Inhibition of contractile vacuole function by brefeldin A

Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells. In the scaly green flagellate Scherffelia dubia, BFA also interfered with the function of the contractile vacuoles (CVs). The CV is an osmoregulatory organelle which periodically expels fluid from the cell in many freshwater protists. Fusion of the CV membrane with the plasma membrane is apparently blocked by BFA in S. dubia. The two CVs of S. dubia swell and finally form large central vacuoles (LCVs). BFA-induced formation of LCVs depends on V-ATPase activity, and can be reversed by hypertonic media, suggesting that water accumulation in the LCVs is driven by osmosis. We suggest that the BFA-induced formation of LCVs represents a prolonged diastole phase. A normal diastole phase takes about 20 s and is difficult to investigate. Therefore, BFA-induced formation of LCVs in S. dubia represents a unique model system to investigate the diastole phase of the CV cycle.     

Acidocalcisomes and the contractile vacuole complex are involved in osmoregulation in Trypanosoma cruzi

Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle. T. cruzi possesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K+ release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation.     

Superoxide dismutase C affects Dictyostelium contractile vacuole biogenesis and function

Dictyostelium cells cope with hypo-osmotic stress with a contractile vacuole (CV) system, which consists of one or two vacuoles that cyclically charge and discharge. Uniquely, a F-Actin remodeling dependent minimal mixing of the CV membrane components with the target plasmalemma during the fusion and the dischargement warrants the integrity of the CV bladder for an efficient next CV cycle. The effect of hypo-osmotic stress on F-Actin remodeling activity, however, is currently not well understood. Dictyostelium cells increase the level of intracellular superoxide level in response to hypo-osmotic stress, which in turn activates redox-sensitive Ras proteins, but not Akt, which is one of the Ras downstream targets and a major regulator of F-Actin remodeling. However, Akt is not insulated from the active Ras in cells lacking Superoxide dismutase C (SodC). We report here that sodC^- cells were compromised in the CV structure and function and the attenuation of Ras/PI3K/Akt signaling in several independent means significantly improved the compromised CV structure but not the function. Interestingly, when sodC^- cells were treated with 5-(N,N-Dimethyl) amiloride hydrochloride (EIPA), an inhibitor of sodium proton exchanger (NHE), both the structure and the function of the CV improved. Thus, a proper CV biogenesis in sodC^- cells was insufficient to restore their CV function, which in turn indicates the presence of an additional target for SodC and EIPA that modulates CV function.     

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi

Acidocalcisomes are dense, acidic organelles with a high concentration of phosphorus present as pyrophosphate and polyphosphate complexed with calcium and other cations. Acidocalcisomes have been linked to the contractile vacuole complex in Chlamydomonas reinhardtii, Dictyostelium discoideum, and Trypanosoma cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement.     

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     

Distribution of alkaline phosphatase in vegetative dictyostelium cells in relation to the contractile vacuole complex

The structure of the contractile vacuole complex of Dictyostelium discoideum has long been a subject of controversy. A model that originated from the work of John Heuser and colleagues described this osmoregulatory organelle as an interconnected array of tubules and cisternae the membranes of which are densely populated with vacuolar proton pumps. A conflicting model described this same organelle as bipartite, consisting of a pump-rich spongiome and a pump-free bladder, the latter membranes being identified by their alkaline phosphatase activity. In the present study we have employed an antiserum specific for Dictyostelium alkaline phosphatase to examine the distribution of this enzyme in vegetative cells. The antiserum labels puncta, probably vesicles, that lie at or near the plasma membrane and are sometimes, but only rarely, enriched near contractile vacuole membranes. We conclude that alkaline phosphatase is not a suitable marker for contractile vacuole membranes. We discuss these results in relation to the two models of contractile vacuole structure and suggest that all data are consistent with the first model.     

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.     

Studies on the contractile vacuole of Poterioochromonas malhamensis Peterfi. II. Antimonate stains the contractile vacuole of Poterioochromonas

Fixation of the chrysophycean flagellate Poterioochromonas malhamensis in the presence of potassium (pyro) antimonate have been carried out. Intense antimonate deposits are characteristic of the various elements of the contractile vacuole as well as a portion of the plasma membrane adjacent to the contractile vacuole. Neither pretreatment of the cells with EGTA or EDTA nor the treatment of sections with these chelators is successful in removing the antimonate precipitates. Despite this, X-ray microanalyses of these deposits indicate the presence of calcium.     

Development of periodic tension in the contractile vacuole complex membrane of paramecium governs its membrane dynamics

The contractile vacuole complex is a membrane-bound osmoregulatory organelle of fresh water protozoa such as Paramecium. In Paramecium it consists of a central vacuole (the contractile vacuole) and 5-10 arms that radially extend from the vacuole into the cytosol (the radial arms). Excess cytosolic water, acquired osmotically, is segregated by the radial arms and enters the vacuole, so that the vacuole swells (the fluid-filling phase). The vacuole then rounds (the rounding phase) and the radial arms sever from the vacuole. The vacuole membrane then fuses with the plasma membrane at the pore region and the pore opens. The vacuole shrinks as its fluid is discharged through the pore (the fluid-discharging phase). The pore closes when the fluid has been discharged. The radial arms then reattach to the vacuole, so that the vacuole swells again as the fluid enters from the arms (the next fluid-filling phase). We found that the vacuole continued to show rounding and slackening even after it together with a small amount of cytosol had been isolated from the cell. Using a microcantilever placed on the surface of the vacuole the tension of the in vitro vacuole increased to 5 x 10(-3)N m(-1) as the vacuole rounds, and its lowest value was 1 x 10(-4)N m(-1) during slackening. We propose a hypothesis that an increase in the spontaneous curvature of the organelle's membrane leads to an increase in membrane tension and thus to the vacuole's rounding, severing of the radial arms from the vacuole, and opening of the pore. Conversely, a decrease in the spontaneous curvature accompanied by a decrease in membrane tension could lead to the closing of the pore and reattachment of the radial arm at the start of the fluid-filling phase.     

Tubular-vesicular transformation in the contractile vacuole system of Dictyostelium

The contractile vacuole complex of Dictyostelium is the paradigm of a membrane system that undergoes tubular-vesicular transitions during its regular cycle of activities. This system acts as an osmoregulatory organelle in freshwater amoebae and protozoa. It collects fluid in a network of tubules and cisternae, and pumps it out of the cell through transient pores in the plasma membrane. Tubules and vacuoles are interconvertible. The tubular channels are associated with the cortical actin network and are capable of moving and fusing. The contractile vacuole complex is separate from vesicles of the endosomal pathway and preserves its identity in a dispersed state during cell division. We outline techniques to visualize the contractile vacuole system by electron and light microscopy. Emphasis is placed on GFP-fusion proteins that allow visualization of the dynamics of the contractile vacuole network in living cells. Proteins that control activities of this specialized organelle in Dictyostelium have been conserved during evolution and also regulate membrane trafficking in man.     

ATP and the autonomy of the contractile vacuole in Amoeba proteus

Contractile vacuole function in amoebae treated with immobilizing (5 mM) and nonimmobilizing (0.125 mM) concentrations of ATP has been studied. In ATP-immobilized amoebae, most vacuolar parameters are accelerated, especially the rate of output which passes from 30 to 70 micron3/sec. This favors the concept of an autonomous vacuole, fully functional in the absence of any bulk contribution to it from remote points of the cell. A lower concentration of ATP (0.125 mM), which does not inhibit movement, causes a still greater acceleration of vacuolar function. Work is in progress to elucidate the site and mode of action of exogenous ATP on Amoeba.     

Regulation of contractile vacuole formation and activity in Dictyostelium

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV–plasma membrane fusion and discharging. disgorgin⁻ cells produce large CVs due to impaired CV–plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin⁻ large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function.     

Identification of contractile vacuole proteins in Trypanosoma cruzi

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.     

A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi

We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl(2) and AgNO(3). glycerol transport was detected. No Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H(+)-PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.     

Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.