Vacuolar Function Research Articles

Osmotic sensitive characteristics of an LmpB mutant strain in cellular slime mould Dictyostelium discoideum

As one of the cellular slime mould mutants produced with restriction enzymes mediated integration (REMI), we isolated a strain that carries an insertion mutation in LmpB gene. LmpB is a lysosomal protein and suggested to be involved in the endosome maturation. Although some regulatory components associated with endosomes are commonly found in the contractile vacuoles, LmpB has not been suggested to be involved in the process of the osmotic regulation. However, when LmpB mutant cells were placed under osmotic stress, they developed morphologically aberrant large vacuoles in the cytoplasm and the viability of the cells was significantly reduced. The result implies that LmpB is not only involved in the endosome maturation, but also involved in the function of contractile vacuoles.

Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae

The vacuole of Saccharomyces cerevisiae has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuolar events have been unable to grow at low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that such severe hypersensitivity to hygromycin B (hhy) is linked to vacuolar defects and performed a genomic screen for the phenotype using a haploid deletion strain library of non-essential genes. Fourteen HHY genes were initially identified and were subjected to bioinformatics analyses. The uncovered hhy mutants were experimentally characterized with respect to vesicular trafficking, vacuole morphology, and growth under various stress and drug conditions. The combination of bioinformatics analyses and phenotypic characterizations implicate defects in vesicular trafficking, vacuole fusion/fission, or vacuole function in all hhy mutants. The collection was enriched for sensitivity to monensin, indicative of vacuolar trafficking defects. Additionally, all hhy mutants showed severe sensitivities to rapamycin and caffeine, suggestive of TOR kinase pathway defects. Our experimental results also establish a new role in vacuolar and vesicular functions for two genes: PAF1, encoding a RNAP II-associated protein required for expression of cell cycle-regulated genes, and TPD3, encoding the regulatory subunit of protein phosphatase 2A. Thus, our results support linkage between severe hypersensitivity to hygromycin B and vacuolar defects.

Autophagy is involved in starvation response and cell death in Blastocystis

Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.

MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces cerevisiae

Magnesium (Mg) is an essential enzyme cofactor and a key structural component of biological molecules, but relatively little is known about the molecular components required for Mg homeostasis in eukaryotic cells. The yeast genome encodes four characterized members of the CorA Mg transporter superfamily located in the plasma membrane (Alr1 and Alr2) or the mitochondrial inner membrane (Mrs2 and Lpe10). We describe a fifth yeast CorA homolog (Mnr2) required for Mg homeostasis. MNR2 gene inactivation was associated with an increase in both the Mg requirement and the Mg content of yeast cells. In Mg-replete conditions, wild-type cells accumulated an intracellular store of Mg that supported growth under deficient conditions. An mnr2 mutant was unable to access this store, suggesting that Mg was trapped in an intracellular compartment. Mnr2 was localized to the vacuole membrane, implicating this organelle in Mg storage. The mnr2 mutant growth and Mg-content phenotypes were dependent on vacuolar proton-ATPase activity, but were unaffected by the loss of mitochondrial Mg uptake, indicating a specific dependence on vacuole function. Overexpression of Mnr2 suppressed the growth defect of an alr1 alr2 mutant, indicating that Mnr2 could function independently of the ALR genes. Together, our results implicate a novel eukaryotic CorA homolog in the regulation of intracellular Mg storage.

New Dynamics in an Old Friend: Dynamic Tubular Vacuoles Radiate Through the Cortical Cytoplasm of Red Onion Epidermal Cells

The textbook image of the plant vacuole sitting passively in the centre of the cell is not always correct. We observed vacuole dynamics in the epidermal cells of red onion (Allium cepa) bulbs, using confocal microscopy to detect autofluorescence from the pigment anthocyanin. The central vacuole was penetrated by highly mobile transvacuolar strands of cytoplasm, which were also visible in concurrent transmitted light images. Tubular vacuoles also extended from the large central vacuole and radiated through the cortical cytoplasm. These tubules were thin, having a diameter of about 1.5 microm, and were connected to the central vacuole as shown by fluorescence recovery after photobleaching (FRAP) experiments. The tubules were bounded by the tonoplast, as revealed by transient expression of green fluorescent protein (GFP) targeted to the vacuolar membrane and through labeling with the dye MDY-64. Expression of endoplasmic reticulum-targeted GFP demonstrated that the vacuolar tubules were distinct from the cortical endoplasmic reticulum. Movement of the tubular vacuoles depended on actin microfilaments, as microfilament disruption blocked tubule movement and caused their collapse into minivacuoles. The close association of the tubules with GFP-tagged actin microfilaments suggests that the tubules are associated with myosin, and that tubules likely move along microfilaments. Tubular vacuoles do not require anthocyanin for their formation, as tubules were also present in white onion cells that lack anthocyanin. The function of these tubular vacuoles remains unknown, but as they greatly increase the surface area of the tonoplast, they might increase transport rates between the cytoplasm and vacuole.

A Simple and Specific Procedure to Permeabilize the Plasma Membrane ofSchizosaccharomyces pombe

Cu(2+)-treatment is a useful technique in selectively permeabilizing the fungal plasma membrane. We describe herein a practical application with Schizosaccharomyces pombe. Incubation of cells with 0.5 mM CuCl(2) at 30 degrees C for 20 min induced efficient leakage of cytosolic constituents. The kinetic characteristics of the calcium and amino acid flux from Cu(2+)-treated S. pombe cells suggested that the Cu(2+) treatment permeabilized the plasma membrane without loss of vacuolar function. As a further application of the method, the amino acid contents of Cu(2+)-treated and untreated cells were also determined. The amino acid pool of Cu(2+)-treated wild-type cells was enriched in basic amino acids but not in acidic amino acids, as is characteristic of the vacuolar amino acid pool of fungi, including Saccharomyces cerevisiae and Neurosporra crassa. The amino acid pool of the S. pombe V-ATPase mutant vma1Delta was also successfully determined. We conclude that the vacuolar amino acid pool of S. pombe can be measured using Cu(2+)-treated cells. The method is simple, inexpensive, and rapid relative to the isolation of vacuolar vesicles, making it useful in estimating vacuolar pools and transport across the vacuolar membrane.

Dynamics of vacuoles and actin filaments in guard cells and their roles in stomatal movement

Vacuoles and actin filaments are important cytoarchitectures involved in guard cell function. The changes in the morphology and number of vacuoles and the regulation of ion channel activity in tonoplast of guard cells are essential for stomatal movement. A number of studies have investigated the regulation of ion channels in animal and plant cells; however, little is known about the regulating mechanism for vacuolar dynamics in stomatal movement. Actin filaments of guard cells are remodelling with the changes in the stomatal aperture; however, the dynamic functions of actin filaments in stomatal movement remain elusive. In this paper, we summarize the recent developments in the understanding of the dynamics of actin filaments and vacuoles of guard cells during stomatal movement. All relevant studies suggest that actin filaments might be involved in stomatal movement by regulating vacuolar dynamics and the ion channels in tonoplast. The future study could be focused on the linker protein mediating the interaction between actin filaments and tonoplast, which will provide insights into the interactive function of actin and vacuole in stomatal movement regulation.

Synthesis and fungicidal activity of 3,5-dichloropyrazin-2(1H)-one derivatives

We synthesized a family of 3,5-dichloropyrazin-2(1H)-one derivatives and assessed their in vitro fungicidal activity against Candida albicans. Compounds 11 and 20 were most active against C. albicans and induced accumulation of reactive oxygen species in this pathogen. Using a genome-wide approach in the yeast Saccharomyces cerevisiae, we demonstrated that genes involved in vacuolar functionality and DNA-related functions play an important role in cellular mechanisms underlying the fungicidal activity of these compounds.

Vacuolar SNAREs Function in the Formation of the Leaf Vascular Network by Regulating Auxin Distribution

In normal leaf development, a two-dimensional pattern of leaf veins is known to form by differentiation of vascular cells from ground meristem cells in a manner that is regulated by the polar flow of auxin. However, the mechanisms regulating the distribution of auxin in the leaf primordium are largely unknown. Here we show that vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), VAM3 and VTI11, are required for the formation of the leaf vascular network in a dosage-dependent manner. This is the first report to show that the pre-vacuolar compartment (PVC)-vacuole traffic pathway is required for the formation of the leaf vascular network. vam3-4, a VAM3-defective mutant, was found to have an immature vascular network. An analysis of the DR5 reporter in vam3-4 indicated that VAM3 is involved in the proper pattern formation of auxin maxima in the leaf primordium. This suggests that the immature vascular network in vam3-4 was mainly determined at the stage of procambium formation in the leaf primordium. The abnormal distribution of auxin maxima was caused by the non-polarized localization of the auxin efflux carrier PIN1 (PIN-FORMED 1) in leaf primordium cells. VAM3 is the first key protein which is required for the proper localization of PIN1 in leaf cells. Finally, we found that PIN1 proteins were constitutively transported to vacuoles in leaf and roots cells. Our findings demonstrate that the PVC-vacuole pathway is required for the formation of auxin maxima, which regulates the polar localization of PIN1, which, in turn, is required for the formation of the leaf vascular network.

Genome-Wide Identification of Genes Required for Yeast Growth Under Imatinib Stress: Vacuolar H+-ATPase Function Is an Important Target of This Anticancer Drug

Imatinib is a highly selective tyrosine kinase inhibitor of the oncogenic kinase Bcr-Abl, the result of a chromosomal abnormality that is associated with chronic myeloid leukaemia (CML). Despite the success of this target-directed therapy, imatinib resistance is an emerging problem, especially in advanced stages of CML. In this study, we explored the yeast Saccharomyces cerevisiae as a model eukaryotic system to better understand the mode of action of imatinib, as well as potential mechanisms of resistance to this drug. Using a systematic approach, we screened a yeast haploid deletion collection with individual knockouts of most nonessential yeast genes, and identified 51 genes that are required for yeast resistance to imatinib. The genes identified are involved mainly in DNA repair and transcription control, cell cycle control and differentiation, vacuolar pH homeostasis, vesicular transport, and protein trafficking. Remarkably, approximately 80% of the genes identified in our screen have human orthologs. The vacuolar pH homeostasis function is associated to our dataset by 13 genes that encode subunits and assembly factors of the yeast vacuolar proton-translocating ATPase (V-ATPase). Further studies using fluorescence microscopy showed that physiological acidification of the vacuole is severely compromised following imatinib treatment of yeast cells, an effect that was found to be dose dependent. Results suggest that imatinib might act as an effective inhibitor of V-ATPase function in yeast, identifying V-ATPase activity and vacuolar function as novel imatinib targets.

Atp6v0d2 Is an Essential Component of the Osteoclast-Specific Proton Pump That Mediates Extracellular Acidification in Bone Resorption

Bone resorption relies on the extracellular acidification function of vacuolar (V-) ATPase proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of osteoclast-specific V-ATPases remains largely unknown. In this study, we found that Atp6v0d2 (d2), an isoform of the d subunit in the V-ATPase, showed 5-fold higher expression than that of Atp6v0d1 (d1) in mature osteoclasts, indicating a potential function in osteoclastic bone resorption. When d2 was depleted at an early stage of RANKL-induced osteoclast differentiation in vitro, formation of multinucleated cells was severely impaired. However, depletion of d2 at a late differentiation stage did not affect osteoclast fusion but did abolish the activity of extracellular acidification and bone resorption of mature osteoclasts. We also showed the association of the two tagged-proteins d2 and a3 when co-expressed in mammalian cells with a co-immunoprecipitation assay. Moreover, glutathione-S-transferase (GST) pull-down assay showed the direct interaction of d2 with the N terminus of Atp6v0a3 (a3), which is the functionally identified osteoclast-specific component of V-ATPase. Therefore, our results show the dual function of d2 as a regulator of cell fusion in osteoclast differentiation and as an essential component of the osteoclast-specific proton pump that mediates extracellular acidification in bone resorption.

On the origin of nuclear vacuoles in spermatozoa a fine structural and cytochemical study in mice.

Morphological and cytochemical changes in nucleoli of spermatids of the mouse during spermiogenesis were examined. Thus, it could be shown that the nucleolar derivatives of the elongation-phase, clusters of coiled fibers that are bordered with spheroidal bodies, show comparable chemical properties as the precursor structure, the fibrillar component, which is known for its transcriptional activity. With progressing development, these nucleolar derivatives continually decrease in size and density, preserving their chemical properties. They persist far beyond into the maturation-phase and will decrease at the time chromatin condensation appears finished. There only remain the spaces in which they formerly existed, now called nuclear vacuoles. A possible function of nuclear vacuoles with respect to chromatin decondensation during the process of fertilization is mentioned.

Phosphoinositide phosphatases and disease

The field of inositol signaling has expanded greatly in recent years. Given the many reviews on phosphoinositide kinases, we have chosen to restrict our discussion to inositol lipid hydrolysis focused on the phosphatases and a brief mention of the lipase isoforms. We also discuss recent discoveries that link mutations in phosphoinositide phosphatases to disease.

S. pombe btn1, the orthologue of the Batten disease geneCLN3, is required for vacuole protein sorting of Cpy1p and Golgi exit of Vps10p

Batten disease is characterised by lysosomal dysfunction. The most common type of the disease is caused by mutations in the membrane protein CLN3, whose function is unknown. We show that the fission yeast orthologue Btn1p, previously implicated in vacuole function, is required for correct sorting of the vacuole hydrolase carboxypeptidase Y (Cpy1p). This is, in part, due to a defect in trafficking of Vps10p, the sorting receptor for Cpy1p, from the Golgi to the trans-Golgi network in btn1Delta cells. Our data also implicate btn1 in other Vps10-independent Cpy1-sorting pathways. Furthermore, btn1 affects the number, intracellular location and structure of Golgi compartments. We show that the prevacuole location of Btn1p is at the Golgi, because Btn1p colocalises predominantly with the Golgi marker Gms1p in compartments that are sensitive to Brefeldin A. Btn1p function might be linked to that of Vps34p, a phosphatidylinositol 3-kinase, because Btn1p acts as a multicopy suppressor of the severe Cpy1p vacuole protein-sorting defect of vps34Delta cells. Together, these results indicate an important role for Btn1p in the Golgi complex, which affects Golgi homeostasis and vacuole protein sorting. We propose a similar role for CLN3 in mammalian cells.

The Forkhead Transcription Factor Foxi1 Is a Master Regulator of Vacuolar H+-ATPase Proton Pump Subunits in the Inner Ear, Kidney and Epididymis

The vacuolar H+-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position −561/−547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H+-ATPase complex at these locations.

In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach

In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast-enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO(2)) affinity chromatography and were subsequently analysed using LC-ESI-MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na(+)/H(+) antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.

Rumen ciliated protozoa decrease generation time and adjust 18S ribosomal DNA copies to adapt to decreased transfer interval, starvation, and monensin

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum, a mix of Entodinium species, Epidinium caudatum, or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25μM) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5μM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture×treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.

The RIM101 pathway has a role inSaccharomyces cerevisiaeadaptive response and resistance to propionic acid and other weak acids

The physiological function of the Saccharomyces cerevisiae RIM101 signaling pathway is extended in this study beyond alkaline pH-induced responses. The transcription factor Rim101p is demonstrated to be required for maximal tolerance to weak acid-induced stress, at pH 4.0, but does not exert protection against low pH itself (range 4.5-2.5), when a strong acid is used as the acidulant. The Rim101p-dependent alterations of the yeast transcriptome following exposure to propionic acid stress (at pH 4.0) include genes of the previously described Rim101p regulon but also new target genes, in particular KNH1, involved in cell wall beta-1,6-glucan synthesis and the uncharacterized ORF YIL029c, both required for maximal propionic acid resistance. Clustering of the genes that provide resistance to propionic acid reveals the enrichment of those involved in protein catabolism through the multivesicular body pathway and in the homeostasis of internal pH and vacuolar function. The analysis of the network of interactions established among all the identified propionic acid resistance determinants shows an enrichment of interactions around the RIM101 gene and highlights the role of proteins involved in Rim101p proteolytic processing. RIM101 expression is shown to be required to counteract propionic acid-induced cytosolic acidification and for proper vacuolar acidification and cell wall structure, these having positive implications for a robust adaptive response and resistance to stress promoted by this food preservative.

The Yeast Vacuolar Membrane Proteome

Transport of solutes between the cytosol and the vacuolar lumen is of crucial importance for various functions of vacuoles, including ion homeostasis; detoxification; storage of different molecules such as amino acids, phosphate, and calcium ions; and proteolysis. To identify proteins that catalyze solute transport across the vacuolar membrane, the membrane proteome of purified Saccharomyces cerevisiae vacuoles was analyzed. Subtractive proteomics was used to distinguish contaminants from true vacuolar proteins by comparing the relative abundances of proteins in pure and crude preparations. A robust statistical analysis combining enrichment ranking with the double boundary iterative group analysis revealed that 148 proteins were significantly enriched in the pure vacuolar preparations. Among these proteins were well characterized vacuolar proteins, such as the subunits of the vacuolar H(+)-ATPase, but also proteins that had not previously been assigned to a cellular location, many of which are likely novel vacuolar membrane transporters, e.g. for nucleosides and oligopeptides. Although the majority of contaminating proteins from other organelles were depleted from the pure vacuolar membranes, some proteins annotated to reside in other cellular locations were enriched along with the vacuolar proteins. In many cases the enrichment of these proteins is biologically relevant, and we discuss that a large group is involved in membrane fusion and protein trafficking to vacuoles and may have multiple localizations. Other proteins are degraded in vacuoles, and in some cases database annotations are likely to be incomplete or incorrect. Our work provides a wealth of information on vacuolar biology and a solid basis for further characterization of vacuolar functions.

Formation of the Food Vacuole in Plasmodium falciparum: A Potential Role for the 19 kDa Fragment of Merozoite Surface Protein 1 (MSP119)

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.