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Biochimica Et Biophysica Acta.... Nov 2020Morphologically, the lipophagy in yeast cell mimics microautophagy, which includes a direct amendment of the vacuolar membrane that engulfs lipid droplets (LDs). The...
Morphologically, the lipophagy in yeast cell mimics microautophagy, which includes a direct amendment of the vacuolar membrane that engulfs lipid droplets (LDs). The molecular mechanism of the membrane modifications that elicits microautophagy still remains elusive. In this study, an analysis of membrane lipid distribution at a nanoscale level showed that PtdIns(4)P is localized in the cytoplasmic leaflet of microautophagic vesicles, which are derived when the vacuole's membrane domains engulfed LDs both in the stationary phase and in acute nitrogen starvation. Furthermore, the PtdIns(4)P-positive raft-like domains engulf LDs through a microautophagic mechanism. When single temperature-conditional mutants of STT4 or PIK1 PtdIns 4-kinases were used, in the vacuole of STT4 and PIK1 mutant cells, microautophagic vesicles drastically decreased at restrictive temperatures, and the labeling density of PtdIns(4)P on the microautophagic vesicles and the sizes of the mutants' microautophagic vesicles also decreased. These results suggest that both Stt4p and Pik1p have important roles in the microautophagy of the vacuole in the stationary phase and under nitrogen starvation conditions.
Topics: 1-Phosphatidylinositol 4-Kinase; Autophagy; Mutation; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 32726584
DOI: 10.1016/j.bbamem.2020.183416 -
Infection and Immunity Mar 2022To colonize mammalian phagocytic cells, the parasite remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The...
To colonize mammalian phagocytic cells, the parasite remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell soluble -ethylmaleimide-sensitive-factor attachment protein receptor proteins to the expansion and functionality of communal vacuoles as well as the replication of the parasite. The differential patterns of recruitment of soluble -ethylmaleimide-sensitive-factor attachment protein receptor to communal vacuoles harboring Leishmania amazonensis and to individual vacuoles housing L. major led us to further investigate the roles of VAMP3 and VAMP8 in the interaction of with its host cell. We show that whereas VAMP8 contributes to the optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither protein has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.
Topics: Animals; Housing; Leishmania; Leishmania mexicana; Macrophages; Mammals; Vacuoles; Vesicle-Associated Membrane Protein 3
PubMed: 35130453
DOI: 10.1128/IAI.00183-21 -
Scientific Reports Jan 2017Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors...
Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.
Topics: Actin Cytoskeleton; Amino Acid Chloromethyl Ketones; Apoptosis; Cathepsin B; Cell Membrane; Cell Nucleus; Cell Nucleus Shape; Cysteine Endopeptidases; Cytochalasin B; Membrane Fusion; Oryza; Permeability; Phalloidine; Protoplasts; Seeds; Vacuoles
PubMed: 28117452
DOI: 10.1038/srep41245 -
MBio Feb 2017is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent phagosome fuses with...
is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent phagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reduced growth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenic cultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading to death. The intracellular Gram-negative bacterium is a significant cause of culture-negative infectious endocarditis, which can be fatal if untreated. The existing treatment strategy requires prolonged antibiotic treatment, with a 10-year mortality rate of 19% in treated patients. Therefore, new clinical therapies are needed and can be achieved by better understanding pathogenesis. Upon infection of host cells, grows within a specialized replication niche, the parasitophorous vacuole (PV). Recent data have linked cholesterol to intracellular growth and PV formation, leading us to further decipher the role of cholesterol during -host interaction. We observed that increasing PV cholesterol concentration leads to increased acidification of the PV and bacterial death. Further, treatment with FDA-approved drugs that alter host cholesterol homeostasis also killed through PV acidification. Our findings suggest that targeting host cholesterol metabolism might prove clinically efficacious in controlling infection.
Topics: Bacteriolysis; Cholesterol; Coxiella burnetii; HeLa Cells; Humans; Hydrogen-Ion Concentration; Vacuoles
PubMed: 28246364
DOI: 10.1128/mBio.02313-16 -
Autophagy Mar 2021, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this...
, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a transposon mutant library, we observed that transposon insertions in led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as -containing vacuoles (CCVs). We thus renamed this effector CvpF for vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, ::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal vacuole biogenesis. ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: -containing vacuole; Cvp: vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.
Topics: Animals; Autophagy; Bacterial Proteins; Bacterial Secretion Systems; Coxiella; Coxiella burnetii; Host-Pathogen Interactions; Humans; Mice; Vacuoles
PubMed: 32116095
DOI: 10.1080/15548627.2020.1728098 -
BioEssays : News and Reviews in... Jun 2018Microautophagy is originally defined as lysosomal (vacuolar) membrane dynamics to directly enwrap and transport cytosolic components into the lumen of the lytic... (Review)
Review
Microautophagy is originally defined as lysosomal (vacuolar) membrane dynamics to directly enwrap and transport cytosolic components into the lumen of the lytic organelle. Molecular details of microautophagy had remained unknown until genetic studies in yeast identified a set of proteins required for the process. Subsequent studies with other experimental model organisms resulted in a series of discoveries that accompanied an expansion of the definition of microautophagy to also encompass endosomal membrane dynamics. These findings, however, still impose puzzling, non-integrated images as to the molecular mechanism of microautophagy. By reviewing recent studies on microautophagy in various experimental systems, we propose the classification of microautophagy into three types, as the basis for developing a comprehensive view of the process.
Topics: Animals; Autophagy; Cytosol; Intracellular Membranes; Lysosomes; Vacuoles
PubMed: 29708272
DOI: 10.1002/bies.201800008 -
Proceedings of the National Academy of... Sep 2019Stomatal movement is essential for plant growth. This process is precisely regulated by various cellular activities in guard cells. F-actin dynamics and vacuole...
Stomatal movement is essential for plant growth. This process is precisely regulated by various cellular activities in guard cells. F-actin dynamics and vacuole morphology are both involved in stomatal movement. The sorting of cargoes by clathrin adaptor protein (AP) complexes from the Golgi to the vacuole is critical for establishing a normal vacuole morphology. In this study, we demonstrate that the medium subunit of the AP3 complex (AP3M) binds to and severs actin filaments in vitro and that it participates in the sorting of cargoes (such as the sucrose exporter SUC4) to the tonoplast, and thereby regulates stomatal closure in Defects in AP3 or SUC4 led to more rapid water loss and delayed stomatal closure, as well as hypersensitivity to drought stress. In mutants, the F-actin status was altered compared to the wild type, and the sorted cargoes failed to localize to the tonoplast. AP3M contains a previously unidentified F-actin binding domain that is conserved in AP3M homologs in both plants and animals. Mutations in the F-actin binding domain of AP3M abolished its F-actin binding activity in vitro, leading to an aberrant vacuole morphology and reduced levels of SUC4 on the tonoplast in guard cells. Our findings indicate that the F-actin binding activity of AP3M is required for the precise localization of AP3-dependent cargoes to the tonoplast and for the regulation of vacuole morphology in guard cells during stomatal closure.
Topics: Actin Cytoskeleton; Adaptor Protein Complex 3; Arabidopsis; Arabidopsis Proteins; Plant Stomata; Vacuoles
PubMed: 31431522
DOI: 10.1073/pnas.1901431116 -
Plant Signaling & Behavior Jul 2013During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a...
During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.
Topics: Beta vulgaris; Cell Membrane; Endocytosis; Sucrose; Vacuoles
PubMed: 23656870
DOI: 10.4161/psb.24793 -
Biophysical Journal Mar 2023Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is...
Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.
Topics: Saccharomyces cerevisiae; Lipidomics; Vacuoles; Saccharomyces cerevisiae Proteins; Lipids
PubMed: 36635960
DOI: 10.1016/j.bpj.2023.01.009 -
Journal of Microbiological Methods Jul 2009Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus....
Current studies in our laboratory demonstrate a functional link between vesicles, vacuoles and aflatoxin biosynthesis in the filamentous fungus, Aspergillus parasiticus. Under aflatoxin inducing conditions in liquid yeast-extract sucrose medium, A. parasiticus undergoes a shift from vacuole biogenesis to accumulation of an enhanced number of vesicles which exhibit significant heterogeneity in size and density. As a first step in conducting a detailed analysis of the role of these organelles in aflatoxin synthesis, we developed a novel method to purify the vesicle and vacuole fraction using protoplasts prepared from cells harvested during aflatoxin synthesis. The method includes the following steps: 1] preparation of protoplasts from mycelia grown for 36 h under aflatoxin inducing conditions; 2] release of vesicles and vacuoles from purified protoplasts in the presence of Triton X-100; and 3] fractionation of the vesicles and vacuoles using a "one-step high density cushion". The vesicle-vacuole fraction showed a 35 fold enrichment in alpha-mannosidase activity (vacuole marker) and non-detectable succinate dehydrogenase and lactate dehydrogenase activities (mitochondrial and cytoplasmic markers, respectively). Confocal laser scanning microscopy with the vacuole dyes MDY-64 and CMAC demonstrated that the fraction contained pure vesicles and vacuoles and was devoid of membranous debris. Transmission electron microscopy (TEM) confirmed that no mitochondria or unbroken protoplasts contaminated the purified fraction. The purified organelles exhibited significant size heterogeneity with a range of sizes similar to that observed in whole cells and protoplasts.
Topics: Aflatoxins; Aspergillus; Cell Fractionation; Cytoplasmic Vesicles; Vacuoles
PubMed: 19358865
DOI: 10.1016/j.mimet.2009.03.014