibiotic effect. The common feature was increase of the cell size likely due to the effect of the inner osmotic pressure on the lowered cell wall strength. In the E. coli population there were besides observed anomalous elongated cells with signs of septation disorder, as well as their structurs, lacking the cytoplasmic liquid fraction. In the B. cereus the inner osmotic pressure mainly enlarged the cell cross section, changing the cell shape from rod to sphere, that was accompanied by significant impairment of the surface structure with liberation of the peptidoglycane fragments to the medium. The particular features of the E. coli K12 TG1 and B. cereus IP 5832 respond to the ampicillin effect were attributed to the differences in the structure of their cell wall, also due to specific properties of the peptidoglycane synthesis and three-dimensional organization.     

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.     

Extraction and analysis of sap from individual wheat leaf cells: the effect of sampling speed on the osmotic pressure of extracted sap

Abstract. A modification to the pressure probe is described which allows very rapid extraction of sap samples from single higher plant cells. The performance of this rapid-sampling probe was assessed and compared with the unmodified probe for cells of both wheat and Tradescantia. Under some conditions, the unmodified probe operated too slowly to avoid dilution of cell sap during the extraction process. This led to values for apparent sample osmotic pressures that were below the turgor pressures for the same cells. The problem was particularly acute in young wheatleaf epidermal cells which are small, elongate and have high turgor pressure. These exhibited rapid water influx when their turgor was depressed during the sampling of their contents (half-time for pressure recovery in wheat cells was less than 1 s while in Tradescantia cells it was 3–5 s). Dilution during sampling was apparently negligible when the rapid sampling probe was used. The study was complemented by a simple model of the way cells dilute during sampling. Quantitative predictions of the model were consistent with our observed findings. The model is used to assess the major factors which determine a cell's susceptibility to dilution during sampling.     

Determination of free space, growth, solute concentrations and parameters of water relations of suspension-cultured tobacco cells

Abstract Methods were developed for measuring water content of the free space of suspension-cultured tobacco cells using ³H- or ¹⁴C-sorbitol. Sorbitol was not taken up by cells in significant quantities over the 3 min taken to label free space. Free space accounted for 50–60% of the water content of cell pellets irrespective of whether ³H- or ¹⁴-C-sorbitol was used. ¹⁴C-inulin labelled 13.5% less of the water in cell pellets than ³H-sorbitol, probably due to inadequate penetration by inulin into the free space in the cell wall matrix and within clumps of cells. Measurement of free space is necessary for measuring growth on a fresh or dry weight basis, solute concentrations and parameters of water relations of cells. Techniques for making these measurements on tobacco cells were also developed in this study. Solutes were measured after extraction from cells by expressing sap or by boiling cells in ethanol. Similar solute concentrations were found using both methods of extraction. By expressing sap from cells grown in culture medium with an osmotic pressure of 0.24 MPa, the cells were found to have an internal osmotic pressure of 0.70 MPa. Glucose, fructose, sucrose, amino acids and K⁺ accounted for 60% of this osmotic pressure. Elastic moduli were estimated using the Boyle-Van't Hoff relationship after suspending cells in solutions with different osmotic pressures and assessing their water content or internal osmotic pressure. For two different lines of tobacco cells, elastic modulus varied between 1 MPa and 5.4 MPa at turgor pressures of 0.15–0.52 MPa (line 1) and between 0.2 MPa and 4.2 MPa at turgor pressures of 0.04–0.26 MPa (line 2).     

Phototropic Response of the Bean Pulvinus: Movement of Water and Ions

Segmental analysis of the laminar pulvinus of Phaseolus vulgaris L. showed that its phototropic curvature is accompanied by efflux of inorganic ions and water from its contracting sector and a comparable influx into its expanding one. All the major ions, except Na⁺, contributed to this transport, suggesting that the response to light involves changes in the driving force, or conductivity of a wide range of solutes. During the curvature, K⁺ and CI^? made the greatest and equivalent contributions to efflux, but only Cl^? exhibited a matching influx into the expanding sector, while K⁺ influx was much less. Use of the cell pressure probe showed that, as the laminar angle of elevation changed between ?40° to +40°, turgor pressure in the expanding motor cells increased by 0.48 MPa and decreased in the contracting cells by 0.32 MPa. Picoliter osmometry of single-cell samples showed that during this movement vacuolar osmotic pressure remained constant. Thus, changes in turgor pressure resulted from changes in apoplastic, rather than the protoplastic osmotic pressure. Volumetric modulus of elasticity of pulvinar motor cells is very low, showing that their walls are very elastic. These properties increase the effectiveness of converting osmotic work into the large-scale, reversible volume changes responsible for leaf movements.