RESUMO
Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.
Assuntos
Vesículas Citoplasmáticas/fisiologia , Proteoma/genética , Proteômica , Virulência , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Vesículas Citoplasmáticas/metabolismo , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Interações Hospedeiro-Parasita , Nefropatias/microbiologia , Nefropatias/veterinária , Lipoproteínas/metabolismo , Renibacterium/citologia , Renibacterium/genética , Renibacterium/patogenicidade , Frações Subcelulares/fisiologia , Virulência/genéticaRESUMO
Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here, we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live-cell imaging to visualize electrical synapses in vivo and an optogenetic assay to confirm that they are functional. In ventral A class (VA) motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of functional electrical synapses.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , AMP Cíclico/farmacologia , Sinapses Elétricas/fisiologia , Proteínas de Homeodomínio/metabolismo , Neurônios Motores/fisiologia , Frações Subcelulares/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Axônios/efeitos dos fármacos , Axônios/fisiologia , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Conexinas/genética , Conexinas/metabolismo , Sinapses Elétricas/efeitos dos fármacos , Junções Comunicantes , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Neurônios Motores/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacosRESUMO
Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.
Assuntos
Algoritmos , Imageamento Tridimensional , Óptica e Fotônica , Tomografia , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Drosophila , Células HeLa , Humanos , Larva/fisiologia , Fígado/diagnóstico por imagem , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Ratos Sprague-Dawley , Razão Sinal-Ruído , Frações Subcelulares/fisiologia , Fatores de Tempo , Peixe-ZebraRESUMO
The notion that graded distributions of signals underlie the spatial organization of biological systems has long been a central pillar in the fields of cell and developmental biology. During morphogenesis, morphogens spread across tissues to guide development of the embryo. Similarly, a variety of dynamic gradients and pattern-forming networks have been discovered that shape subcellular organization. Here we discuss the principles of intracellular pattern formation by these intracellular morphogens and relate them to conceptually similar processes operating at the tissue scale. We will specifically review mechanisms for generating cellular asymmetry and consider how intracellular patterning networks are controlled and adapt to cellular geometry. Finally, we assess the general concept of intracellular gradients as a mechanism for positional control in light of current data, highlighting how the simple readout of fixed concentration thresholds fails to fully capture the complexity of spatial patterning processes occurring inside cells.
Assuntos
Padronização Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Frações Subcelulares/fisiologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologiaRESUMO
Earthworms have the ability to accumulate of heavy metals, however, there was few studies that addressed the metals in earthworm at subcellular levels in fields. The distributions of metals (Cd, Cu, Zn, and Pb) in subcellular fractions (cytosol, debris, and granules) of earthworm Metaphire californica were investigated. The relationship between soil metals and earthworms were analyzed to explain its high plasticity to inhabit in situ contaminated soil of Hunan Province, south China. The concentration of Cd in subcellular compartments showed the same pattern as Cu in the order of cytosol > debris > granules. The distribution of Zn and Pb in earthworms indicated a similar propensity for different subcellular fractions that ranked as granules > debris > cytosol for Zn, and granules > cytosol > debris for Pb. The internal metal concentrations in earthworms increased with the soil metals (p<0.05). Significant positive correlations were found between soil Cd and Cd concentrations in cytosol and debris (p<0.01). Moreover, the soil Pb concentration significantly influenced the Pb concentrations in cytosol and debris (p<0.01), similar to that of Cd. The soil Cu concentrations was only associated with the Cu in granules (p<0.05). Soil Zn concentrations correlated with the Zn concentrations in each subcellular fraction (p<0.05). Our results provide insights into the variations of metals partitioning in earthworms at subcellular levels and the relationships of soil metals, which could be one of the detoxification strategies to adapt the long-term contaminated environment.
Assuntos
Monitoramento Ambiental , Metais Pesados/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Frações Subcelulares/fisiologia , Animais , China , Metais Pesados/metabolismo , Poluentes do Solo/metabolismoRESUMO
The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, therefore, plays a key role in apoptosis execution. However, the detailed molecular mechanisms of these events remain unclear. To get more insights into the mechanisms of nuclear events during apoptosis we set up a rapid fractionation protocol for the separation of the cytoplasmic and nuclear fractions of cells undergoing cisplatin-induced apoptosis. Importantly, nuclear accumulation of effector caspase-3 as well as initiator caspase-2, -8 and -9 was observed using the developed protocol and immunofluorescence microscopy. The detection of caspases and their cleavage products in the nucleus occurred within the same time interval after cisplatin treatment and took place shortly before nuclear fragmentation. The entry of initiator caspases to the nucleus was independent of caspase-3. Given that all three initiator caspases had catalytic activity in the nuclei, our findings indicate that initiator caspases might participate in the proteolysis of nuclear components during apoptosis, promoting its disintegration and apoptotic cell death.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Caspases/fisiologia , Caspase 2/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Fracionamento Celular/métodos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cisplatino/farmacologia , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Fragmentação do DNA , Células HeLa , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Frações Subcelulares/fisiologiaRESUMO
AF6/afadin is an F-actin scaffold protein that plays essential roles in the organization of cell-cell junctions of polarized epithelia. Afadin comprises two major isoforms produced by alternative splicing - a longer isoform l-afadin, having the F-actin-binding domain, and a shorter isoform s-afadin, harboring the amino acid sequences unique to the isoform but lacking the F-actin-binding domain. We recently identified functional differences between l- and s-afadin isoforms in the regulation of axon branching in primary cultured cortical neurons; the former potentiates and the latter blocks axon branching. Previous biochemical reports indicate differences in tissue and cell-type distributions of isoforms, and it was shown that l-afadin is ubiquitously expressed in various tissues and cell-types, while s-afadin is predominantly expressed in neuronal tissues and cultured neurons. However, the spatial expression pattern of s-afadin across neuronal tissues or within neurons has not been revealed because no antibody specific for s-afadin is yet available. In this study, we report the generation and characterization of an antibody that specifically distinguishes s-afadin from l-afadin, and its application to investigate the expression profile of s-afadin in primary cultured neurons and tissue cryosections of adult mouse brain and retina. We describe differential regional and subcellular localization patterns of l- and s-afadin isoforms in the mouse central nervous system.
Assuntos
Processamento Alternativo/fisiologia , Córtex Cerebral/citologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neurônios/ultraestrutura , Isoformas de Proteínas/metabolismo , Frações Subcelulares/ultraestrutura , Animais , Ontologias Biológicas , Células Cultivadas , Sistema Nervoso Central , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Neurônios/metabolismo , Gravidez , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Retina/citologia , Frações Subcelulares/fisiologiaRESUMO
BACKGROUND: The therapeutic efficacy of adipose-derived stem cells (ASCs) has been investigated for numerous clinical indications, including autoimmune and neurodegenerative diseases. Less is known using the crude adipose product called stromal vascular fraction (SVF) as therapy, although our previous studies demonstrated greater efficacy at late-stage disease compared to ASCs in the experimental autoimmune encephalomyelitis (EAE) mouse, a model of multiple sclerosis. In this study, SVF cells and ASCs were administered during the pathogenic progression, designated as early disease, to elucidate immunomodulatory mechanisms when high immune cell activities associated with autoimmune signaling occur. These implications are essential for clinical translation when considering timing of administration for cell therapies. METHODS: We investigated the effects of SVF cells and ASCs by analyzing the spleens, peripheral blood, and central nervous system tissues throughout the course of EAE disease following administration of SVF cells, ASCs, or vehicle. In vitro, immunomodulatory potentials of SVF cells and ASCs were measured when exposed to EAE-derived splenocytes. RESULTS: Interestingly, treatment with SVF cells and ASCs transiently enhanced the severity of disease directly after administration, substantiating this critical immunomodulatory signaling. More importantly, it was only the EAE mice treated with SVF cells that were able to overcome the advancing pathogenesis and showed improvements by the end of the study. The frequency of lesions in spinal cords following SVF treatment correlated with diminished activities of the T helper type 1 cells, known effector cells of this disease. Co-cultures with splenocytes isolated from EAE mice revealed transcripts of interleukin-10 and transforming growth factor-ß, known promoters of regulatory T cells, that were greatly expressed in SVF cells compared to ASCs, and expression levels of signaling mediators related to effector T cells were insignificant in both SVF cells and ASCs. CONCLUSION: This is the first evidence, to date, to elucidate a mechanism of action of SVF treatment in an inflammatory, autoimmune disease. Our data supports key immunomodulatory signaling between cell therapies and T cells in this T cell-mediated disease. Together, treatment with SVF mediated immunomodulatory effects that diminished effector cell activities, promoted regulatory T cells, and reduced neuroinflammation.
Assuntos
Tecido Adiposo/citologia , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Células Estromais/fisiologia , Frações Subcelulares/fisiologia , Células Th1/patologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Técnicas de Cocultura , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores Imunológicos/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Células Estromais/ultraestruturaRESUMO
Renal transplantation is currently the most effective treatment for end-stage renal disease, which represents one of the major current public health problems. However, the number of available donor kidneys is drastically insufficient to meet the demand, causing prolonged waiting lists. For this reason, tissue engineering offers great potential to increase the pool of donated organs for kidney transplantation, by way of seeding cells on supporting scaffolding material. Biological scaffolds are prepared by removing cellular components from the donor organs using a decellularization process with detergents, enzymes or other cell lysing solutions. Extracellular matrix which makes up the scaffold is critical to directing the cell attachment and to creating a suitable environment for cell survival, proliferation and differentiation. Researchers are now studying whole intact scaffolds produced from the kidneys of animals or humans without adversely affecting extracellular matrix, biological activity and mechanical integrity. The process of recellularization includes cell seeding strategies and the choice of the cell source to repopulate the scaffold. This is the most difficult phase, due to the complexity of the kidney. Indeed, no studies have provided sufficient results of complete renal scaffold repopulation and differentiation. This review summarizes the research that has been conducted to obtain decellularized kidney scaffolds and to repopulate the scaffolds, evaluating the best cell sources, the cell seeding methods and the cell differentiation in kidney scaffolds.
Assuntos
Matriz Extracelular/fisiologia , Rim , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Diferenciação Celular , Matriz Extracelular/química , Humanos , Rim/citologia , Rim/patologia , Técnicas de Cultura de Órgãos , Células-Tronco/citologia , Células-Tronco/fisiologia , Frações Subcelulares/química , Frações Subcelulares/fisiologiaRESUMO
Spatial-temporal regulation among proteins forms dynamic networks in cells. Coexistence in common cell compartments can improve biological reliability of the protein-protein interactions. However, this is usually overlooked by most proteomic studies and leads to unrealistic discoveries. In this paper, we systematically characterize the interaction localization diversity in the human protein interactome using the localization coefficient, a novel metric proposed for assessing how diversely the interactions localize among cell compartments. Our analysis reveals the following: (1) the subcellular networks of the nucleus, cytosol, and mitochondrion are dense but the interactions tend to localize in specific cell compartments, whereas the subnetworks of the secretory-pathway, membrane, and extracellular region are sparse but the interactions are diversely localized; (2) the housekeeping proteins tend to appear in multiple compartments, while the tissue-specific proteins present a relatively flat profile of localization breadth; (3) the autophagy proteins tend to diversely localize in multiple compartments, especially those with high connectivity, compared with the apoptosis proteins; (4) the proteins targeted by small-molecule drugs show no preference for compartments, whereas the proteins directed by antibody-based drugs tend to belong to transmembrane regions with a strong diversity. In summary, our analysis provides a comprehensive view of the subcellular localization for interacting proteins, demonstrates that localization diversity is an important feature of protein interactions, and shows its ability to highlight meaningful biological functions.
Assuntos
Compartimento Celular , Mapas de Interação de Proteínas/fisiologia , Proteoma/análise , Frações Subcelulares/química , Humanos , Espaço Intracelular/química , Mapeamento de Interação de Proteínas , Proteômica/métodos , Análise Espaço-Temporal , Frações Subcelulares/fisiologiaRESUMO
Myelin formation has been identified as a modulator of neural plasticity. New tools are required to investigate the mechanisms by which environmental inputs and neural activity regulate myelination patterns. In this study, we demonstrate a microfluidic compartmentalized culture system with integrated electrical stimulation capabilities that can induce neural activity by whole cell and focal stimulation. A set of electric field simulations was performed to confirm spatial restriction of the electrical input in the compartmentalized culture system. We further demonstrate that electrode localization is a key consideration for generating uniform the stimulation of neuron and oligodendrocytes within the compartments. Using three configurations of the electrodes we tested the effects of subcellular activation of neural activity on distal axon myelination with oligodendrocytes. We further investigated if oligodendrocytes have to be exposed to the electrical field to induce axon myelination. An isolated stimulation of cell bodies and proximal axons had the same effect as an isolated stimulation of distal axons co-cultured with oligodendrocytes, and the two modes had a non-different result than whole cell stimulation. Our platform enabled the demonstration that electrical stimulation enhances oligodendrocyte maturation and myelin formation independent of the input localization and oligodendrocyte exposure to the electrical field.
Assuntos
Axônios/fisiologia , Estimulação Elétrica , Neurônios/fisiologia , Oligodendroglia/fisiologia , Animais , Microfluídica , Frações Subcelulares/fisiologiaRESUMO
While various material factors have been shown to influence cell behaviors, recent studies started to pay attention to the effects of some material cues on "subcellular" geometry of cells, such as self-deformation of cell nuclei. It is particularly interesting to examine whether a self deformation happens discontinuously like a first-order transition and whether subcellular geometry influences significantly the extent of stem cell differentiation. Herein we prepared a series of micropillar arrays of poly(lactide-co-glycolide) and discovered a first-order transition of nuclear shape as a function of micropillar height under the examined section area and interspacing of the pillars. The deformed state of the nuclei of mesenchymal stem cells (MSCs) was well maintained even after osteogenic or adipogenic induction for several days. The nuclear deformation on the micropillar arrays was accompanied with smaller projected areas of cells, but led to an enhanced osteogenesis and attenuated adipogenesis of the MSCs, which is different from the previously known relationship between morphology and differentiation of stem cells on flat substrates. Hence, the present study reveals that the geometry of cell nuclei may afford a new cue to regulate the lineage commitment of stem cells on the subcellular level.
Assuntos
Materiais Biocompatíveis/química , Diferenciação Celular/fisiologia , Mecanotransdução Celular/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Frações Subcelulares/fisiologia , Frações Subcelulares/ultraestrutura , Animais , Animais Recém-Nascidos , Tamanho Celular , Células Cultivadas , Força Compressiva/fisiologia , Módulo de Elasticidade/fisiologia , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Propriedades de SuperfícieRESUMO
Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines.
Assuntos
Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Gentamicinas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Frações Subcelulares/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Gentamicinas/efeitos adversos , Células LLC-PK1 , Chaperonas Moleculares/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Frações Subcelulares/metabolismo , Suínos , Proteína Supressora de Tumor p53/genéticaRESUMO
BK channels are important for the regulation of many cell functions. The significance of plasma membrane BK channels in the control of action potentials, resting membrane potential, and neurotransmitter release is well established; however, the composition and functions of mitochondrial and nuclear BK (nBK) channels are largely unknown. In this chapter, we summarize the recent findings on the subcellular localization, biophysical, and pharmacological properties of mitochondrial and nBK channels and discuss their molecular identity and physiological functions.
Assuntos
Potenciais de Ação/fisiologia , Núcleo Celular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Mitocôndrias/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Frações Subcelulares/fisiologiaRESUMO
The SNARE complex composed of VAMP727, SYP22, VTI11 and SYP51 is critical for protein trafficking and PSV biogenesis in Arabidopsis. This SNARE complex directs the fusion between the prevacuolar compartment (PVC) and the vacuole, and thus mediates protein trafficking to the vacuole. In this study, we examined the role of AtNHX5 and AtNHX6 in regulating this SNARE complex and its function in protein trafficking. We found that AtNHX5 and AtNHX6 were required for seed production, protein trafficking and PSV biogenesis. We further found that the nhx5 nhx6 syp22 triple mutant showed severe defects in seedling growth and seed development. The triple mutant had short siliques and reduced seed sets, but larger seeds. In addition, the triple mutant had numerous smaller protein storage vacuoles (PSVs) and accumulated precursors of the seed storage proteins in seeds. The PVC localization of SYP22 and VAMP727 was repressed in nhx5 nhx6, while a significant amount of SYP22 and VAMP727 was trapped in the Golgi or TGN in nhx5 nhx6. AtNHX5 and AtNHX6 were co-localized with SYP22 and VAMP727. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for the transport of the storage proteins, indicating the importance of exchange activity in protein transport. AtNHX5 or AtNHX6 did not interact physically with the SNARE complex. Taken together, AtNHX5 and AtNHX6 are required for the PVC localization of the SNARE complex and hence its function in protein transport. AtNHX5 and AtNHX6 may regulate the subcellular localization of the SNARE complex by their transport activity.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Transporte Proteico/fisiologia , Proteínas SNARE/fisiologia , Sementes/metabolismo , Proteínas Qa-SNARE/fisiologia , Proteínas Qb-SNARE/fisiologia , Frações Subcelulares/fisiologia , Vacúolos/fisiologiaRESUMO
The OsLpa1 gene (LOC_Os57400) was identified to be involved in phytic acid (PA) metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter::GUS transgenic plants (GUS: ß-glucuronidase); GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts (OsLpa1.1, OsLpa1.2, OsLpa1.3) are produced due to alternative splicing; quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa all tissues. OsLpa1.2 is predominant in germinating seeds (about 5 times that of OsLpa1.1), but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF (day after flowering) seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein (GFP) in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.
Assuntos
Processamento Alternativo/fisiologia , Oryza/fisiologia , Ácido Fítico/metabolismo , Proteínas de Plantas/fisiologia , Frações Subcelulares/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/ultraestrutura , Plantas Geneticamente Modificadas/fisiologia , Análise Espaço-Temporal , Frações Subcelulares/ultraestrutura , Distribuição TecidualRESUMO
The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) modulates the expression of many genes, primarily through activation of NF-κB. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-α-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-α-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-α on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-α was transient and independent of NF-κB. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-α treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing.
Assuntos
Proteínas Nucleares/metabolismo , Splicing de RNA/fisiologia , RNA/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , DNA Polimerase II/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Frações Subcelulares/fisiologia , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The Auger-electrons emitted by (99m)Tc have been recently associated with the induction of thyroid stunning in in vivo experiments in mice, making the dosimetry at the sub-cellular level of (99m)Tc a pertinent and pressing subject. The S-values for (99m)Tc were calculated using MCNP6, which was first validated for studies at the sub-cellular scale and for low energies electrons. The calculation was then performed for (99m)Tc within different cellular compartments in a single mouse thyroid follicle model, considering the radiative and non-radiative transitions of the (99m)Tc radiation spectrum. It was shown that the contribution of the (99m)Tc Auger and low energy electrons to the absorbed dose to the follicular cells' nucleus is important, being at least of the same order of magnitude compared to the emitted photons' contribution and cannot be neglected. The results suggest that Auger-electrons emitted by (99m)Tc play a significant role in the occurrence of the thyroid stunning effect in mice.
Assuntos
Elétrons , Modelos Estatísticos , Radiometria/métodos , Frações Subcelulares/fisiologia , Tecnécio/análise , Glândula Tireoide/fisiologia , Absorção de Radiação/fisiologia , Animais , Simulação por Computador , Camundongos , Modelos Biológicos , Método de Monte Carlo , Doses de Radiação , Glândula Tireoide/ultraestruturaRESUMO
Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Rastreamento de Células/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Caenorhabditis elegans/embriologia , Forma Celular/fisiologia , Rastreamento de Células/instrumentação , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Fenótipo , Software , Frações Subcelulares/fisiologia , Imagem com Lapso de Tempo/métodosRESUMO
This paper presents a framework for modelling biological tissues based on discrete particles. Cell components (e.g. cell membranes, cell cytoskeleton, cell nucleus) and extracellular matrix (e.g. collagen) are represented using collections of particles. Simple particle to particle interaction laws are used to simulate and control complex physical interaction types (e.g. cell-cell adhesion via cadherins, integrin basement membrane attachment, cytoskeletal mechanical properties). Particles may be given the capacity to change their properties and behaviours in response to changes in the cellular microenvironment (e.g., in response to cell-cell signalling or mechanical loadings). Each particle is in effect an 'agent', meaning that the agent can sense local environmental information and respond according to pre-determined or stochastic events. The behaviour of the proposed framework is exemplified through several biological problems of ongoing interest. These examples illustrate how the modelling framework allows enormous flexibility for representing the mechanical behaviour of different tissues, and we argue this is a more intuitive approach than perhaps offered by traditional continuum methods. Because of this flexibility, we believe the discrete modelling framework provides an avenue for biologists and bioengineers to explore the behaviour of tissue systems in a computational laboratory.