The Response of Contractile and Non-Contractile Vacuoles of Paramecium calkinsi to Widely Varying Salinities

ABSTRACT. Paramecium calkinsi from tidal marshes survive a wide salinity range. Fluid output of contractile vacuoles of these cells decreased as salinity of the medium to which they were acclimated increased, and both pulse rate and vacuole volume were used to regulate output. When cells were first exposed to more dilute medium, contractile vacuoles greatly increased volume so that fluid output increased even though pulse rate decreased. In cells shifted to a more concentrated medium, contractile vacuole output decreased by decreasing pulse rate. The contractile vacuole is surrounded by a set of collecting structures which change form as the salinity changes. Distensible ampullae are found in media of low salinity and collecting canals are found in media of high salinity. When cells are shifted from high salinity to low, the number of ampullae increases and the number of canals decreases. When cells are shifted from low salinity to high, the number of ampullae decreases and the number of canals decreases. Other non-contracting vacuoles also appear in response to a hypoosmotic shock. These include vacuoles within the cell as well as "blisters" on the surface. The number and frequency of blisters increases with the size of the hypoosmotic shock. They detach from cells without resulting in any visible loss of cytoplasm. Non-contractile vacuoles may play a role in sequestering and removing excess water that the contractile vacuoles cannot handle.     

Membrane Dynamics of the Contractile Vacuole Complex of Paramecium1

ABSTRACT. Membrane dynamics of the contractile vacuole complex of Paramecium were investigated using conventional electron microscopy of cells so that the vacuoles were serial-sectioned longitudinally and transversely. During systole, vacuolar membrane collapses first into flattened cisternae which undergo further modification into a mass of interconnected small membrane tubules. These tubules retain their connections with the radiating microtubular ribbons; consequently they are found only in the poleward hemisphere. Permanent connections between ampullae and the collapsed vacuole membrane could not be verified nor was a sphincter-like mechanism for closing such a junction observed. Membranes of the ampullae and the collecting canals also collapse to varying extents into arrays of tubules that remain bound to microtubular ribbons during diastole. Thus vacuole, ampullae, and collecting canal membranes all assume tubular forms when internal volume is at a minimum. Having failed to observe a microfilamentous encasement of the vacuole, we suggest that an alternative mechanism for the “contractile” function should be sought. One such is based on fluid volume increase and fluid flow within transiently interconnected tubular membrane systems that cycle between a tubular and a planar membrane form as internal volume is periodically increased and reduced. The driving force for this mechanism might best be sought in the molecular structure of the membranes of the contractile vacuole complex.     

ELECTRON MICROSCOPY OF A CONTRACTILE-VACUOLE MUTANT OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYTA) DEFECTIVE IN THE LATE STAGES OF DIASTOLE1

Electron microscopy of a “vacuole-less” mutant of Chlamydomonas moewusii Gerloff revealed the presence of small anterior vacuoles. These vacuoles behaved like contractile vacuoles in wild-type cells, but they were apparently unable to complete diastole and discharge their contents. When wild-type and mutant cells were incubated in hypertonic medium, small coated vacuoles persisted in the region where contractile vacuoles form. When these cells were transferred to hypotonic medium, the vacuoles appeared to fill and fuse to form larger vacuoles Shortly after the appearance of full expanded contractile vacuoles, collapsed vacuoles were observed in wild-type cells suggesting the completion of diastole and the onset of systole. In mutant cells, the initial steps of filling and fusion to form larger vacuoles apparent interactions of vacuoles with the plasma membrane were not observed. New contractile vacuoles accumulated around the nucleus. When fusion of the contractile vacuole with the plasma membrane was blocked by EGTA, a similar accumulation of large vacuoles occurred. Our observations suggest that the contractile-vacuole mutant of C. Moewusii produces vacuoles which can accumulate excess water as part of the mechanism of osmoregulation but which cannot complete diastole.     

The Role of the Contractile Vacuole in the Osmoregulation of Tetrahymena pyriformis*

The relationship of cell size and contractile vacuole efflux to osmotic stress was studied in Tetrahymena pyriformis strain W, after transfer into fresh solutions iso- or hypoosmotic to the growth medium. Microscopic measurements of the cell and contractile vacuole dimensions, made with an image-sharing ocular at 27 C, allowed the calculation of the cell size and shape and the vacuolar efflux rate which provide a measure of osmoregulation. The contractile vacuole cycles have no homeostatic oscillations. In 0.03–0.10 osmolar solutions, the cell size and shape are constant while the vacuolar efflux rate has an inverse linear dependence upon extracellular osmolarity. Regression analyses indicate that for cells with systole faster than 0.1 sec (the major part of the population), it is only the final diastolic volume of the contractile vacuole that is related to osmotic stress while the frequency of systole is independent of osmotic stress and has a constant period of 7.7 ± 0.2 sec. Therefore, osmotic stress upon Tetrahymena is regulated by a corresponding change in the filling rate of its contractile vacuole to allow an unaltered cell size and shape. Kinetic measurements of vacuoles during diastole fit the model (dV/dt = K_1-K₂A), where (dV/dt) is the vacuolar filling rate and (A) is the vacuolar surface area. This dependence of vacuolar volume upon its surface area may be ascribed either to elastic components of the vacuolar membrane or to an increasing leakiness of this membrane during diastole. Mitochondrial inhibitors were used to observe the energy requirements of vacuolar operation and of intracellular secretion of water.     

Behavior of the contractile vacuole of Tetrahymena pyriformis W: a redescription with comments on the terminology.

The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.     

Behavior of the Contractile Vacuole of Tetrahymena pyriformis W: A Redescription with Comments on the Terminology

SYNOPSIS. The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.     

Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum

mAbs specific for calmodulin were used to examine the distribution of calmodulin in vegetative Dictyostelium cells. Indirect immunofluorescence indicated that calmodulin was greatly enriched at the periphery of phase lucent vacuoles. The presence of these vacuoles in newly germinated (non-feeding) as well as growing cells, and the response of the vacuoles to changes in the osmotic environment, identified them as contractile vacuoles, osmoregulatory organelles. No evidence was found for an association of calmodulin with endosomes or lysosomes, nor was calmodulin enriched along cytoskeletal filaments. When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml. Several high molecular weight calmodulin-binding proteins were enriched in the same region of the gradient. One of the calmodulin-binding polypeptides (molecular mass approximately 150 kD) cross-reacted with an antiserum specific for Acanthamoeba myosin IC. By indirect immunofluorescence, this protein was also enriched on contractile vacuole membranes. These results suggest that a calmodulin-binding unconventional myosin is associated with contractile vacuoles in Dictyostelium; similar proteins in yeast and mammalian cells have been implicated in vesicle movement.     

Effect of Osmotic Shock on Protein Synthesis of Oyster Hemocytes In Vitro

Because marine bivalves are osmoconformers, their cells may be exposed to widely fluctuating osmolality in some habitats. In vitro studies were conducted to evaluate the effect of changes in salinity on protein synthesis of oyster hemocytes. Increasing salinity from a control value of 20–25 ppt to 32–98 ppt decreased the rate of incorporation of amino acid into protein, but did not qualitatively alter the pattern of protein synthesis. On the other hand, decreasing salinity to 3.5–4 ppt not only decreased the rate of protein synthesis, but also altered the types of protein produced. At least a third of the cells remained viable at low salinity and resumed the control pattern of protein synthesis within hours after return to the normal medium. The response to hypoosmotic shock was different from the response to a hyperthermic shock, each stressor inducing expression of a characteristic set of proteins. Preferential synthesis of these proteins may represent an adaptation to preserve or restore oyster cell functions under adverse conditions.     

Hypoosmotic Shock Induces Increases in Cytosolic Ca2+ in Tobacco Suspension-Culture Cells

Hypoosmotic shock treatment increased cytosolic Ca2+ ion concentration ([Ca2+]cyt) in tobacco (Nicotiana tabacum) suspension-culture cells. [Ca2+]cyt measurements were made by genetically transforming these cells to express apoaequorin and by reconstituting the Ca2+-dependent photoprotein, aequorin, in the cytosol by incubation with chemically synthesized coelenterazine. Measurement of Ca2+-dependent luminescence output thus allowed the direct monitoring of [Ca2+]cyt changes. When cells were added to a hypoosmotic medium, a biphasic increase in [Ca2+]cyt was observed; an immediate small elevation (phase 1) was observed first, followed by a rapid, large elevation (phase 2). Phase 1 [Ca2+]cyt was stimulated by the V-type ATPase inhibitor bafilomycin A1. Phase 2 was inhibited by the protein kinase inhibitor K-252a and required the continued presence of the hypoosmotic stimulus to maintain it. Although Ca2+ in the medium was needed to produce phase 2, it was not needed to render the cells competent to the hypoosmotic stimulus. If cells were subject to hypoosmotic shock in Ca2+- depleted medium, increases in luminescence could be induced up to 20 min after the shock by adding Ca2+ to the medium. These data suggest that hypoosmotic shock-induced [Ca2+]cyt elevation results from the activity of a Ca2+ channel in the plasma membrane or associated hypoosmotic sensing components that require Ca2+- independent phosphorylation and a continued stimulus to maintain full activity.     

Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro

In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.     

The monomeric clathrin assembly protein, AP180, regulates contractile vacuole size in Dictyostelium discoideum

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium.     

Comparative analysis of the structure of the vacuolar system of the bladder granular cells in the frog and of the contractile vacuole complex of protozoans

A comparative morphological study of the vacuolar system of the frog urinary bladder epithelial cells and of the contractile vacuole complex of Paramecium caudatum enabled us to reveal some common structural elements in these: spongial channels and general vacuole reservoir. The structural similarity of these organoids seems to be the base of their analogous functions in the cell. Detection of vacuoles of various forms in different areas of granular cells may point to a possible migration of the vacuoles around their cytoplasm. Localization of spheric vacuoles in the innermost contact with the plasma membrane, and dilution of the intercellular space in this epithelial part may suggest an expulsion of the vacuole content in the basolateral part of the cell. The "contractile" vacuoles of granular cells are related to other intracellular structure: the Golgi apparatus, coated vesicles, microtubules, microfilaments.     

Food ingestion and egestion in mating reactive populations of Paramecium primaurelia.

The study of food ingestion and egestion carried out on Paramecium primaurelia mating reactive cells shows that, after their transfer into a medium with suspended particles, the complementary mating type cells exhibit very significant differences in the food vacuole formation and egestion rate. Under the same external environmental conditions, the mating type II cells form and egest a higher number of food vacuoles when compared with mating type I cells. The higher rate of food vacuole formation shown by the mating type II cells is related to their faster growth rate.     

AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.     

Osmoregulatory mutants that affect the function of the contractile vacuole inChlamydomonas reinhardtii

Summary Four independent osmoregulatory mutants,osml, osm3,osm4, and osm7, were isolated on the basis of their requirement for growth medium of high osmotic strength. In normal low-osmoticstrength medium, in contrast to wild-type cells, the mutants grow poorly or not at all; in distilled water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timing of various stages of the CV cycle showed thatosm1 was affected primarily in the early stage of the cycle when the CV begins to grow,osm3 primarily in midcycle when vacuoles fuse to form the CV proper,osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, andosm4 during contraction of the CV. At the electron microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and dilated ER cisternae. Although electron microscopy showed general abnormalities of the contractile vacuoles consistent with the videomicroscopic observations of living cells, no obvious vacuole membrane abnormalities were seen which would explain the mutational defects. The mutations help define the separate processes that contribute to the coordinated CV cycle inChlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.Abbreviations CV contractile vacuole - TAP Tris-acetate-phosphate medium - TAP+L medium supplemented with lactose - TAP+S medium supplemented with sucrose or other sugar     

A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.     

Intracellular glycerol in Dunaliella is depleted by intracellular metabolism in response to hypoosmotic stress by dilution

Abstract When Dunaliella tertiolecta cells are subjected to a dilution stress (hypoosmotic shock) the intracellular glycerol is metabolised biochemically, but it does not leak into the medium. However, Dunaliella cells have a certain 'threshold' to withstanding a hypoosmotic shock beyond which cell damage occurs and then glycerol is leaked into the medium. Both in the light or the dark, the glycerol metabolism was inhibited by micromolar concentration of carbonylcyanide m - chlorophenyl - hydrazone (CCCP) suggesting that the required ATP for the glycerol dissimilation is dependent upon an energized membrane, and most of which can be supplied through the oxidative phosphorylation.     

A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.     

The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii

Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used Chlamydomonas reinhardtii as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in Chlamydomonas is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed.     

Role of water channels in the regulation of the volume of principal cells of rat kidney collecting ducts in hypoosmotic medium

The effect of plasma membrane water permeability on the rate of changes in the volume of principal cells of collecting ducts of the outer substantia medullaris under conditions of hypoosmotic shock has been studied. Changes in cell volume were studied by the fluorescent method. It was shown that the hypotonic shock induced a rapid increase in the cell volume with the characteristic time that depended on plasma membrane water permeability. The decrease in volume occurred much more slowly, and the rate of volume decrease directly correlated with the rate of swelling. The inhibition of potassium transport by barium chloride decreased the rate of volume restoration, without affecting substantially the duration of the swelling phase. The inhibition of mercury-sensitive water channels by mercury caused a significant increase in the time of both cell swelling and volume restoration. It was concluded that the state of water channels largely determines the rate of the regulatory response of epithelial cells of collecting ducts to hypoosmotic shock and affects the exchange of cell osmolites.