Vacuolar Fluid Research Articles

Magnetic tweezers for manipulation of magnetic particles in single cells

Magnetic tweezers gain increasing interest for applications in biology. Here, a setup of magnetic tweezers is introduced using micropatterned conducting lines on transparent glass slides. Magnetic particles of 1 μm diameter were injected in barley cell vacuoles using a microinject system under microscopic control. Time dependent tracking of the particles after application of a magnetic field was used to determine the viscosity of vacuolar sap in vivo relative to water and isolated vacuolar fluid. The viscosity of vacuolar sap in cells was about 2-fold higher than that of extracted vacuolar fluid and 5 times higher than that of water.

NMR studies of water compartmentation in carrot parenchyma tissue during drying and freezing

An analysis is presented of the effect of drying and freezing on the distribution and pulse spacing dependence of water proton transverse relaxation times in carrot parenchyma tissue. The relaxation behaviour can be interpreted by treating the effects of changing subcellular morphology with a numerical cell model and combining this with a two-site proton exchange model to take account of solutes dissolved in the vacuolar fluid. In this way it is shown that drying removes water primarily from the vacuolar compartment, causing cell shrinkage and concentration of dissolved solutes, with little change in the volume of intercellular air spaces. Freezing causes initial ice crystal formation in the vacuolar compartment which concentrates the dissolved solutes. However, even at 248 K substantial quantities of nonfreezing water are associated with the cell walls and dissolved biopolymers. Comparison is made with previous studies on apple tissue.

INFLUENCE OF APOPLASTIC SOLUTES ON THE TURGOR OF TOMATO PERICARP CELLS

The pressure microprobe was used to measure cell turgor (ψp) in tomato pericarp tissue, and also to sample vacuolar fluid for the measurement of cell osmotic potential (ψs) in a nanoleter freezing point osmometer. In fresh tissue, cell ψs agreed well with the ψs of frozen-thawed whole tissue measured with a vapor pressure osmometer. Under a wide range of ripeness conditions however, and for both intact fruit and discs of fruit tissue, fruit cell turgor was consistently lower than expected, based on the values of cell ψs. When tissue discs were hydrated in aerated distilled water, disc fresh weight increased substantially (20 - 50+%), and both cell turgor and tissue ψs increased. Cell ψs however, remained relatively constant. These and other observations suggest that the turgor increase during hydration was largely due to losses of solute from the apoplastic space, partly by direct losses from the tissue, and partly by cell solute accumulation.

Direct Measurement of Turgor and Osmotic Potential in Individual Epidermal Cells

The pressure probe, which is routinely used to measure the turgor potential (Psi(p)) of individual epidermal cells in Tradescantia virginiana (L.), has also been used to sample small volumes of vacuolar fluid from these same cells (as low as 0.02 nl) for measurement of cellular solute (osmotic) potential (Psi(s)) in a micro freezing point osmometer. The water potential components Psi(p) and Psi(o) have been used to calculate the total water potential of individual epidermal cells (Psi(cell)) which has then been directly compared to the total leaf water potential (Psi(leaf)) measured psychrometrically. The relation of Psi(leaf) and Psi(cell) to leaf transpiration indicates that in T. virginiana, a relatively straightforward relation exists between the level of water flow through the leaf tissue, and the DeltaPsi within the leaf, between two points along the water flow pathway. Substantial agreement was found between the two independent, in situ methods of measuring Psi when extrapolated to zero transpiration conditions. These results are discussed with respect to the thermodynamics of water transport in plant tissues.

Untersuchungen an den kontraktilen vakuolen von Homalozoon vermiculare und Dileptus anser

Summary Homalozoon vermiculare and Dileptus anser are elongate predatory ciliates which contain many (5–21 in Homalozoon, 6–15 in Dileptus) contractile vacuoles. In both cases the total number was greater in longer animals. The behaviour of these structures was investigated by analysis of video recordings. The vacuoles appear simple, there are no collecting canals. No ampullae are visible after expulsion of vacuolar fluid has taken place. Vesicles are not involved in vacuolar filling. The average diastolic diameter attained by contractile vacuoles of H. vermiculare was 9.5 μm (range 4–15μm) and in D. anser 4.0 μm (range 2–5μm). The frequency of contractions in H. vermiculare varied greatly averaging 1 contraction per 45 s, and individual vacuoles had a wide range of cycle period, e.g., 38–53 s. The vacuoles of D. anser, in contrast, have cycle periods varying within quite a narrow range and had a mean cycle duration of 25 s. The frequency of contractions in H. vermiculare show gradual, sinusoidal fluctuations in the duration of the cycle. In both organisms the vacuoles towards the front and back ends of the cell contract less frequently than those towards the centre of the cell. There is a direct correlation between vacuolar contraction frequency and the width of the body. However, there is no direct correlation between vacuolar output and the volume of the intervacuolar cytoplasm, or the intervacuolar cell surface area. There is an apparent gradient in the functional activity (output per vacuole per volume of cytoplasm) of the contractile vacuoles of these organisms, with a maximum encountered immediately behind the cytopharynx.

Osmoregulation in the fresh water sponge, Spongilla lacustris

AbstractExternal medium passed through the canal system of small Spongilla lacustris at the rate of 70 body volumes per hour, allowing for little or no modification of its composition by the animal. Consequently any osmotic gradient between medium and animal was experienced at the cell level. Forty or more contractile vacuoles arranged around the nucleus in each basal pinacocyte removed fluid at the rate of 24.7 ± 1.0 μm3/cell/min in 1.3 milliosmolal (mosm) medium. Output of the contractile vacuoles depended in a nearly linear fashion on the osmotic concentration of the medium, decreasing to zero at 12–16 mosm. The contractile vacuoles responded to the osmotic concentration of the medium, not to ionic strength or the concentration ratios of particular ions. Osmotic concentration of the vacuolar fluid can be estimated as 10–40 mosm, leading to the hypothesis that contractile vacuoles of sponges function by isotonic water separation.

Cytoplasm Exudate from Cut Phloem

CYTOPLASM structure in sieve elements and particularly at sieve pores is of critical importance in any consideration of the translocation mechanism. The mass flow theory1 assumes a degenerate sieve element protoplast in which vacuolar fluid fills the sieve element and is continuous through open sieve pores. A fluid flow of sucrose solution is thought to occur through sieve tubes due to an osmotic pressure gradient. Other theories propose that metabolic energy provides the motive force required to move nutrients over long distances through plants. These ‘active’ theories assume that the sieve elements have cytoplasmic contents similar to parenchyma cells but with exceptionally large end wall pores which are filled with cytoplasm. This interpretation is consistent with de Vries protoplasmic streaming theory2, with Mason and Maskell's activated diffusion theory3, with Kursanov's molecular transfer theory4 and Spanner's electrokinetic theory5.

Amoeba proteus : Studying the Contractile Vacuole by Micropuncture

Direct measurements of the freezing point depression of the protoplasm and of the fluid from the contractile vacuole of fresh-water amoebae showed that the fluid in the vacuole is distinctly hypoosmotic to the protoplasm. Fourteen samples of protoplasm from amoebae, placed in a medium with a milliosmolality of 7, had an average osmolality of 101 milliosmoles with a range of 73 to 116. Eleven samples of vacuolar fluid had an average osmolality of 32 milliosmoles, with a range of 24 to 38. It is suggested that the fluid may be isoosmotic to the protoplasm when secreted and that salt is subsequently reabsorbed, leaving the vacuolar fluid hypoosmotic to the protoplasm.

CONTRACTILE VACUOLES1

SummaryAlthough comparatively little has been added to our knowledge of the structure of contractile vacuoles since the publication of Lloyd's (1928) review,1 the work of Gelei has thrown doubt upon previous interpretations of the functioning of the feeder canals and central vesicle of Paramecium. Moreover, much work with methods of osmic impregnation has suggested that the vacuolar membrane contains fats or lipoids.The idea that marine and parasitic Protozoa rarely or never have contractile vacuoles is incorrect; contractile vacuoles occur in a large number of marine and parasitic ciliates. However, the rate of output of fluid is much lower in marine than in fresh‐water ciliates of the same size. In some Protozoa–and probably in most fresh‐water ones–the water to be evacuated enters mainly by the general body surface. However, in a few cases the water taken in with the food provides the greater share. Nothing is known of the composition of the vacuolar fluid apart from its hydrogen‐ion concentration. Thus circumstantial evidence suggests that in many fresh‐water Protozoa osmoregulation is one of the functions of the contractile vacuole.The osmoregulatory theory has been given support by the results of many experiments. The rate of vacuolar output is usually decreased by an increase, and increased by a decrease, in the concentration of (suitable) solutes in the external medium. More direct evidence is provided by the use of agents, such as cyanide, which inhibit vacuolar activity. When vacuolar activity is stopped the body of fresh‐water Peritricha swells; and removal of the cyanide leads to recovery of the contractile vacuole and shrinkage of the body. A similar effect of cyanide has been found in euryhaline marine Peritricha placed in dilute sea water. It is concluded that the contractile vacuole controls the body volume by baling out water as fast as this comes in by osmosis through the body surface. In fact the contractile vacuole may be compared with the bilge pump of a rather leaky ship. However, it is clearly necessary that an osmoregulatory organ should conserve the internal salts of the organism; therefore according to the osmoregulatory theory the contractile vacuole should separate solutes from water. It is not known whether any other part of the body, such as the body surface, is concerned in osmoregulation. It should also be emphasized that the contractile vacuole does not maintain the internal concentration of the organism absolutely constant in spite of changes in the external osmotic pressure. The contractile vacuole merely has an ameliorating effect, so that the internal changes are less drastic.The contractile vacuole might perform other functions besides osmoregulation, and an excretory function has often been suggested for it. It is believed that in some cases granules disappear in the neighbourhood of the contractile vacuole during diastole, but it is by no means certain that their substance is removed in solution in the vacuolar fluid. There is no chemical evidence to show that the contractile vacuole eliminates nitrogenous waste matter. In certain Protozoa which take in large quantities of fluid food at one “mouthful”, the contractile vacuole gets rid of the excess water. The function of the contractile vacuoles of marine and parasitic Protozoa is unknown.Much speculation must enter into any discussion of the mechanisms of diastole and systole. The inadequacy of all other theories gives indirect support to the view that diastole involves an active secretion of water, and retention of solutes, by the vacuolar membrane. This view alone meets the requirements of the osmoregulatory theory of vacuolar function. Systole may be brought about by body turgor, but it appears from a consideration of multi‐vacuoled species that there must in addition be some mechanism which ensures that a vacuole does not discharge until it reaches a certain size. Many abnormalities of the ultimate vacuolar diameter may be attributed to a change in the structure of the surface membrane of the body.Many other external conditions influence vacuolar activity. Rise of temperature leads to an increase of frequency and to a decrease in ultimate diameter. The effects of neutral salts, hydrogen‐ion concentration, and heavy water are probably only indirect. Experiments on the effects of glandular extracts are so far inconclusive. Feeding and division rate are said to influence vacuolar frequency.According to the secretion theory, which is supported in this review, vacuolar activity involves the performance of osmotic work, the energy for which must ultimately be derived from cell respiration. It is not yet known whether the adverse effect of cyanide on vacuolar activity is due to inhibition of cell respiration, or to some independent poisoning action on the vacuolar mechanism. 1 Biological Reviews

The pH of gastric vacuoles

1. Actinosphaerium Eichhornii may incorporate in its gastric vacuoles, ciliates (usually hypotrichs) rotifers and nematodes, which remain alive after incorporation. 2. The pH of the vacuolar fluid in a newly formed vacuole containing live food is, for a period of five or ten minutes, neutral or slightly acid, i.e. 6·6 to 7·0. 3. With the increase in hydrogen-ion concentration, the activity of live prey decreases, until it becomes motionless. The length of time elapsing before this occurs is variable. In the case of ciliates this occupies but a few minutes, while rotifers may live for several hours. It is possible, therefore, that the acid functions as a killing fluid within the vacuole. 4. The pH of all gastric vacuoles containing food in the process of active digestion is 4·3 ± 0·1. 5. Old vacuoles, containing indigestible residuum usually have a pH value of 5·4 to 5·6. More rarely these may register 6·8 to 7·0.

Expulsion of Injected Solute by Contractile Vacuole of Amoeba.

Although it has long been the presumption that the contractile vacuole expels substances in solution in the endoplasm of the protozoon, the actual taking up and expulsion of a definite solute by the organoid has never been demonstrated. This now has been accomplished by means of the micrurgical apparatus. If a moderate amount of saturated aqueous solution of picric acid is injected into an ameba (Amoeba dubia) the course taken by the solute may be traced by its yellow color. The effect on the cytoplasm has already been described by Pollack. Though a part of the colored region is often injured by the pipette and thereupon pinched off by the ameba, a certain quantity of the solute diffuses into the remaining endoplasm before this occurs. This is taken up by the vacuole, the intensity of the yellow color of the vacuolar fluid increasing in proportion to the fading out of color in the endoplasm. When a 2% solution of picric acid in 95% alcohol is injected, diffusion throughout the endoplasm is much more rapid, and the yellow color appears more quickly in the vacuolar fluid. In the small percentage of cases where the injected region is not pinched off and the entire amount of the solute is retained, the fluid in the enlarging vacuole becomes an intense yellow. Such vacuoles progressively become very flaccid. Contact with the least obstacle, or stress exerted by endoplasmic currents easily causes their deformation. Their limp membranes may temporarily infold deeply, and the vacuoles often appear bean-shaped, long ovoid, or pyriform.

Application of the murexide test to Amoeba verrucosa and Paramecium caudatum

Observations made on the distended contractile vacuoles of Amoeba verrucosa subsequent to the application of the murexide test give no optical evidence of the presence of uric acid in the vacuolar fluid. This result is not in accord with the generally accepted conclusions of Griffiths1 who reported the production of “prismatic crystals of murexide” on application of this test to Amoeba proteus, Vorticella and Paramecium. Mass cultures of Amoeba verrucosa, killed in 50 per cent. alcohol, show the contractile vacuoles fixed at partial or complete expansion in a large percentage of cases, but a critical examination of the fluid of the vacuoles has never revealed the presence of uric acid crystals precipitated there by this method. Dark field examination of such animals shows the fluid to be structureless. Treatment with nitric acid and ammonia in the manner described by Griffiths colors both cytoplasm and pellicle a lemon yellow. Furthermore, control slides, consisting of a small quantity of pure uric acid c...

Studies on the contractile vacuoles of Amoeba verrucosa and Paramecium caudatum

The contractile vacuole of Amoeba verrucosa is formed by the union of a variable number of lesser globules. Subsequent to each contraction a new series of contributary globules may appear in the same location as the preceding vacuole, but this is not invariably the case. Random formation of new vacuoles is common, either at some distance from the original organelle, or close by. The walls of two functioning vacuoles may lie in contact for some time without confluence. The wall of the vacuole is easily indented with a blunt microneedle. A sharp-pointed microneedle will induce artificial systole by perforation of the wall of the vacuole. A large vacuole, or a group of contributary globules, freed into the water by ripping open a cell under slight pressure, retain their identity in this medium for some time. A quick upward thrust of a blunt needle against a large vacuole apparently causes its rupture into the surrounding endoplasm, for an increase in fluidity is observed in this area. This would imply the miscibility of vacuolar fluid and endoplasm. The contractile vacuoles of Paramecium caudatum become dilated on the addition of Grubler's Alizarin blue to the culture. The distended vacuoles together with their feeders become set, gradually taking on a vivid blue color. This stain is specific for the vacuolar walls, and, although it appears long before ciliary action ceases, should be classified as a sub-mortem rather than a vital stain. These stained and distended vacuoles do not collapse when removed from the animal but retain their shape and may be manipulated and dissected with microneedles.