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Vacuolar Membrane (vacuolar + membrane)
Selected AbstractsIntracellular pH homeostasis in the filamentous fungus Aspergillus nigerFEBS JOURNAL, Issue 14 2002Stephan J. A. Hesse Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+ -alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ,pH over the cytoplasmic membrane of 6.6,6.7 was reached (pHex 0.7,0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ,pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ,pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m -chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ,pH. Nonlethal levels of the metabolic inhibitor azide (N3,, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively). [source] Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genesFEMS YEAST RESEARCH, Issue 1 2001Anna Rita Bellu Abstract In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes. [source] Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiaeGENES TO CELLS, Issue 6 2008Keisuke Obara Vps34, the sole PtdIns 3-kinase in yeast, is essential for autophagy. Here, we show that the lipid-kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3)P. The protein-kinase activity of Vps15, a regulatory subunit of the PtdIns 3-kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3)P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3)P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3)P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3)P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3)P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3)P in autophagy. [source] THE ROLE OF CALCIUM IN FLOW-STIMULATED BIOLUMINESCENCE OF THE RED TIDE DINOFLAGELLATE LINGULODINIUM POLYEDRUMJOURNAL OF PHYCOLOGY, Issue 2000P. Von Dassow Many marine planktonic dinoflagellates emit flashes of light in response to either laminar or turbulent flows as well as direct mechanical stimulation. The production of a flash of light is known to be mediated by a proton-mediated action potential across the vacuolar membrane; the mechanotransduction process initiating this action potential is unknown. Here we report on an investigation into the role of Ca+2 in the mechanotransduction process regulating bioluminescence in the red tide dinoflagellate Lingulodinium polyedrum. Calcium ionophores and low concentrations of the membrane-disrupting agent digitonin stimulated bioluminescence only when calcium was present in the media or added with the agent, indicating that the flash-triggering vacuolar action potential is specifically stimulated by a calcium influx. A variety of known calcium channel blockers or antagonists inhibited mechanically stimulated bioluminescence but did not affect cellular bioluminescent capacity. In many cases the inhibitory affect occurred after only a brief exposure. In addition, gadolinium (Gd+3), a blocker of many stretch-activated ion channels, caused potent inhibition of mechanically stimulated bioluminescence. The order of potency of the transition metals tested was La+3 > Gd+3 > Co+2 > Mn+2 > Ni+2, similar to their potency as blockers of known calcium channels. Experiments with a quantified shear flow demonstrated that flow-stimulated bioluminescence depended on the level of extracellular calcium. Future work will elucidate the signaling pathway involving calcium-mediated flow-stimulated mechanotransduction. Our goal is to use bioluminescence as a proxy for the initial cellular mechanotransduction events triggered by fluid flow. [source] Pathogen trafficking pathways and host phosphoinositide metabolismMOLECULAR MICROBIOLOGY, Issue 6 2009Stefan S. Weber Summary Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila, Brucella abortus, Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells. [source] NRAMP genes function in Arabidopsis thaliana resistance to Erwinia chrysanthemi infectionTHE PLANT JOURNAL, Issue 2 2009Diego Segond Summary AtNRAMP3 and AtNRAMP4 are two Arabidopsis metal transporters sharing about 50% sequence identity with mouse NRAMP1. The NRAMP1/Slc11A1 metal ion transporter plays a crucial role in the innate immunity of animal macrophages targeted by intracellular bacterial pathogens. AtNRAMP3 and AtNRAMP4 localize to the vacuolar membrane. We found that AtNRAMP3 is upregulated in leaves challenged with the bacterial pathogens Pseudomonas syringae and Erwinia chrysanthemi, whereas AtNRAMP4 expression is not modified. Using single and double nramp3 and nramp4 mutants, as well as lines ectopically expressing either of these genes, we show that AtNRAMP3 and, to a lesser extent, AtNRAMP4 are involved in Arabidopsis thaliana resistance against the bacterial pathogen E. chrysanthemi. The susceptibility of the double nramp3 nramp4 mutant is associated with the reduced accumulation of reactive oxygen species and ferritin (AtFER1), an iron storage protein known to participate in A. thaliana defense. Interestingly, roots from infected plants accumulated transcripts of AtNRAMP3 as well as the iron-deficiency markers IRT1 and FRO2. This finding suggests the existence of a shoot-to-root signal reminiscent of an iron-deficiency signal activated by pathogen infection. Our data indicate that the functions of NRAMP proteins in innate immunity have been conserved between animals and plants. [source] New insights into protein export in malaria parasitesCELLULAR MICROBIOLOGY, Issue 5 2010Silvia Haase Summary In order to survive and promote its virulence the malaria parasite must export hundreds of its proteins beyond an encasing vacuole and membrane into the host red blood cell. In the last few years, several major advances have been made that have significantly contributed to our understanding of this export process. These include: (i) the identification of sequences that direct protein export (a signal sequence and a motif termed PEXEL), which have allowed predictions of the exportomes of Plasmodium species that are the cause of malaria, (ii) the recognition that the fate of proteins destined for export is already decided within the parasite's endoplasmic reticulum and involves the PEXEL motif being recognized and cleaved by the aspartic protease plasmepsin V and (iii) the discovery of the Plasmodium translocon of exported proteins (PTEX) that is responsible for the passage of proteins across the vacuolar membrane. We review protein export in Plasmodium and these latest developments in the field that have now provided a new platform from which trafficking of malaria proteins can be dissected. [source] Intracellular parasitism with Toxoplasma gondii stimulates mammalian-target-of-rapamycin-dependent host cell growth despite impaired signalling to S6K1 and 4E-BP1CELLULAR MICROBIOLOGY, Issue 6 2009Yubao Wang Summary The Ser/Thr kinase mammalian-target-of-rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin-sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth-associated mTOR substrates 4E-BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E-BP1 in response to insulin, although the S6K1 response was blunted. Parasite-induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR-inducing pathways mediated by phosphatidylinositol 3-kinase,Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E-BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR-dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite-induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma -infected cell provides a unique example of non-canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E-BP1. [source] Functions and effectors of the Salmonella pathogenicity island 2 type III secretion systemCELLULAR MICROBIOLOGY, Issue 8 2003Scott R. Waterman Summary Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI-1 and SPI-2 to transfer effector proteins into host cells. A major function of the SPI-1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI-2 is to facilitate the replication of intracellular bacteria within membrane-bound Salmonella -containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI-2-dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase-dependent killing, the induction of a delayed apoptosis-like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI-2-dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments. [source] Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivoCELLULAR MICROBIOLOGY, Issue 9 2001Suzana P. Salcedo We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA, mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo. [source] Candida albicans ABG1 gene is involved in endocytosisFEMS YEAST RESEARCH, Issue 2 2009Verónica Veses Abstract The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis. [source] | ||||||||||