-
The Journal of Biological Chemistry Dec 2021Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and... (Review)
Review
Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.
Topics: Animals; Bacterial Proteins; Homeostasis; Host-Pathogen Interactions; Humans; Inflammation; Legionella pneumophila; Legionnaires' Disease; Type IV Secretion Systems; Ubiquitination; Vacuoles
PubMed: 34695417
DOI: 10.1016/j.jbc.2021.101340 -
Plant & Cell Physiology Jul 2018Plant vacuoles display many versatile functions. Vacuoles in vegetative tissues are generally involved in protein degradation, and are called lytic vacuoles. However,... (Review)
Review
Plant vacuoles display many versatile functions. Vacuoles in vegetative tissues are generally involved in protein degradation, and are called lytic vacuoles. However, vegetative vacuoles in specialized cells can accumulate large concentrations of proteins, such as those in idioblast myrosin cells along veins in the order Brassicales, which store large amounts of myrosinases (thioglucoside glucohydrolase and thioglucoside glucohydrolase). Myrosinases cleave the bond between sulfur and glucose in sulfur-rich compounds (glucosinolates) to produce toxic compounds (isothiocyanates) when plants are damaged by pests. This defense strategy is called the myrosinase-glucosinolate system. Recent studies identified atypical myrosinases, PENETRATION 2 (PEN2) and PYK10, along with key components for development of myrosin cells. In this review, we discuss three topics in the myrosinase-glucosinolate system. First, we summarize the complexity and importance of the myrosinase-glucosinolate system, including classical myrosinases, atypical myrosinases and the system that counteracts the myrosinase-glucosinolate system. Secondly, we describe molecular machineries underlying myrosin cell development, including specific reporters, cell lineage, cell differentiation and cell fate determination. The master regulators for myrosin cell differentiation, FAMA and SCREAM, are key transcription factors involved in guard cell differentiation. This indicates that myrosin cells and guard cells share similar transcriptional networks. Finally, we hypothesize that the myrosinase-glucosinolate system may have originated in stomata of ancestral Brassicales plants and, after that, plants co-opted this defense strategy into idioblasts near veins at inner tissue layers.
Topics: Animals; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Biological Evolution; Brassicaceae; Cell Differentiation; Glucosinolates; Glycoside Hydrolases; Herbivory; Plant Cells; Transcription Factors; Vacuoles
PubMed: 29897512
DOI: 10.1093/pcp/pcy082 -
The Journal of Cell Biology Dec 2020The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology;...
The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology; however, cellular mechanisms used to alleviate NPC assembly stress are not well defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched NE-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to remodel the NE and promote vacuole-dependent degradation of specific nucleoporins in nup116ΔGLFG cells. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. These results therefore define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.
Topics: Gene Deletion; Intermediate Filament Proteins; Nuclear Pore; Nuclear Pore Complex Proteins; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles
PubMed: 33053148
DOI: 10.1083/jcb.202001165 -
Australian Veterinary Journal Dec 2016Since its initial detection in Norway in 1998, atypical scrapie ('atypical/Nor98 scrapie') has been reported in sheep in the majority of European countries (including in...
BACKGROUND
Since its initial detection in Norway in 1998, atypical scrapie ('atypical/Nor98 scrapie') has been reported in sheep in the majority of European countries (including in regions free of classical scrapie) and in the Falkland Islands, the USA, Canada, New Zealand and Australia.
CASE SERIES
The diagnosis in Australia of atypical scrapie in four Merino and one Merino-cross sheep showing clinical signs of neurological disease was based on the detection of grey matter neuropil vacuolation (spongiform change) in the brain (particularly in the molecular layer of the cerebellar cortex) and associated abnormal prion protein (PrP ) deposition in both grey and white matter. Changes were minimal in the caudal brainstem, the predilection site for lesions of classical scrapie.
CONCLUSION
The distinctive lesion profile of atypical scrapie in these five sheep highlights the diagnostic importance of routine histological evaluation of the cerebellum for evidence of neuropil vacuolation and associated PrP deposition in adult sheep with suspected neurological disease.
Topics: Animals; Australia; Brain; Female; Neuropil; PrPSc Proteins; Scrapie; Sheep; Vacuoles
PubMed: 27807855
DOI: 10.1111/avj.12529 -
Current Genetics Apr 2018All eukaryotes require the transition metal, iron, a redox active element that is an essential cofactor in many metabolic pathways, as well as an oxygen carrier. Iron... (Review)
Review
All eukaryotes require the transition metal, iron, a redox active element that is an essential cofactor in many metabolic pathways, as well as an oxygen carrier. Iron can also react to generate oxygen radicals such as hydroxyl radicals and superoxide anions, which are highly toxic to cells. Therefore, organisms have developed intricate mechanisms to acquire iron as well as to protect themselves from the toxic effects of excess iron. In fungi and plants, iron is stored in the vacuole as a protective mechanism against iron toxicity. Iron storage in the vacuole is mediated predominantly by the vacuolar metal importer Ccc1 in yeast and the homologous transporter VIT1 in plants. Transcription of yeast CCC1 expression is tightly controlled primarily by the transcription factor Yap5, which sits on the CCC1 promoter and activates transcription through the binding of Fe-S clusters. A second mechanism that regulates CCC1 transcription is through the Snf1 signaling pathway involved in low-glucose sensing. Snf1 activates stress transcription factors Msn2 and Msn4 to mediate CCC1 transcription. Transcriptional regulation by Yap5 and Snf1 are completely independent and provide for a graded response in Ccc1 expression. The identification of multiple independent transcriptional pathways that regulate the levels of Ccc1 under high iron conditions accentuates the importance of protecting cells from the toxic effects of high iron.
Topics: Basic-Leucine Zipper Transcription Factors; Cation Transport Proteins; DNA-Binding Proteins; Gene Expression Regulation, Fungal; Iron; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Transcription Factors; Vacuoles
PubMed: 29043483
DOI: 10.1007/s00294-017-0767-7 -
Methods in Molecular Biology (Clifton,... 2019Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by...
Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.
Topics: Animals; Cell Line; Cell Survival; Flow Cytometry; Gene Expression; Genes, Reporter; Host-Pathogen Interactions; Humans; Legionella; Legionella pneumophila; Legionellosis; Macrophages; Mice; RNA Interference; Type IV Secretion Systems; Vacuoles
PubMed: 30694495
DOI: 10.1007/978-1-4939-9048-1_14 -
Journal of Cell Science Nov 2023The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum...
The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input.
Topics: Vacuoles; Tetrahymena thermophila; Endosomes; Proteins; Mitosis
PubMed: 37902010
DOI: 10.1242/jcs.261511 -
Autophagy Apr 2024During starvation in the yeast vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There...
During starvation in the yeast vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep-learning computational approach to visualize and quantify these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 ( cells) or Npc2 ( cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that and cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells. BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza--Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; , yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, -(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; , yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD, optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.
Topics: Saccharomyces cerevisiae; Vacuoles; Deep Learning; Saccharomyces cerevisiae Proteins; Microscopy, Fluorescence; Autophagy; Lipid Droplets; Tomography, X-Ray
PubMed: 37908116
DOI: 10.1080/15548627.2023.2270378 -
FEBS Letters Sep 2022Autophagy fulfills a crucial role in plant cellular homeostasis by recycling diverse cellular components ranging from protein complexes to whole organelles. Autophagy... (Review)
Review
Autophagy fulfills a crucial role in plant cellular homeostasis by recycling diverse cellular components ranging from protein complexes to whole organelles. Autophagy cargos are shuttled to the vacuole for degradation, thereby completing the recycling process. Canonical autophagy requires the lipidation and insertion of ATG8 proteins into double-membrane structures, termed autophagosomes, which engulf the cargo to be degraded. As such, the autophagy pathway actively contributes to intracellular membrane trafficking. Yet, the autophagic process is not fully considered a bona fide component of the canonical membrane trafficking pathway. However, recent findings have started to pinpoint the interconnection between classical membrane trafficking pathways and autophagy. This review details the latest advances in our comprehension of the interplay between these two pathways. Understanding the overlap between autophagy and canonical membrane trafficking pathways is important to illuminate the inner workings of both pathways in plant cells.
Topics: Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Plants; Vacuoles
PubMed: 35593306
DOI: 10.1002/1873-3468.14404 -
The Journal of Infectious Diseases Mar 2022The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small...
The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.
Topics: Bacterial Proteins; Cytoplasm; Cytosol; Hemolysin Proteins; Humans; Listeria monocytogenes; Listeriosis; Vacuoles
PubMed: 32582947
DOI: 10.1093/infdis/jiaa367