Contractile Vacuole Function Research Articles

A conserved pressure-driven mechanism for regulating cytosolic osmolarity

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of 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 distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

Rediscovered as a potential eukaryotic epigenetic mark, DNA N6-adenine methylation (6mA) varies across species in abundance and its relationships with transcription. Here we characterize AMT1—representing a distinct MT-A70 family methyltransferase—in the ciliate Tetrahymena thermophila. AMT1 loss-of-function leads to severe defects in growth and development. Single Molecule, Real-Time (SMRT) sequencing reveals that AMT1 is required for the bulk of 6mA and all symmetric methylation at the ApT dinucleotides. The detection of hemi-methylated ApT sites suggests a semi-conservative mechanism for maintaining symmetric methylation. AMT1 affects expression of many genes; in particular, RAB46, encoding a Rab family GTPase involved in contractile vacuole function, is likely a direct target. The distribution of 6mA resembles H3K4 methylation and H2A.Z, two conserved epigenetic marks associated with RNA polymerase II transcription. Furthermore, strong 6mA and nucleosome positioning in wild-type cells is attenuated in ΔAMT1 cells. Our results support that AMT1-catalyzed 6mA is an integral part of the transcription-associated epigenetic landscape. AMT1 homologues are generally found in protists and basal fungi featuring ApT hyper-methylation associated with transcription, which are missing in animals, plants, and true fungi. This dichotomy of 6mA functions and the underlying molecular mechanisms may have implications in eukaryotic diversification.

Osmotolerance in the Cryptophyceae: Jacks-of-all-trades in the Chroomonas Clade

No detailed studies have been performed to date on osmotolerance in cryptophytes, although one species, Chroomonas africana, had previously been reported to grow in freshwater as well as seawater. This study focused on osmotolerance in Chroomonas. Growth at different osmolalities and parameters of contractile vacuole function were examined and compared across a high-resolution phylogeny. Two evolutionary lineages in the Chroomonas clade proved to be euryhaline. Ranges of osmotolerance depended not only on osmolality, but also on culture medium. All cryptophytes contained contractile vacuoles. In the euryhaline strain CCAP 978/08 contractile vacuoles could be observed even at an osmolality beyond that of seawater. In addition the cells accumulated floridoside, an osmoprotectant likely originating from the red algal carbohydrate metabolism of the complex rhodoplast. Further evidence for functional contractile vacuoles also in marine cryptophytes was provided by identification of contractile vacuole-specific genes in the genome of Guillardia theta.

An Acanthamoeba castellanii metacaspase associates with the contractile vacuole and functions in osmoregulation

Acanthamoeba castellanii is a free-living protozoan. Some strains are opportunistic pathogens. A type-I metacaspase was identified in A. castellanii (Acmcp) and was shown to be expressed through the encystation process. The model organism, Dictyostelium discoideum, has been used here as a model for studying these caspase-like proteins. Separate cell lines expressing a GFP-tagged version of the full length Acmcp protein, as well as a deletion proline region mutant of Acmcp protein (GFP-Acmcp-dpr), have been introduced into D. discoideum. Both mutants affect the cellular metabolism, characterized by an increase in the growth rate. Microscopic imaging revealed an association between Acmcp and the contractile vacuole system in D. discoideum. The treatment of cells with selected inhibitors in different environments added additional support to these findings. This evidence shows that Acmcp plays an important role in contractile vacuole regulation and mediated membrane trafficking in D. discoideum. Additionally, the severe defect in contractile vacuole function in GFP-Acmcp-dpr mutant cells suggests that the proline-rich region in Acmcp has an essential role in binding this protein with other partners to maintain this process. Furthermore, Yeast two-hybrid system identified there are weak interactions of the Dictyostelium contractile vacuolar proteins, including Calmodulin, RabD, Rab11 and vacuolar proton ATPase, with Acmcp protein. Taken together, our findings suggest that A. castellanii metacaspase associate with the contractile vacuole and have an essential role in cell osmoregulation, which contributes to its attractiveness as a possible target for treatment therapies against A. castellanii infection.

A Mechanism of Intracellular P2X Receptor Activation

P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen.

The System of Contractile Vacuoles in the Green Alga Mesostigma viride (Streptophyta)

The contractile vacuole (CV) is an osmoregulatory organelle which is found in many protists. We have investigated the structure and function of the CV in the green alga Mesostigma viride by light (video) and serial section electron microscopy. Mesostigma is the only known flagellate streptophyte (charophycean green algae and land plants) and therefore of great importance for our understanding of the evolution of streptophytes. The entire CV system of Mesostigma has been reconstructed 3-dimensionally for three cells. Based on light microscopy cells contain an average of 8 CVs. The maximal diameter of a CV in Mesostigma was 1.5 microm and the expulsion interval 24.1s in 10 mosM medium. Video microscopy revealed the system of CVs to be very dynamic with individual CVs connecting temporarily or fusing completely with each other. Electron microscopy confirmed these observations and showed coated vesicles to be predominantly associated with large CVs. No expulsion pore was observed by electron microscopy. Instead we encountered close contact zones of approximately 150 nm diameter, which we propose to be the site of water expulsion. A model for the function of CVs in Mesostigma is presented.

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.

Presence of aquaporin and V‐ATPase on the contractile vacuole of Amoeba proteus

The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90]. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.

Copine A Is Required for Cytokinesis, Contractile Vacuole Function, and Development inDictyostelium

Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA- knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA- cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA- cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA- cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA- cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development.

Inhibition of Contractile Vacuole Function by Brefeldin A

Inhibition of Contractile Vacuole Function by Brefeldin A

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.

Localization of Pyrophosphatase and V‐ATPase in Chlamydomonas reinhardtii

Abstract:Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V‐ATPase activities. By immunogold labelling we have shown that H+‐pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V‐ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V‐ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 − 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,‐head of V‐ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate‐sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely‐packed 6 − 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5‐ and 6‐fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V0 body of the V‐ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function.

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

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.

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.

Inhibition of contractile vacuole function in vivo by antibodies against myosin-I.

Myosin-I is thought to supply the force for movement of cell membranes relative to actin filaments (reviewed in refs 1, 2), but confirmation of this hypothesis has been difficult because of the presence of multiple isoforms of myosin-I and other unconventional myosins in most cells. We report here the first evidence that a myosin-I isoform is essential for a specific class of intracellular membrane movements in vivo. In Acanthamoeba, the contractile vacuole is an autonomous structure which fuses with the plasma membrane to control the water content of the cell. Because myosin-IC is the only myosin-I isoform concentrated in the contractile vacuole complex, and a protein antigenically related to myosin-IC is located on or near the Dictyostelium (slime mould) contractile vacuole, we thought this organelle might provide the best opportunity to demonstrate a relationship between myosin-I and membrane motility. Antibodies that inhibit the activity of Acanthamoeba myosin-IC in vitro interfere with expulsion of excess water by the contractile vacuole in vivo, leading to overfilling of this organelle and cell lysis. Myosin-IC may generate the force required to contract the vacuole and may also be involved in transfer of water to the contractile vacuole during refilling.

Contractile vacuole activity in Paramecium aurelia: Dependence upon intracellular sodium

1. 1. The dependence of contractile vacuole activity upon the levels of cytoplasmic Na+ were studied in Paramecium aurelia by means of drugs capable of increasing or decreasing membrane permeability to Na+. 2. 2. The Na+ ionophore monensin produced an abrupt and reversible decrease in the frequency of contraction. This effect was partly dependent upon the presence of Na+ in the medium. 3. 3. The diuretic amiloride, known to hinder Na+ influx in many cells, increased vacuole activity, although it did not prevent a decline in frequency induced by a sudden rise in the transmembrane gradient of Na+. 4. 4. The results suggest an inverse relationship between Na+ levels in the cytoplasm and the frequency of contractile vacuole function.

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.

Dimensionality and contractile vacuole function in ciliated protozoa

AbstractIt is believed by some (Günther, '75a) that the dimensional theory of biological similarity is somehow not appropriate at the microscopic and cellular levels. The results of the present study of contractile vacuoles of ciliated protozoa, ranging in length from 25 to 150 μm, sugggests, however, that the theory is appropiate. The data demonstrate that 1)the expected strong negative correlation exisots between frequency of contraction and cell size, and 2) that the expected positive correlation exists between volume output of the vacuole and cell size. An additional experiment indicates the improbability of Tucker's hypothesis ('71) that the epiplasmic layer provides a barrier to water flux through the cell cortex.

THE ENERGETICS OF FRESHWATER ALGAE; ENERGY REQUIREMENTS FOR BIOSYNTHESIS AND VOLUME REGULATION

SUMMARYFreshwater algae have an intracellular osmolarity in excess of that of the medium. The tendency for water to enter the cells is resisted, in cells with a mechanically functional cell wall, by the ability of the wall to withstand a turgor pressure equivalent to the intracellular osmolarity; in cells lacking such a mechanically functional wall the water which enters is expelled by a contractile vacuole (or its functional equivalent). A comparison of the energy costs of cell volume regulation by these two mechanisms has been carried out making energy costings based on the minimum thermodynamic requirement and on plausible mechanisms for the two processes (wall synthesis and contractile vacuole function). This analysis yielded the following conclusions.(1) Wall synthesis is a ‘growth’ process, while contractile vacuole operation is a’maintenance’process; other things being equal, a cell wall is a preferable mechanism of volume regulation in non‐growing cells.(2) For a 5 μm radius cell with a specific growth rate of 8 × 10−6 s−1 and a 10 μm radius cell with a rate of 4 × 10−6 s−1 the energy input rate for contractile vacuole operation is considerably lower than for wall synthesis on the basis of minimum thermodynamic requirement; the plausible mechanism approach makes the two energy costs quite similar. Decreased growth rates favours the wall over the contractile vacuole mechanism.(3) Increased cell size (10 μm radius rather than 5 μm) has similar effects on the energy costs of both volume regulation mechanisms, provided a (cell organic weight)−0.32 dependence of intrinsic growth rate is incorporated into the computations.(4) Increased intracellular osmolarity (at constant extracellular osmolarity) causes a directly proportional increase in energy input rate for the cell wall mechanism, and a more than proportional increase in energy cost for the contractile vacuole mechanism.(5) A non‐spherical shape increases the energy cost (at constant cell volume) for both the cell wall and the contractile vacuole mechanisms.These conclusions may be used in the interpretation of such phenomena as the lower osmolarity of wall‐less than walled cells and the tendency for the resting stages of normally wall‐less and flagellate cells to have walls. It appears that the majority of freshwater algae have genetic capabilities to produce both walls and contractile vacuoles for cell volume regulation (e.g, the production of walled cysts by organisms which are normally flagellate, and the production of wall‐less motile spores and gametes by normally walled organisms).