RESUMEN
DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helps maintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discovered that lysosomes process topoisomerase 1 cleavage complexes (TOP1cc) DNA lesions in vertebrates. Selective degradation of TOP1cc by autophagy directs DNA damage repair and cell survival at clinically relevant doses of topoisomerase 1 inhibitors. TOP1cc are exported from the nucleus to lysosomes through a transient alteration of the nuclear envelope and independent of the proteasome. Mechanistically, the autophagy receptor TEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by the p97 ATPase and mediating the delivery of TOP1cc to lysosomes in an MRE11-nuclease- and ATR-kinase-dependent manner. We found an evolutionarily conserved role for selective autophagy in DNA repair that enables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients.
Asunto(s)
Autofagia , Supervivencia Celular , Daño del ADN , Reparación del ADN , ADN-Topoisomerasas de Tipo I , Lisosomas , Proteínas de la Membrana , Animales , Humanos , Ratones , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Inestabilidad Genómica , Lisosomas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.
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Células Asesinas Naturales/fisiología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Axones , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Células Asesinas Naturales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Regeneración Nerviosa , Neuronas/citología , Neuronas Aferentes/inmunología , Neuronas Aferentes/metabolismo , Proteínas Asociadas a Matriz Nuclear/fisiología , Proteínas de Transporte Nucleocitoplasmático/fisiología , Dolor , Traumatismos de los Nervios Periféricos/inmunología , Enfermedades del Sistema Nervioso Periférico , Nervio Ciático , Células Receptoras Sensoriales/metabolismoRESUMEN
Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte-specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti-inflammatory energy substrates. Instead, Atg7-deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2-mediated upregulation of Ephx1. This shift reduced secretion of IL-10 from adipose tissues, which was dependent on the cytochrome P450-EPHX pathway, and lowered circulating levels of IL-10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat-gut crosstalk through an autophagy-dependent regulation of anti-inflammatory oxylipins via the cytochrome P450-EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.
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Ácidos Grasos no Esterificados , Oxilipinas , Humanos , Adipocitos/metabolismo , Autofagia/fisiología , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/farmacología , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos no Esterificados/farmacología , Inflamación/genética , Inflamación/metabolismo , Interleucina-10/genética , Oxilipinas/metabolismoRESUMEN
Neutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation.
Asunto(s)
Autofagia , Diferenciación Celular , Ácidos Grasos no Esterificados/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Adaptación Biológica , Animales , Análisis por Conglomerados , Metabolismo Energético , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Metabolismo de los Lípidos , Lipólisis , Mielopoyesis , Neutrófilos/ultraestructura , Oxidación-Reducción , Ácido Pirúvico/metabolismoRESUMEN
The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.
Asunto(s)
Colon/citología , Colon/patología , Células Epiteliales/clasificación , Células Epiteliales/citología , Salud , Enfermedades Inflamatorias del Intestino/patología , Canales Iónicos/metabolismo , Animales , Biomarcadores/análisis , Colitis Ulcerosa/genética , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Células Epiteliales/microbiología , Células Epiteliales/patología , Predisposición Genética a la Enfermedad/genética , Células Caliciformes/citología , Células Caliciformes/metabolismo , Células Caliciformes/patología , Humanos , Concentración de Iones de Hidrógeno , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Péptidos Natriuréticos/metabolismo , Proteínas/metabolismo , Análisis de la Célula Individual , Células Madre/citología , Células Madre/metabolismo , Células Madre/patología , Uniones Estrechas/metabolismo , Transcripción Genética , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.
Asunto(s)
Proliferación Celular , Exosomas , Glutamina/deficiencia , Sistema de Señalización de MAP Quinasas , Neoplasias , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Exosomas/genética , Exosomas/metabolismo , Exosomas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
It is increasingly being recognised that the interplay between commensal and pathogenic bacteria can dictate the outcome of infection. Consequently, there is a need to understand how commensals interact with their human host and influence pathogen behaviour at epithelial surfaces. Neisseria meningitidis, a leading cause of sepsis and meningitis, exclusively colonises the human nasopharynx and shares this niche with several other Neisseria species, including the commensal Neisseria cinerea. Here, we demonstrate that during adhesion to human epithelial cells N. cinerea co-localises with molecules that are also recruited by the meningococcus, and show that, similar to N. meningitidis, N. cinerea forms dynamic microcolonies on the cell surface in a Type four pilus (Tfp) dependent manner. Finally, we demonstrate that N. cinerea colocalises with N. meningitidis on the epithelial cell surface, limits the size and motility of meningococcal microcolonies, and impairs the effective colonisation of epithelial cells by the pathogen. Our data establish that commensal Neisseria can mimic and affect the behaviour of a pathogen on epithelial cell surfaces.
Asunto(s)
Adhesión Bacteriana , Células Epiteliales/microbiología , Fimbrias Bacterianas/metabolismo , Neisseria cinerea/crecimiento & desarrollo , Neisseria meningitidis/crecimiento & desarrollo , Células A549 , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Neisseria cinerea/patogenicidad , Neisseria meningitidis/patogenicidadRESUMEN
BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
Asunto(s)
Anemia Diseritropoyética Congénita/genética , Predisposición Genética a la Enfermedad , Glicoproteínas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Anemia Diseritropoyética Congénita/patología , Femenino , Regulación de la Expresión Génica/genética , Pruebas Genéticas , Genética de Población , Humanos , Masculino , Complejos Multiproteicos/genética , Mutación/genéticaRESUMEN
BACKGROUND: Why some but not all patients with severe aortic stenosis (SevAS) develop otherwise unexplained reduced systolic function is unclear. We investigate the hypothesis that reduced creatine kinase (CK) capacity and flux is associated with this transition. METHODS: We recruited 102 participants to 5 groups: moderate aortic stenosis (ModAS) (n=13), SevAS, left ventricular (LV) ejection fraction ≥55% (SevAS-preserved ejection fraction, n=37), SevAS, LV ejection fraction <55% (SevAS-reduced ejection fraction, n=15), healthy volunteers with nonhypertrophied hearts with normal systolic function (normal healthy volunteer, n=30), and patients with nonhypertrophied, non-pressure-loaded hearts with normal systolic function undergoing cardiac surgery and donating LV biopsy (non-pressure-loaded heart biopsy, n=7). All underwent cardiac magnetic resonance imaging and 31P magnetic resonance spectroscopy for myocardial energetics. LV biopsies (AS and non-pressure-loaded heart biopsy) were analyzed for CK total activity, CK isoforms, citrate synthase activity, and total creatine. Mitochondria-sarcomere diffusion distances were calculated by using serial block-face scanning electron microscopy. RESULTS: In the absence of failure, CK flux was lower in the presence of AS (by 32%, P=0.04), driven primarily by reduction in phosphocreatine/ATP (by 17%, P<0.001), with CK kf unchanged (P=0.46). Although lowest in the SevAS-reduced ejection fraction group, CK flux was not different from the SevAS-preserved ejection fraction group (P>0.99). Accompanying the fall in CK flux, total CK and citrate synthase activities and the absolute activities of mitochondrial-type CK and CK-MM isoforms were also lower (P<0.02, all analyses). Median mitochondria-sarcomere diffusion distances correlated well with CK total activity (r=0.86, P=0.003). CONCLUSIONS: Total CK capacity is reduced in SevAS, with median values lowest in those with systolic failure, consistent with reduced energy supply reserve. Despite this, in vivo magnetic resonance spectroscopy measures of resting CK flux suggest that ATP delivery is reduced earlier, at the moderate AS stage, where LV function remains preserved. These findings show that significant energetic impairment is already established in moderate AS and suggest that a fall in CK flux is not by itself a necessary cause of transition to systolic failure. However, because ATP demands increase with AS severity, this could increase susceptibility to systolic failure. As such, targeting CK capacity and flux may be a therapeutic strategy to prevent and treat systolic failure in AS.
Asunto(s)
Estenosis de la Válvula Aórtica/sangre , Creatina Quinasa/sangre , Metabolismo Energético/fisiología , Volumen Sistólico/fisiología , Disfunción Ventricular Izquierda/sangre , Función Ventricular Izquierda/fisiología , Adenosina Trifosfato/sangre , Adulto , Anciano , Anciano de 80 o más Años , Estenosis de la Válvula Aórtica/diagnóstico , Estenosis de la Válvula Aórtica/fisiopatología , Biomarcadores/sangre , Femenino , Humanos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.
Asunto(s)
Anemia Diseritropoyética Congénita , Anemia Diseritropoyética Congénita/diagnóstico , Anemia Diseritropoyética Congénita/genética , Células Eritroides , Eritropoyesis , Glicoproteínas/genética , Humanos , Proteínas Nucleares/genéticaRESUMEN
Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.
Asunto(s)
Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/fisiología , Microtúbulos/metabolismo , Animales , Animales Modificados Genéticamente , Cuerpos Basales/metabolismo , Cuerpos Basales/fisiología , Proteínas de Unión a Calmodulina , Centriolos/genética , Cilios/genética , Cilios/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Interfase/fisiología , Complejos Multiproteicos/metabolismo , Mutación/fisiología , Multimerización de Proteína/fisiología , Estabilidad Proteica , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/ultraestructuraRESUMEN
Recently reported DNA nanoflowers are an interesting class of organic-inorganic hybrid materials which are prepared using DNA polymerases. DNA nanoflowers combine the high surface area and scaffolding of inorganic Mg2P2O7 nanocrystals with the targeting properties of DNA, whilst adding enzymatic stability and enhanced cellular uptake. We have investigated conditions for chemically modifying the inorganic core of these nanoflowers through substitution of Mg2+ with Mn2+, Co2+ or Zn2+ and have characterized the resulting particles. These have a range of novel nanoarchitectures, retain the enzymatic stability of their magnesium counterparts and the Co2+ and Mn2+ DNA nanoflowers have added magnetic properties. We investigate conditions to control different morphologies, DNA content, hybridization properties, and size. Additionally, we show that DNA nanoflower production is not limited to Ф29 DNA polymerase and that the choice of polymerase can influence the DNA length within the constructs. We anticipate that the added control of structure, size and chemistry will enhance future applications.
Asunto(s)
Cobalto/química , ADN Polimerasa Dirigida por ADN/química , ADN/síntesis química , Manganeso/química , Nanopartículas del Metal/química , Oligonucleótidos/síntesis química , Zinc/química , Fagos de Bacillus/enzimología , Nanotecnología/métodosRESUMEN
The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies.
Asunto(s)
Acuaporina 1/metabolismo , Arterias , Permeabilidad Capilar , Endotelio Vascular , Microscopía Óptica no Lineal , Animales , Arterias/diagnóstico por imagen , Arterias/metabolismo , Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.
Asunto(s)
Membrana Celular/metabolismo , Citocinesis/fisiología , Proteínas del Citoesqueleto/metabolismo , Flagelos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Membrana Celular/genética , Proteínas del Citoesqueleto/genética , Flagelos/genética , Técnicas de Silenciamiento del Gen , Leishmania/genética , Leishmania/metabolismo , Proteínas Protozoarias/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host-pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus-pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.
Asunto(s)
Adhesión Bacteriana , Células Epiteliales/microbiología , Proteínas Fimbrias/análisis , Neisseria cinerea/fisiología , Adhesinas Bacterianas/análisis , Adhesinas Bacterianas/genética , Línea Celular , Proteínas Fimbrias/genética , Eliminación de Gen , Humanos , Neisseria meningitidisRESUMEN
The secretion of cell wall polysaccharides through the trans-Golgi network (TGN) is required for plant cell elongation. However, the components mediating the post-Golgi secretion of pectin and hemicellulose, the two major cell wall polysaccharides, are largely unknown. We identified evolutionarily conserved YPT/RAB GTPase Interacting Protein 4a (YIP4a) and YIP4b (formerly YIP2), which form a TGN-localized complex with ECHIDNA (ECH) in Arabidopsis thaliana. The localization of YIP4 and ECH proteins at the TGN is interdependent and influences the localization of VHA-a1 and SYP61, which are key components of the TGN. YIP4a and YIP4b act redundantly, and the yip4a yip4b double mutants have a cell elongation defect. Genetic, biochemical, and cell biological analyses demonstrate that the ECH/YIP4 complex plays a key role in TGN-mediated secretion of pectin and hemicellulose to the cell wall in dark-grown hypocotyls and in secretory cells of the seed coat. In keeping with these observations, Fourier transform infrared microspectroscopy analysis revealed that the ech and yip4a yip4b mutants exhibit changes in their cell wall composition. Overall, our results reveal a TGN subdomain defined by ECH/YIP4 that is required for the secretion of pectin and hemicellulose and distinguishes the role of the TGN in secretion from its roles in endocytic and vacuolar trafficking.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Polisacáridos/metabolismo , Red trans-Golgi/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Pared Celular/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Filogenia , Plantas Modificadas Genéticamente , Unión Proteica , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)-mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Aumento de la Célula , Ácidos Indolacéticos/metabolismo , Tallos de la Planta/citología , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Tomografía con Microscopio Electrónico , Aparato de Golgi/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Tallos de la Planta/crecimiento & desarrollo , Transporte de Proteínas/fisiología , Proteínas de Transporte Vesicular/genéticaRESUMEN
Inorganic polyphosphate (polyP) is a ubiquitous polymer that controls fundamental processes. To overcome the absence of a genetically tractable mammalian model, we developed an inducible mammalian cell line expressing Escherichia coli polyphosphate kinase 1 (EcPPK1). Inducing EcPPK1 expression prompted polyP synthesis, enabling validation of polyP analytical methods. Virtually all newly synthesized polyP accumulates within the nucleus, mainly in the nucleolus. The channeled polyP within the nucleolus results in the redistribution of its markers, leading to altered rRNA processing. Ultrastructural analysis reveals electron-dense polyP structures associated with a hyper-condensed nucleolus resulting from an exacerbation of the liquid-liquid phase separation (LLPS) phenomena controlling this membraneless organelle. The selective accumulation of polyP in the nucleoli could be interpreted as an amplification of polyP channeling to where its physiological function takes place. Indeed, quantitative analysis of several mammalian cell lines confirms that endogenous polyP accumulates within the nucleolus.