RESUMEN
Macroautophagy, hereafter autophagy, plays a crucial role in the degradation of harmful or unwanted cellular components through a double-membrane autophagosome. Upon autophagosome fusion with the vacuole, the degraded materials are subsequently recycled to generate macromolecules, contributing to cellular homeostasis, metabolism, and stress tolerance in plants. A hallmark during autophagy is the formation of isolation membrane structure named as phagophore, which undergoes multiple steps to become as a complete double-membrane autophagosome. Methodologies have been developed in recent years to observe and quantify the autophagic process, which greatly advance knowledge of autophagosome biogenesis in plant cells. In this chapter, we will introduce two methods to dissect the autophagosome-related structures in the Arabidopsis plant cells, including the correlative light and electron microscopy, to map the ultrastructural feature of autophagosomal structures, and time-lapse imaging to monitor the temporal recruitment of autophagy machinery during autophagosome formation.
Asunto(s)
Arabidopsis , Autofagosomas , Autofagia , Células Vegetales , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Arabidopsis/metabolismo , Arabidopsis/ultraestructura , Autofagia/fisiología , Células Vegetales/metabolismo , Células Vegetales/ultraestructura , Imagen de Lapso de Tiempo/métodos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Microscopía Electrónica/métodosRESUMEN
The retinal pigment epithelium (RPE) is an essential component of the retina that plays multiple roles required to support visual function. These include light onset- and circadian rhythm-dependent tasks, such as daily phagocytosis of photoreceptor outer segments. Mitochondria provide energy to the highly specialized and energy-dependent RPE. In this study, we examined the positioning of mitochondria and how this is influenced by the onset of light. We identified a population of mitochondria that are tethered to the basal plasma membrane pre- and post-light onset. Following light onset, mitochondria redistributed apically and interacted with melanosomes and phagosomes. In a choroideremia mouse model that has regions of the RPE with disrupted or lost infolding of the plasma membrane, the positionings of only the non-tethered mitochondria were affected. This provides evidence that the tethering of mitochondria to the plasma membrane plays an important role that is maintained under these disease conditions. Our work shows that there are subpopulations of RPE mitochondria based on their positioning after light onset. It is likely they play distinct roles in the RPE that are needed to fulfil the changing cellular demands throughout the day.
Asunto(s)
Membrana Celular , Luz , Mitocondrias , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/metabolismo , Animales , Mitocondrias/metabolismo , Ratones , Membrana Celular/metabolismo , Ratones Endogámicos C57BL , Melanosomas/metabolismo , Ritmo Circadiano/fisiología , Fagosomas/metabolismoRESUMEN
The understanding of the inactivation process of ingested bacteria by phagocytes is a key focus in the field of host-pathogen interactions. Dictyostelium is a model organism that has been at the forefront of uncovering the mechanisms underlying this type of interaction. In this study, we describe an assay designed to measure the inactivation of Klebsiella aerogenes in the phagosomes of Dictyostelium discoideum.
Asunto(s)
Dictyostelium , Dictyostelium/microbiología , Dictyostelium/fisiología , Interacciones Huésped-Patógeno , Fagosomas/microbiología , Fagosomas/metabolismo , FagocitosisRESUMEN
Phagocytosis is an important immune response that protects the host from pathogen invasion. Rit1 GTPase is known to be involved in diverse cellular processes. However, its role in FcγR-mediated phagocytosis remains unclear. Our live-cell imaging analysis revealed that Rit1 was localized to the membranes of F-actin-rich phagocytic cups in RAW264 macrophages. Rit1 knockout and expression of the GDP-locked Rit1 mutant suppressed phagosome formation. We also found that TBC1D10B, a GAP for the Rab family GTPases, colocalizes with Rit1 in the membranes of phagocytic cups. Expression and knockout studies have shown that TBC1D10B decreases phagosome formation in both Rab-GAP activity-dependent and -independent manners. Notably, the expression of the GDP-locked Rit1 mutant or Rit1 knockout inhibited the dissociation of TBC1D10B from phagocytic cups. In addition, the expression of the GTP-locked Rit1 mutant promoted the dissociation of TBC1D10B in phagocytic cups and restored the rate of phagosome formation in TBC1D10B-expressing cells. These data suggest that Rit1-TBC1D10B signaling regulates FcγR-mediated phagosome formation in macrophages.
Asunto(s)
Proteínas Activadoras de GTPasa , Macrófagos , Fagocitosis , Fagosomas , Receptores de IgG , Transducción de Señal , Animales , Receptores de IgG/metabolismo , Ratones , Fagosomas/metabolismo , Macrófagos/metabolismo , Fagocitosis/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Células RAW 264.7 , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Actinas/metabolismoRESUMEN
One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process, named phagocytosis, for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this, he gave us the basis for our modern understanding of inflammation and the innate immune response. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In this chapter, we present a general view of our current knowledge on phagocytosis performed mainly by professional phagocytes through antibody and complement receptors and discuss aspects that remain incompletely understood.
Asunto(s)
Fagocitosis , Fagosomas , Humanos , Animales , Fagosomas/metabolismo , Fagocitos/inmunología , Fagocitos/metabolismo , Transducción de Señal , Inmunidad InnataRESUMEN
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used by Mtb to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), Mtb remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of Mtb is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for Mtb molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame sapM mutants in both the attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and Mtb. Characterization of these mutants demonstrated that SapM is required for PMA in BCG and Mtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in a Mtb mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and Mtb, and that it can function independent of the known mechanism for Mtb molecules to leave the phagosome.
Asunto(s)
Proteínas Bacterianas , Mycobacterium bovis , Mycobacterium tuberculosis , Fagosomas , Fagosomas/microbiología , Fagosomas/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Humanos , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Animales , RatonesRESUMEN
Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms1. During phagocytosis, innate immune receptors and associated signalling proteins are localized to the maturing phagosome compartment, forming an immune information processing hub brimming with microorganism-sensing features2-8. Here we developed proximity labelling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria. By comparing the protein composition of phagosomes containing evolutionarily and biochemically distinct microorganisms, we unexpectedly identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein Rpl20b as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of Rpl20b by macrophages cross-regulates production of distinct cytokines including interleukin-10 (IL-10) induced by the activation of other innate immune receptors. Thus, this study establishes PhagoPL as a useful approach to quantifying the collection of proteins enriched in phagosomes during host-microorganism interactions, exemplified by identifying PD-L1 as a receptor that binds to fungi.
Asunto(s)
Antígeno B7-H1 , Proteínas Fúngicas , Fagosomas , Proteínas Ribosómicas , Saccharomyces cerevisiae , Animales , Femenino , Humanos , Masculino , Ratones , Antígeno B7-H1/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Interacciones Microbiota-Huesped , Inmunidad Innata , Interleucina-10/metabolismo , Ligandos , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Ratones Endogámicos BALB C , Fagocitosis , Fagosomas/química , Fagosomas/metabolismo , Fagosomas/microbiología , Unión Proteica , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.
Asunto(s)
Legionella pneumophila , Fagosomas , Proteínas SNARE , Ubiquitinación , Proteínas de Unión al GTP rab , Legionella pneumophila/metabolismo , Humanos , Fagosomas/metabolismo , Fagosomas/microbiología , Proteínas SNARE/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Animales , Proteínas Qa-SNARE/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Vacuolas/metabolismo , Vacuolas/microbiología , Células HEK293 , Ratones , Proteínas de Unión a GTP rab7/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.
Asunto(s)
Proteínas Bacterianas , Lisosomas , Macrófagos , Streptococcus pyogenes , Estreptolisinas , Streptococcus pyogenes/inmunología , Humanos , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Lisosomas/metabolismo , Lisosomas/microbiología , Estreptolisinas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Fagosomas/microbiología , Fagosomas/metabolismo , Células THP-1 , Fagocitosis , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/metabolismo , Catepsina B/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas , Macrófagos , Mycobacterium tuberculosis , Fagosomas , Anticuerpos de Dominio Único , Humanos , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/metabolismo , Concentración de Iones de Hidrógeno , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Fagosomas/metabolismo , Anticuerpos de Dominio Único/metabolismoRESUMEN
Legionella pneumophila strains harboring wild-type rpsL such as Lp02rpsLWT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The bacterial factor directly responsible for inducing such cell death and the host factor involved in initiating the signaling cascade that leads to lysosome damage remain unknown. Similarly, host factors that may alleviate cell death induced by these bacterial strains have not yet been investigated. Using a genome-wide CRISPR/Cas9 screening, we identified Hmg20a and Nol9 as host factors important for restricting strain Lp02rpsLWT in BMDMs. Depletion of Hmg20a protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by Hmg20a was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02rpsLWT in BMDMs. Our results establish that L. pneumophila exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.
Asunto(s)
Legionella pneumophila , Lisosomas , Macrófagos , Fagosomas , Proteínas de Unión al GTP rab , Proteínas de Unión a GTP rab7 , Legionella pneumophila/metabolismo , Legionella pneumophila/genética , Animales , Proteínas de Unión al GTP rab/metabolismo , Ratones , Fagosomas/metabolismo , Fagosomas/microbiología , Lisosomas/metabolismo , Lisosomas/microbiología , Macrófagos/microbiología , Macrófagos/metabolismo , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Sumoilación , Ratones Endogámicos C57BL , Endosomas/metabolismo , Endosomas/microbiologíaRESUMEN
Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.
Asunto(s)
Señalización del Calcio , Calcio , Neutrófilos , Fagocitosis , Fagosomas , Humanos , Calcio/metabolismo , Fagosomas/metabolismo , Neutrófilos/metabolismo , Citosol/metabolismo , Membrana Celular/metabolismoRESUMEN
Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4+ large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.
Asunto(s)
Antígenos de Neoplasias , Carcinogénesis , Macrófagos Peritoneales , Animales , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Femenino , Ratones , Carcinogénesis/patología , Carcinogénesis/inmunología , Carcinogénesis/metabolismo , Humanos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Reactividad Cruzada/inmunología , Línea Celular Tumoral , Fagosomas/metabolismo , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Actinas/metabolismoRESUMEN
Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.
Asunto(s)
Dineínas , Cinesinas , Microtúbulos , Proteínas tau , Cinesinas/metabolismo , Cinesinas/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Dineínas/metabolismo , Dineínas/genética , Animales , Microtúbulos/metabolismo , Fagosomas/metabolismo , Transporte Biológico , Ratones , Humanos , Endocitosis/fisiologíaRESUMEN
Efficient degradation of phagocytic cargo in lysosomes is crucial to maintain cellular homeostasis and defending cells against pathogens. However, the mechanisms underlying the degradation and recycling of macromolecular cargo within the phagolysosome remain incompletely understood. We previously reported that the phagolysosome containing the corpse of the polar body in C. elegans tubulates into small vesicles to facilitate corpse clearance, a process that requires cargo protein degradation and amino acid export. Here we show that degradation of hexosylceramides by the prosaposin ortholog SPP-10 and glucosylceramidases is required for timely corpse clearance. We observed accumulation of membranous structures inside endolysosomes of spp-10-deficient worms, which are likely caused by increased hexosylceramide species. spp-10 deficiency also caused alteration of additional sphingolipid subclasses, like dihydroceramides, 2-OH-ceramides, and dihydrosphingomyelins. While corpse engulfment, initial breakdown of corpse membrane inside the phagolysosome and lumen acidification proceeded normally in spp-10-deficient worms, formation of the cargo-containing vesicles from the corpse phagolysosome was reduced, resulting in delayed cargo degradation and phagolysosome resolution. Thus, by combining ultrastructural studies and sphingolipidomic analysis with observing single phagolysosomes over time, we identified a role of prosaposin/SPP-10 in maintaining phagolysosomal structure, which promotes efficient resolution of phagocytic cargos.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Fagosomas , Animales , Caenorhabditis elegans/metabolismo , Fagosomas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Saposinas/metabolismo , Lisosomas/metabolismo , Fagocitosis , Ceramidas/metabolismoRESUMEN
Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative disease pathogenesis. Phagocytic glia are responsible for regulating the load of pathological proteins in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. A forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings uncover new mechanisms that enhance our understanding of the beneficial and harmful effects of phagocytic glia in HD and other neurodegenerative diseases.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de Drosophila , Drosophila , Proteína Huntingtina , Enfermedad de Huntington , Neuroglía , Animales , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Enfermedad de Huntington/genética , Neuroglía/metabolismo , Neuroglía/patología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Femenino , Masculino , Fagocitosis/fisiología , Lisosomas/metabolismo , Fagosomas/metabolismo , Animales Modificados Genéticamente , Priones/metabolismo , Priones/genética , Neuronas/metabolismoRESUMEN
During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.
Asunto(s)
Eferocitosis , Macrófagos , ARN Polimerasa II , Elongación de la Transcripción Genética , Animales , Humanos , Masculino , Ratones , Apoptosis , Citoesqueleto/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz/deficiencia , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Eferocitosis/genética , Concentración de Iones de Hidrógeno , Macrófagos/inmunología , Macrófagos/metabolismo , Neuronas/metabolismo , Fagosomas/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Pez Cebra/embriología , Pez Cebra/genética , Factores de TiempoRESUMEN
INTRODUCTION: Gallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). METHODS: A cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. RESULTS: Our results demonstrated that GA at 50 µM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. CONCLUSIONS: In summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.
Asunto(s)
Ácido Gálico , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Fagosomas , Transducción de Señal , Serina-Treonina Quinasas TOR , Vibrio vulnificus , Ácido Gálico/farmacología , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Vibrio vulnificus/efectos de los fármacos , Línea Celular , Citocinas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , FemeninoRESUMEN
Pseudomonas aeruginosa often colonizes immunocompromised patients, causing acute and chronic infections. This bacterium can reside transiently inside cultured macrophages, but the contribution of the intramacrophic stage during infection remains unclear. MgtC and OprF have been identified as important bacterial factors when P. aeruginosa resides inside cultured macrophages. In this study, we showed that P. aeruginosa mgtC and oprF mutants, particular the latter one, had attenuated virulence in both mouse and zebrafish animal models of acute infection. To further investigate P. aeruginosa pathogenesis in zebrafish at a stage different from acute infection, we monitored bacterial load and visualized fluorescent bacteria in live larvae up to 4 days after infection. Whereas the attenuated phenotype of the oprF mutant was associated with a rapid elimination of bacteria, the mgtC mutant was able to persist at low level, a feature also observed with the wild-type strain in surviving larvae. Interestingly, these persistent bacteria can be visualized in macrophages of zebrafish. In a short-time infection model using a macrophage cell line, electron microscopy revealed that internalized P. aeruginosa wild-type bacteria were either released after macrophage lysis or remained intracellularly, where they were localized in vacuoles or in the cytoplasm. The mgtC mutant could also be detected inside macrophages, but without causing cell damage, whereas the oprF mutant was almost completely eliminated after phagocytosis, or localized in phagolysosomes. Taken together, our results show that the main role of OprF for intramacrophage survival impacts both acute and persistent infection by this bacterium. On the other hand, MgtC plays a clear role in acute infection but is not essential for bacterial persistence, in relation with the finding that the mgtC mutant is not completely eliminated by macrophages.