RESUMEN
1258A is a new line of B.napus with Nsa cytoplasmic male sterility (CMS) with potential applications in hybrid rapeseed breeding. Sterile cytoplasm was obtained from XinJiang Sinapis arvensis through distant hybridization and then backcrossed with 1258B for many generations. However, the characteristics and molecular mechanisms underlying pollen abortion in this sterile line are poorly understood. In this study, a cytological analysis revealed normal microsporogenesis and uninucleate pollen grain formation. Pollen abortion was due to non-programmed cell death in the tapetum and the inability of microspores to develop into mature pollen grains. Sucrose, soluble sugar, and adenosine triphosphate (ATP) contents during microspore development were lower than those of the maintainer line, along with an insufficient energy supply, reduced antioxidant enzyme activity, and substantial malondialdehyde (MDA) accumulation in the anthers. Transcriptome analysis revealed that genes involved in secondary metabolite biosynthesis, glutathione metabolism, phenylpropane biosynthesis, cyanoamino acid metabolism, starch and sucrose metabolism, and glycerolipid metabolism may contribute to pollen abortion. The down regulation of nine cytochrome P450 monooxygenases genes were closely associated with pollen abortion. These results suggest that pollen abortion in 1258A CMS stems from abnormalities in the chorioallantoic membranes, energy deficiencies, and dysfunctional antioxidant systems in the anthers. Our results provide insight into the molecular mechanism underlying pollen abortion in Nsa CMS and provide a theoretical basis for better heterosis utilization in B.napus.
Asunto(s)
Brassica napus/genética , Citoplasma/genética , Hibridación Genética/genética , Proteínas de Plantas/genética , Transcriptoma/genética , Citosol/fisiología , Flores/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/genética , Ontología de Genes , Fitomejoramiento/métodos , Infertilidad Vegetal/genética , Polen/genética , Almidón/genéticaRESUMEN
Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.
Asunto(s)
Muerte Celular , Citoesqueleto/fisiología , Citosol/fisiología , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor de Adenosina A2A/genética , Agonistas del Receptor de Adenosina A2/farmacología , Animales , Línea Celular Tumoral , Femenino , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Receptor de Adenosina A2A/metabolismoRESUMEN
Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Endocitosis/fisiología , Priones/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Membrana Celular , Proteínas del Citoesqueleto/genética , Citosol/fisiología , Regulación Fúngica de la Expresión Génica , Glutamina/química , Mecanotransducción Celular , Conformación Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Sustancias ViscoelásticasRESUMEN
Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.
When it comes to studying the nervous system, neurons often steal the limelight; yet, they can only work properly thanks to an ensemble cast of cell types whose roles are only just emerging. For example, 'glial cells' their name derives from the Greek word for glue were once thought to play only a passive, supporting function in nervous tissues. Now, growing evidence shows that they are, in fact, integrated into neural circuits: their activity is influenced by neurons, and, in turn, they help neurons to function properly. The role of glial cells is becoming clear in the retina, the thin, light-sensitive layer that lines the back of the eye and relays visual information to the brain. There, beautifully intricate Müller glial cells display fine protrusions (or 'processes') that intermingle with synapses, the busy space between neurons where chemical messengers are exchanged. These messengers can act on Müller cells, triggering cascades of molecular events that may influence the structure and function of glia. This is of particular interest during development: as Müller cells mature, they are exposed to chemicals released by more fully formed retinal neurons. Tworig et al. explored how neuronal messengers can influence the way Müller cells grow their processes. To do so, they tracked mouse retinal glial cells 'live' during development, showing that they were growing fine, highly dynamic processes in a region rich in synapses just as neurons and glia increased their communication. However, using drugs to disrupt this messaging for a short period did not seem to impact how the processes grew. Extending the blockade over a longer timeframe also did not change the way Müller cells developed, with the cells still acquiring their characteristic elaborate process networks. Taken together, these results suggest that the structural maturation of Müller glial cells is not impacted by neuronal signaling, giving a more refined understanding of how glia form in the retina and potentially in the brain.
Asunto(s)
Calcio/metabolismo , Células Ependimogliales/fisiología , Transmisión Sináptica , Animales , Calcio/análisis , Fenómenos Fisiológicos Celulares , Citosol/química , Citosol/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Retina/citología , Retina/crecimiento & desarrolloRESUMEN
Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H+ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H+ transport activity, namely, H+-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H+ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H+-ATPases mediate H+ extrusion, whereas most members of other six proteins mediate H+ influx, thus contributing to cytosolic pH homeostasis by directly modulating H+ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H+-coupled substrate influx, whereas other intra-family members facilitate H+-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H+-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H+ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H+ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.
Asunto(s)
Citosol/fisiología , Transporte Iónico/fisiología , ATPasas de Translocación de Protón/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Homeostasis/fisiología , Concentración de Iones de Hidrógeno , Plantas , ATPasas de Translocación de Protón/genética , ProtonesRESUMEN
DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.
Asunto(s)
Metilación de ADN , Dermatitis/genética , Epidermis/fisiopatología , Nucleotidiltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Aberraciones Cromosómicas , Citosol/fisiología , ADN (Citosina-5-)-Metiltransferasa 1/genética , Dermatitis/inmunología , Dermatitis/patología , Humanos , Inmunidad Innata/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Queratinocitos/inmunología , Queratinocitos/metabolismo , Queratinocitos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Nucleotidiltransferasas/genéticaRESUMEN
Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element-induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.
Asunto(s)
Adenosina Desaminasa/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Interferón Tipo I/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Adenosina Desaminasa/metabolismo , Elementos Alu , Sistemas CRISPR-Cas , Citosol/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Humanos , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Intrones , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Edición de ARN , Proteínas de Unión al ARN/metabolismo , Células THP-1RESUMEN
Ischemic stroke is the second leading cause of mortality and disability globally. Neuronal damage following ischemic stroke is rapid and irreversible, and eventually results in neuronal death. In addition to activation of cell death signaling, neuroinflammation is also considered as another pathogenesis that can occur within hours after cerebral ischemia. Under physiological conditions, subcellular organelles play a substantial role in neuronal functionality and viability. However, their functions can be remarkably perturbed under neurological disorders, particularly cerebral ischemia. Therefore, their biochemical and structural response has a determining role in the sequel of neuronal cells and the progression of disease. However, their effects on cell death and neuroinflammation, as major underlying mechanisms of ischemic stroke, are still not understood. This review aims to provide a comprehensive overview of the contribution of each organelle on these pathological processes after ischemic stroke.
Asunto(s)
Accidente Cerebrovascular Isquémico/patología , Enfermedades Neuroinflamatorias/prevención & control , Neuronas/patología , Orgánulos/fisiología , Animales , Muerte Celular , Citosol/fisiología , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Humanos , Accidente Cerebrovascular Isquémico/complicaciones , Accidente Cerebrovascular Isquémico/etiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Enfermedades Neuroinflamatorias/etiología , Peroxisomas/fisiología , Ribosomas/fisiologíaRESUMEN
Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.
Asunto(s)
Membrana Celular/fisiología , Retículo Endoplásmico/fisiología , Homeostasis/fisiología , Animales , Anoctaminas/metabolismo , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Citosol/fisiología , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Iones/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distrofia Muscular de Cinturas/metabolismoRESUMEN
Enhanced growth and proliferation of cancer cells are accompanied by profound changes in cellular metabolism. These metabolic changes are also common under physiological conditions, and include increased glucose fermentation accompanied by elevated cytosolic pH (pHc)1,2. However, how these changes contribute to enhanced cell growth and proliferation is unclear. Here, we show that elevated pHc specifically orchestrates an E2F-dependent transcriptional programme to drive cell proliferation by promoting cyclin D1 expression. pHc-dependent transcription of cyclin D1 requires the transcription factors CREB1, ATF1 and ETS1, and the histone acetyltransferases p300 and CBP. Biochemical characterization revealed that the CREB1-p300/CBP interaction acts as a pH sensor and coincidence detector, integrating different mitotic signals to regulate cyclin D1 transcription. We also show that elevated pHc contributes to increased cyclin D1 expression in malignant pleural mesotheliomas (MPMs), and renders these cells hypersensitive to pharmacological reduction of pHc. Taken together, these data demonstrate that elevated pHc is a critical cellular signal regulating G1 progression, and provide a mechanism linking elevated pHc to oncogenic activation of cyclin D1 in MPMs, and possibly other cyclin D1~dependent tumours. Thus, an increase of pHc may represent a functionally important, early event in the aetiology of cancer that is amenable to therapeutic intervention.
Asunto(s)
Proliferación Celular , Ciclina D1/biosíntesis , Citosol/metabolismo , Línea Celular Tumoral , Biología Computacional , Ciclina D1/genética , Citosol/patología , Citosol/fisiología , Factores de Transcripción E2F/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Concentración de Iones de Hidrógeno , Masculino , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Mesotelioma/patología , Metabolómica , Mitosis/fisiología , Fracciones Subcelulares/metabolismo , Factores de TranscripciónRESUMEN
Heat shock induces a conserved transcriptional program regulated by heat shock factor 1 (Hsf1) in eukaryotic cells. Activation of this heat shock response is triggered by heat-induced misfolding of newly synthesized polypeptides, and so has been thought to depend on ongoing protein synthesis. Here, using the budding yeast Saccharomyces cerevisiae, we report the discovery that Hsf1 can be robustly activated when protein synthesis is inhibited, so long as cells undergo cytosolic acidification. Heat shock has long been known to cause transient intracellular acidification which, for reasons which have remained unclear, is associated with increased stress resistance in eukaryotes. We demonstrate that acidification is required for heat shock response induction in translationally inhibited cells, and specifically affects Hsf1 activation. Physiological heat-triggered acidification also increases population fitness and promotes cell cycle reentry following heat shock. Our results uncover a previously unknown adaptive dimension of the well-studied eukaryotic heat shock response.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/genética , Citosol/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Concentración de Iones de Hidrógeno , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
MAIN CONCLUSION: The nuclear-localized CAX-interacting protein VvCXIP4 is exported to the cytosol after a Ca2+ pulse, to activate the tonoplast-localized Ca2+/H+ exchanger VvCAX3. Vacuolar cation/H+ exchangers (CAXs) have long been recognized as 'housekeeping' components in cellular Ca2+ and trace metal homeostasis, being involved in a range of key cellular and physiological processes. However, the mechanisms that drive functional activation of the transporters are largely unknown. In the present study, we investigated the function of a putative grapevine CAX-interacting protein, VvCXIP4, by testing its ability to activate VvCAX3, previously characterized as a tonoplast-localized Ca2+/H+ exchanger. VvCAX3 contains an autoinhibitory domain that drives inactivation of the transporter and thus, is incapable of suppressing the Ca2+-hypersensitive phenotype of the S. cerevisiae mutant K667. In this study, the co-expression of VvCXIP4 and VvCAX3 in this strain efficiently rescued its growth defect at high Ca2+ levels. Flow cytometry experiments showed that yeast harboring both proteins effectively accumulated higher Ca2+ levels than cells expressing each of the proteins separately. Bimolecular fluorescence complementation (BiFC) assays allowed visualization of the direct interaction between the proteins in tobacco plants and in yeast, and also showed the self-interaction of VvCAX3 but not of VvCXIP4. Subcellular localization studies showed that, despite being primarily localized to the nucleus, VvCXIP4 is able to move to other cell compartments upon a Ca2+ stimulus, becoming prone to interaction with the tonoplast-localized VvCAX3. qPCR analysis showed that both genes are more expressed in grapevine stems and leaves, followed by the roots, and that the steady-state transcript levels were higher in the pulp than in the skin of grape berries. Also, both VvCXIP4 and VvCAX3 were upregulated by Ca2+ and Na+, indicating they share common regulatory mechanisms. However, VvCXIP4 was also upregulated by Li+, Cu2+ and Mn2+, and its expression increased steadily throughout grape berry development, contrary to VvCAX3, suggesting additional physiological roles for VvCXIP4, including the regulation of VvCAXs not yet functionally characterized. The main novelty of the present study was the demonstration of physical interaction between CXIP and CAX proteins from a woody plant model by BiFC assays, demonstrating the intracellular mobilization of CXIPs in response to Ca2+.
Asunto(s)
Transporte Biológico/fisiología , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/fisiología , Núcleo Celular/fisiología , Citosol/fisiología , Vitis/genética , Vitis/fisiología , Frutas/fisiología , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/fisiología , Proteínas de Plantas/fisiologíaRESUMEN
The existence of nuclear pore complexes in the nuclear envelope has led to the assumption that ions move freely from the cytosol into the nucleus, and that the molecular mechanisms at the plasma membrane that regulate cytosolic pH also regulate nuclear pH. Furthermore, studies to measure pH in the nucleus have produced contradictory results, since it has been found that the nuclear pH is either similar to the cytosol or more alkaline than the cytosol. However, most studies of nuclear pH have lacked the rigor needed to understand pH regulation in the nucleus. A major problem has been the lack of in situ titrations in the nucleus and cytosol, since the intracellular environment is different in the cytosol and nucleus and the behavior of fluorescent pH probes is different in these environments. Here we present a method that uses the fluorescence of SNARF-1 that labels both cytosol and nucleus. Using ratio imaging microscopy, regions of interest corresponding to the nucleus and cytosol to perform steady-state pH measurements followed by in situ titrations, to correctly assign pH in those cellular domains.
Asunto(s)
Núcleo Celular/fisiología , Citosol/fisiología , Concentración de Iones de Hidrógeno , Microscopía Fluorescente/métodos , Benzopiranos/química , Línea Celular , Núcleo Celular/química , Fenómenos Fisiológicos Celulares , Citosol/química , Colorantes Fluorescentes/química , Humanos , Membrana Nuclear/fisiología , ProtonesRESUMEN
Various stress conditions induce the nuclear translocation of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), but its nuclear function in plant stress responses remains elusive. Here we show that GAPC interacts with a transcription factor to promote the expression of heat-inducible genes and heat tolerance in Arabidopsis. GAPC accumulates in the nucleus under heat stress. Overexpression of GAPC enhances heat tolerance of seedlings and the expression of heat-inducible genes whereas knockout of GAPCs has opposite effects. Screening of Arabidopsis transcription factors identifies nuclear factor Y subunit C10 (NF-YC10) as a GAPC-binding protein. The effects of GAPC overexpression are abolished when NF-YC10 is deficient, the heat-induced nuclear accumulation of GAPC is suppressed, or the GAPC-NF-YC10 interaction is disrupted. GAPC overexpression also enhances the binding ability of NF-YC10 to its target promoter. The results reveal a cellular and molecular mechanism for the nuclear moonlighting of a glycolytic enzyme in plant response to environmental changes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Citosol/metabolismo , Citosol/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/fisiología , Plantones/genética , Plantones/metabolismo , Plantones/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.
Asunto(s)
Citosol/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Individual de Molécula/métodos , Citoplasma/metabolismo , Citoplasma/fisiología , Citosol/metabolismo , Eremothecium/metabolismo , Aprendizaje Automático , Nanopartículas , Orientación Espacial/fisiologíaRESUMEN
Endocrine cells of the pancreatic islet interact with their microenvironment to maintain tissue homeostasis. Communication with local macrophages is particularly important in this context, but the homeostatic functions of human islet macrophages are not known. In this study, we show that the human islet contains macrophages in perivascular regions that are the main local source of the anti-inflammatory cytokine interleukin-10 (IL-10) and the metalloproteinase MMP9. Macrophage production and secretion of these homeostatic factors are controlled by endogenous purinergic signals. In obese and diabetic states, macrophage expression of purinergic receptors MMP9 and IL-10 is reduced. We propose that in those states, exacerbated ß-cell activity due to increased insulin demand and increased cell death produce high levels of ATP that downregulate purinergic receptor expression. Loss of ATP sensing in macrophages may reduce their secretory capacity.
Asunto(s)
Islotes Pancreáticos/citología , Macrófagos/fisiología , Purinas/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Calcio/metabolismo , Citocinas , Citosol/química , Citosol/fisiología , Diabetes Mellitus/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Islotes Pancreáticos/diagnóstico por imagen , Ratones , Receptores Purinérgicos/metabolismo , Transducción de Señal , TranscriptomaRESUMEN
Cell polarity is an important cellular process that cells use for various cellular functions such as asymmetric division, cell migration, and directionality determination. In asymmetric cell division, a mother cell creates multiple polarities of various proteins simultaneously within her membrane and cytosol to generate two different daughter cells. The formation of multiple polarities in asymmetric cell division has been found to be controlled via the regulatory system by upstream polarity of the membrane to downstream polarity of the cytosol, which is involved in not only polarity establishment but also polarity positioning. However, the mechanism for polarity positioning remains unclear. In this study, we found a general mechanism and mathematical structure for the multiple streams of polarities to determine their relative position via conceptional models based on the biological example of the asymmetric cell division process of C. elegans embryo. Using conceptional modeling and model reductions, we show that the positional relation of polarities is determined by a contrasting role of regulation by upstream polarity proteins on the transition process of diffusion dynamics of downstream proteins. We analytically prove that our findings hold under the general mathematical conditions, suggesting that the mechanism of relative position between upstream and downstream dynamics could be understood without depending on a specific type of bio-chemical reaction, and it could be the universal mechanism in multiple streams of polarity dynamics of the cell.
Asunto(s)
Polaridad Celular/fisiología , Modelos Biológicos , Animales , División Celular Asimétrica/fisiología , Transporte Biológico/fisiología , Tipificación del Cuerpo/fisiología , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/fisiología , Membrana Celular/fisiología , Movimiento Celular/fisiología , Citosol/fisiología , Conceptos Matemáticos , Transducción de Señal/fisiologíaRESUMEN
Iron-sulfur (Fe-S) clusters play an essential role in plants as protein cofactors mediating diverse electron transfer reactions. Because they can react with oxygen to form reactive oxygen species (ROS) and inflict cellular damage, the biogenesis of Fe-S clusters is highly regulated. A recently discovered group of 2Fe-2S proteins, termed NEET proteins, was proposed to coordinate Fe-S, Fe and ROS homeostasis in mammalian cells. Here we report that disrupting the function of AtNEET, the sole member of the NEET protein family in Arabidopsis thaliana, triggers leaf-associated Fe-S- and Fe-deficiency responses, elevated Fe content in chloroplasts (1.2-1.5-fold), chlorosis, structural damage to chloroplasts and a high seedling mortality rate. Our findings suggest that disrupting AtNEET function disrupts the transfer of 2Fe-2S clusters from the chloroplastic 2Fe-2S biogenesis pathway to different cytosolic and chloroplastic Fe-S proteins, as well as to the cytosolic Fe-S biogenesis system, and that uncoupling this process triggers leaf-associated Fe-S- and Fe-deficiency responses that result in Fe over-accumulation in chloroplasts and enhanced ROS accumulation. We further show that AtNEET transfers its 2Fe-2S clusters to DRE2, a key protein of the cytosolic Fe-S biogenesis system, and propose that the availability of 2Fe-2S clusters in the chloroplast and cytosol is linked to Fe homeostasis in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Azufre/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Citosol/fisiología , Transporte de Electrón , Homeostasis , Proteínas Hierro-Azufre/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The gastric pathogen Helicobacter pylori requires a noncanonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here, we reconstituted a complete chemotransduction signaling complex in vitro with TlpD and the chemotaxis (Che) proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found that H. pylori is resistant to killing by millimolar concentrations of HOCl and responds to HOCl in the micromolar range by increasing its smooth-swimming behavior, leading to chemoattraction to HOCl sources. We show related protein domains from Salmonella enterica and Escherichia coli possess similar reactivity toward HOCl. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy that H. pylori uses to aid in colonizing and persisting in inflamed gastric tissue.
Asunto(s)
Quimiotaxis/fisiología , Helicobacter pylori/metabolismo , Receptores de Formil Péptido/metabolismo , Proteínas Bacterianas/metabolismo , Blanqueadores , Células Quimiorreceptoras/metabolismo , Factores Quimiotácticos/metabolismo , Citosol/metabolismo , Citosol/fisiología , Helicobacter pylori/fisiología , Ácido Hipocloroso , Oxidación-Reducción , Receptores de Formil Péptido/fisiología , Transducción de SeñalRESUMEN
Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella-containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi-function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion-associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra-cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.