-
PLoS Pathogens Jul 2023A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the...
A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.
Topics: Animals; Plasmodium falciparum; Vacuoles; Protein Transport; Protozoan Proteins; Erythrocytes; Parasites; Peptide Hydrolases
PubMed: 37523385
DOI: 10.1371/journal.ppat.1011006 -
Life Science Alliance Sep 2022Membrane contact sites are functional nodes at which organelles reorganize metabolic pathways and adapt to changing cues. In , the nuclear envelope subdomain surrounding...
Membrane contact sites are functional nodes at which organelles reorganize metabolic pathways and adapt to changing cues. In , the nuclear envelope subdomain surrounding the nucleolus, very plastic and prone to expansion, can establish contacts with the vacuole and be remodeled in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a fully new remodeling event at this nuclear subdomain: the nuclear envelope partitions into its regular contact with the vacuole and a dramatic internalization within the nucleus. This leads to the nuclear engulfment of a globular, cytoplasmic portion. In spite of how we discovered it, the phenomenon is likely DNA damage-independent. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona fide drivers of this event. Mechanistically, we suggest that the engulfment of the cytoplasm triggers a suction phenomenon that enhances the docking of proton pump-containing vesicles with the vacuolar membrane, which we show matches a boost in autophagy. Thus, our findings unveil an unprecedented remodeling of the nucleolus-surrounding membranes with impact on metabolic adaptation.
Topics: Autophagy; Cytoplasm; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 35568434
DOI: 10.26508/lsa.202101160 -
Life Science Alliance Oct 2022The ribosomal DNA (rDNA) array of has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly...
The ribosomal DNA (rDNA) array of has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct presentations that arise as spatial adaptations to changes in the nuclear morphology triggered during mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of nuclear components yet colocalizes with the vacuole. We show that the rDNA-associated nuclear envelope (NE) often reshapes into a ladle to accommodate the vacuole in the SUL, with the nucleus becoming bilobed and doughnut-shaped. Finally, we demonstrate that the formation of the rDNA loop and the SUL require TORC1, membrane synthesis and functional vacuoles, yet is independent of nucleus-vacuole junctions and rDNA-NE tethering.
Topics: Anaphase; DNA, Ribosomal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 35961781
DOI: 10.26508/lsa.202101161 -
International Journal of Molecular... Jan 2023Vacuolar processing enzymes (VPEs) are plant cysteine proteases that are subjected to autoactivation in an acidic pH. It is presumed that VPEs, by activating other... (Review)
Review
Vacuolar processing enzymes (VPEs) are plant cysteine proteases that are subjected to autoactivation in an acidic pH. It is presumed that VPEs, by activating other vacuolar hydrolases, are in control of tonoplast rupture during programmed cell death (PCD). Involvement of VPEs has been indicated in various types of plant PCD related to development, senescence, and environmental stress responses. Another pathway induced during such processes is autophagy, which leads to the degradation of cellular components and metabolite salvage, and it is presumed that VPEs may be involved in the degradation of autophagic bodies during plant autophagy. As both PCD and autophagy occur under similar conditions, research on the relationship between them is needed, and VPEs, as key vacuolar proteases, seem to be an important factor to consider. They may even constitute a potential point of crosstalk between cell death and autophagy in plant cells. This review describes new insights into the role of VPEs in plant PCD, with an emphasis on evidence and hypotheses on the interconnections between autophagy and cell death, and indicates several new research opportunities.
Topics: Apoptosis; Plants; Vacuoles; Autophagy; Cell Death
PubMed: 36674706
DOI: 10.3390/ijms24021198 -
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 -
Biochemical and Biophysical Research... Feb 2022Candida albicans is an important opportunistic fungus in the clinic. In recent years, with the widespread use of antibiotics, drug-resistant strains have been isolated...
Candida albicans is an important opportunistic fungus in the clinic. In recent years, with the widespread use of antibiotics, drug-resistant strains have been isolated in the clinic, so finding new drug targets has become an urgent problem to be solved. The vacuole and mitochondria patch (vCLAMP) and the ER-mitochondria encounter structure (ERMES) are new types of inner membrane junction systems in Saccharomyces cerevisiae. However, the functions in maintaining cell survival of the two structures have not yet been elucidated in C. albicans. In this study, VAM6 and MDM34 knockout mutants (vam6Δ/Δmet-MDM34) were constructed using an induction system regulated by the MET3 promoter. PI-positive assays showed that deletion of vCLAMP and ERMES led to abnormal growth of C. albicans. Furthermore, the vam6Δ/Δmet-MDM34 mutant exhibited obvious mitochondrial fragmentation, mtDNA damage, reduced ATP levels, and abnormal mitochondrial membrane potential, indicating its important role in maintaining the structures and functions of mitochondria. Moreover, deletion of vCLAMP and ERMES inhibited filamentous growth. Overall This study shows that vCLAMP and ERMES play important roles in maintaining the survival of C. albicans cells.
Topics: Candida albicans; Cell Survival; Endoplasmic Reticulum; Fungal Proteins; Hyphae; Intracellular Membranes; Mitochondria; Vacuoles
PubMed: 34999259
DOI: 10.1016/j.bbrc.2021.12.116 -
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 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2023As a generally-recognized-as-safe microorganism, is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology.... (Review)
Review
As a generally-recognized-as-safe microorganism, is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in . by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, . has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in . are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Golgi Apparatus; Metabolic Engineering; Vacuoles
PubMed: 37401597
DOI: 10.13345/j.cjb.221030