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Cell Reports Jan 2021Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish...
Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.
Topics: Cell Nucleus; Glucose; Phosphate Transport Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Vacuoles
PubMed: 33472077
DOI: 10.1016/j.celrep.2020.108637 -
Methods in Molecular Biology (Clifton,... 2023Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S....
Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells; internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid, and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.
Topics: Animals; Humans; Salmonella enterica; Vacuoles; Epithelial Cells; Salmonella typhimurium; Gentamicins; Bacterial Proteins; Mammals
PubMed: 37365470
DOI: 10.1007/978-1-0716-3338-0_14 -
Journal of Cataract and Refractive... May 2020To develop an advanced test methodology for quantification of scattered light from intraocular lenses (IOLs) and to evaluate the correlation between IOL vacuole...
PURPOSE
To develop an advanced test methodology for quantification of scattered light from intraocular lenses (IOLs) and to evaluate the correlation between IOL vacuole characteristics and measured scattered light.
SETTING
U.S. Food and Drug Administration, Optical Therapeutics and Medical Nanophotonics Laboratory, Silver Spring, Maryland, USA.
DESIGN
Experimental and analytical study.
METHODS
Twenty-four IOLs containing vacuoles were evaluated using a digital microscopy approach for identifying and characterizing the vacuoles present. A scanning light scattering profiler (SLSP) was used to evaluate and quantify the amount of scattered light from each IOL and from a 25th control IOL without any vacuoles. A variety of IOLs and vacuoles were also modeled in a Zemax simulation of the SLSP, and the simulated scattered light was modeled.
RESULTS
The scattered light as measured with SLSP was well correlated with vacuole characteristics, specifically density and size, as measured under the digital microscope for the 24 vacuole-containing IOLs. Additional correlations were found between vacuole sizes, orientations, and the angle at which light was scattered most severely. These correlations were also present in the Zemax model.
CONCLUSIONS
Vacuole optical characteristics can be well correlated with measured scatter, demonstrating an ability to predict scattered light based solely on microscope evaluation. Furthermore, the quantitative amount of scatter predicted with Zemax simulations trended closely with the experimentally measured trends.
Topics: Humans; Lenses, Intraocular; Maryland; Scattering, Radiation; Vacuoles; Vision, Ocular
PubMed: 32358273
DOI: 10.1097/j.jcrs.0000000000000167 -
Infection, Genetics and Evolution :... Jun 2021Listeria monocytogenes is a pathogen causing serious or mortal infections in human risk populations. Its infectivity is in part due to its ability to infect diverse...
Listeria monocytogenes is a pathogen causing serious or mortal infections in human risk populations. Its infectivity is in part due to its ability to infect diverse eukaryotic cells. Since several bacteria can enter into yeast cells, including Candida albicans, the aims of this work were to evaluate if L. monocytogenes was able to harbor, retaining its viability, within C. albicans cells and to evaluate the effect of temperature and an antibiotic as stressing factors in its rate of entry into yeast cells. Both microorganisms were co-incubated in BHI broth during 48 h and the entry of bacteria into yeast cells was evaluated at different times. Then, yeasts free of extracellular bacteria were obtained seeding samples of the co-culture on YGC agar, which contains chloramphenicol, to obtain extracellular bacteria-free yeasts. These extracellular bacteria free yeasts were used to search for bacterial DNA in total yeast DNA and to evaluate the viability of intra-yeast bacteria. Finally, the effect of temperature and of chloramphenicol as inducers of stress on the rate of bacterial entry into yeast cells were investigated. After co-culturing both microorganisms, wet mount optical microscopy showed the presence of moving bacteria within yeasts and transmission electron microscopy confirmed the presence of intra-yeast bacteria. PCR allowed to amplify L. monocytogenes iap gene in C. albicans total DNA obtained from yeasts free of extracellular bacteria. Moreover, the SYTO 9 green fluorescence observed in bacterial cells within vacuoles of yeasts suggests that intra-yeast bacteria remain viable. Furthermore, the entry of L. monocytogenes into yeasts cells was favored by the presence of stressing factors (chloramphenicol and temperature). Therefore, yeasts may be reservoirs of viable L. monocytogenes and might spread them to the following generations of yeasts.
Topics: Candida albicans; DNA, Bacterial; Disease Reservoirs; Listeria monocytogenes; Vacuoles
PubMed: 33639305
DOI: 10.1016/j.meegid.2021.104779 -
Microbiology (Reading, England) Jan 2019Coxiella burnetii is an obligate intracellular pathogen that causes acute and chronic Q fever. C. burnetii grows within a eukaryotic host cell in a vacuole highly...
Coxiella burnetii is an obligate intracellular pathogen that causes acute and chronic Q fever. C. burnetii grows within a eukaryotic host cell in a vacuole highly similar to a phagolysosome. Found worldwide, this environmentally stable pathogen is maintained in nature via chronic infection of ruminants. Aerosol-mediated infection of humans results in infection and usurpation of alveolar macrophages through mechanisms using a bacterial Type 4B Secretion System and secreted effector proteins. Advances in axenic culture and genetic systems are changing our understanding of the pathogen's physiology and intimate molecular manipulations of host cells during infection.
Topics: Acids; Bacterial Proteins; Bacterial Secretion Systems; Coxiella burnetii; Genome, Bacterial; Humans; Hydrogen-Ion Concentration; Phylogeny; Q Fever; Vacuoles
PubMed: 30422108
DOI: 10.1099/mic.0.000707 -
ACS Chemical Biology Jun 2022Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method...
Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in . Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the endoplasmic reticulum-vacuole-tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.
Topics: Cholesterol; Intermediate Filament Proteins; Lipid Metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sphingolipids; Vacuoles
PubMed: 35667650
DOI: 10.1021/acschembio.2c00120 -
Autophagy Sep 2023Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and...
Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and fusing with lysosomes/vacuoles, after which the materials are degraded for recycling purposes. Autophagy is associated with increased cell survival under different stress conditions. AP-lysosome/vacuole fusion is a critical step in autophagy. Some mutant cells can accumulate phagophores under autophagy-induction conditions. Autophagy is interrupted when accumulated phagophores cannot fuse with lysosomes/vacuoles, resulting in a significant decrease in cell survivability. However, phagophore-lysosome/vacuole fusion has been reported in related mammalian cells and yeast mutant cells. This observation indicates that it is possible to restore a partial autophagy process after interruption. Furthermore, these findings indicate that phagophore closure is not a prerequisite for its fusion with the lysosome/vacuole in the mutant cells. The phagophore-lysosome/vacuole fusion strategy can significantly rescue defective autophagy due to failed phagophore closure. This commentary discusses the fusion of phagophores and lysosomes/vacuoles and implications of such fusion events.: AB: autophagic body; AL: autolysosome; AP: autophagosome; ATG: autophagy related; EM: electron microscopy; ESCRT: endosomal sorting complex required for transport; ET: electron tomography; FIB: focus ion beam; IM: inner membrane; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; OM; outer membrane; STX17: syntaxin 17; TEM: transmission electron microscopy; TM: transmembrane domain; Vps: vacuolar protein sorting; WT: wild-type.
Topics: Animals; Autophagosomes; Vacuoles; Saccharomyces cerevisiae; Autophagy; Lysosomes; Membrane Fusion; Mammals
PubMed: 37083184
DOI: 10.1080/15548627.2023.2205272 -
Current Biology : CB Aug 2023Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water...
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.
Topics: Cytosol; Dictyostelium; Osmolar Concentration; Water-Electrolyte Balance; Vacuoles; Eukaryota; Water
PubMed: 37478864
DOI: 10.1016/j.cub.2023.06.061 -
Critical Reviews in Microbiology Jun 2015The contractile vacuole complex (CVC) of freshwater protists sequesters the excess of water and ions (Ca(2+)) for exocytosis cycles at the pore. Sequestration is based... (Review)
Review
The contractile vacuole complex (CVC) of freshwater protists sequesters the excess of water and ions (Ca(2+)) for exocytosis cycles at the pore. Sequestration is based on a chemiosmotic proton gradient produced by a V-type H(+)-ATPase. So far, many pieces of information available have not been combined to a comprehensive view on CVC biogenesis and function. One main function now appears as follows. Ca(2+)-release channels, type inositol 1,4,5-trisphosphate receptors (InsP3R), may serve for fine-tuning of local cytosolic Ca(2+) concentration and mediate numerous membrane-to-membrane interactions within the tubular spongiome meshwork. Such activity is suggested by the occurrence of organelle-specific soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) and Ras-related in brain (Rab) proteins, which may regulate functional requirements. For tubulation, F-Bin-amphiphysin-Rvs (F-BAR) proteins are available. In addition, there is indirect evidence for the occurrence of H(+)/Ca(2+) exchangers (to sequester Ca(2+)) and mechanosensitive Ca(2+)-channels (for signaling the filling sate). The periodic activity of the CVC may be regulated by the mechanosensitive Ca(2+)-channels. Such channels are known to colocalize with and to be functionally supported by stomatins, which were recently detected in the CVC. A Kif18-related kinesin motor protein might control the length of radial arms. Two additional InsP3-related channels and several SNAREs are associated with the pore. De novo organelle biogenesis occurs under epigenetic control during mitotic activity and may involve the assembly of γ-tubulin, centrin, calmodulin and a never in mitosis A-type (NIMA) kinase - components also engaged in mitotic processes.
Topics: Eukaryotic Cells; Exocytosis; Organelle Biogenesis; Signal Transduction; Vacuoles
PubMed: 23919298
DOI: 10.3109/1040841X.2013.821650 -
Biochemical Society Transactions Apr 2016Eukaryotic cells rely on a set of membrane-enclosed organelles to perform highly efficient reactions in an optimized environment. Trafficking of molecules via vesicular... (Review)
Review
Eukaryotic cells rely on a set of membrane-enclosed organelles to perform highly efficient reactions in an optimized environment. Trafficking of molecules via vesicular carriers and membrane contact sites (MCS) allow the coordination between these compartments, though the precise mechanisms are still enigmatic. Among the cellular organelles, the lysosome/vacuole stands out as a central hub, where multiple pathways merge. Importantly, the delivered material is degraded and the monomers are recycled for further usage, which explains its wide variety of roles in controlling cellular metabolism. We will highlight recent advances in the field by focusing on the yeast vacuole as a model system to understand lysosomal function in general.
Topics: Binding Sites; Cell Nucleus; Organelles; Signal Transduction; Vacuoles
PubMed: 27068965
DOI: 10.1042/BST20150277