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1.
Adv Exp Med Biol ; 1131: 7-26, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646505

RESUMO

Measuring free Ca2+ concentration ([Ca2+]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators coupled with the relative ease of transfecting cell lines with biological Ca2+ sensors have led to the situation where cellular and subcellular [Ca2+] is examined by many non-specialists. In this chapter, we evaluate the most used Ca2+ indicators and highlight what their major advantages and disadvantages are. We stress the potential pitfalls of non-ratiometric techniques for measuring Ca2+ and the clear advantages of ratiometric methods. Likely improvements and new directions for Ca2+ measurement are discussed.


Assuntos
Cálcio , Citosol , Organelas , Animais , Cálcio/metabolismo , Técnicas Citológicas , Citosol/química , Citosol/metabolismo , Humanos , Organelas/química , Organelas/metabolismo
3.
Anticancer Res ; 39(7): 3395-3404, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262861

RESUMO

Although modern anticancer drugs have made great progress in disease treatment, the occurrence of drug resistance often leads to treatment failure. Understanding the molecular basis of resistance mechanisms is important to determine prognosis and develop strategies for circumvention. In this context, subcellular vesicles released by cancer cells have been identified to mediate cellular resistance by various mechanisms. Such extracellular vesicles (EVs) can be subdivided into exosomes and ectosomes based on their size, cargo, and mechanism of formation. The unveiling of EV-targeted treatment options depends on a sound knowledge on EV biology including biogenesis, release, targeting to recipient cells, and uptake. In this review, we focus on EVs as mediators of cancer drug resistance with a particular emphasis on the distinction of exosomes and ectosomes.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Vesículas Extracelulares , Neoplasias/tratamento farmacológico , Antineoplásicos/uso terapêutico , Humanos , Organelas
4.
Environ Pollut ; 251: 910-920, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31234257

RESUMO

The toxic sensitivity in different physiological levels of chromium (Cr) contaminated soils with environmentally equivalent concentrations (EEC) was fully unknown. The earthworm Eisenia fetida was exposed to a Cr-contaminated soil at the EEC level (referred to as Cr-CS) to characterize the induced toxicity at the whole body, organ, tissue, subcellular structure and metabolic levels. The results showed that the survival rate, weight and biodiversity of the gut microorganisms (organ) had no significant difference (p > 0.05) between control and Cr-CS groups. Qualitative histopathological and subcellular evaluations from morphology showed earthworms obvious injuries. The organelle injuries combined with the metabolic changes provided additional evidence that the Cr-CS damaged the nucleus and probably disturbed the nucleic acid metabolism of earthworms. 2-hexyl-5-ethyl-3-furansulfonate, dimethylglycine, betaine and scyllo-inositol were sensitive and relatively quantitative metabolites that were recommended as potential biomarkers for Cr-CS based on their significant weights in the multivariate analysis model. In addition, the relative abundance of Burkholderiaceae, Enterobacteriaceae and Microscillaceae of the earthworm guts in the Cr-CS group significantly increased, particularly for Burkholderiaceae (increased by 13.1%), while that of Aeromonadaceae significantly decreased by 5.6% in contrast with the control group. These results provided new insights into our understanding of the toxic effects of the EEC level of Cr contaminated soil from different physiological levels of earthworms and extend our knowledge on the composition and sensitivity of the earthworm gut microbiota in Cr contaminated soil ecosystems. Furthermore, these toxic responses from gut microorganisms to metabolites of earthworms provided important data to improve the adverse outcome pathway and toxic mechanism of the Cr-CS if the earthworm genomics and proteomics would be also gained in the future.


Assuntos
Cromo/toxicidade , Poluição Ambiental/análise , Oligoquetos/metabolismo , Oligoquetos/microbiologia , Poluentes do Solo/toxicidade , Solo/química , Animais , Bacteroidetes/crescimento & desenvolvimento , Burkholderiaceae/crescimento & desenvolvimento , Cromo/análise , Enterobacteriaceae/crescimento & desenvolvimento , Organelas/efeitos dos fármacos , Poluentes do Solo/análise
5.
Genes Dev ; 33(13-14): 799-813, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171700

RESUMO

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Complexo Repressor Polycomb 1/química , Animais , Linhagem Celular , Escherichia coli/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Organelas/metabolismo , Mutação Puntual , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Domínios Proteicos , Células Sf9
6.
Nat Neurosci ; 22(7): 1099-1109, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235907

RESUMO

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.


Assuntos
Membranas Intracelulares/ultraestrutura , Corpos de Lewy/ultraestrutura , Doença por Corpos de Lewy/patologia , Lipídeos de Membrana/análise , Organelas/ultraestrutura , Doença de Parkinson/patologia , alfa-Sinucleína/análise , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Hipocampo/química , Hipocampo/ultraestrutura , Humanos , Imagem Tridimensional , Corpos de Lewy/química , Doença por Corpos de Lewy/metabolismo , Mesencéfalo/química , Mesencéfalo/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica/métodos , Microscopia de Fluorescência , Doença de Parkinson/metabolismo , Substância Negra/química , Substância Negra/ultraestrutura , Sequenciamento Completo do Exoma
7.
Anal Chim Acta ; 1073: 79-89, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31146839

RESUMO

We investigated the effect of oxidative stress (OS) on lipidomic perturbations in the subcellular fractions and exosomes of human embryonic kidney (HEK) 293 cells using asymmetrical flow field-flow fractionation (AF4) and nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). We treated HEK 293 cells with hydrogen peroxide (H2O2) and fractionated the cell lysates using AF4 to determine the change in size and population of the subcellular fractions and exosomes, and to obtain narrow size fractions for lipid analysis. A total of 438 lipids from 642 identified species-including oxidized lipids-were quantified. The relative amount of secreted exosomes increased by 28% during OS, whereas the amount of subcellular species decreased by 35%. There was a significant increase in the level of oxidized phospholipids in the mitochondrion-enriched subcellular fractions, but not in the exosomes. Most high-abundance triacylglycerol (TG) species increased in the stressed cells, whereas they decreased in the exosomes. During OS, ceramides involved in the apoptotic mitochondrial pathway were accumulated in the subcellular fractions, whereas their levels were unaffected in the exosomes. The present study demonstrated that AF4 and nUHPLC-ESI-MS/MS can be used to investigate lipid alterations in subcellular and extracellular species during OS, and the pathological relationships in diseases caused by reactive oxygen species.


Assuntos
Exossomos/metabolismo , Fracionamento por Campo e Fluxo , Lipídeos/análise , Organelas/metabolismo , Estresse Oxidativo , Cromatografia Líquida de Alta Pressão , Exossomos/química , Células HEK293 , Humanos , Organelas/química , Espectrometria de Massas em Tandem
8.
Nat Commun ; 10(1): 2878, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253789

RESUMO

Brassica napus, an allotetraploid crop, is hypothesized to be a hybrid from unknown varieties of Brassica rapa and Brassica oleracea. Despite the economic importance of B. napus, much is unresolved regarding its phylogenomic relationships, genetic structure, and diversification. Here we conduct a comprehensive study among diverse accessions from 183 B. napus (including rapeseed, rutabaga, and Siberian kale), 112 B. rapa, and 62 B. oleracea and its wild relatives. Using RNA-seq of B. napus accessions, we define the genetic diversity and sub-genome variance of six genetic clusters. Nuclear and organellar phylogenies for B. napus and its progenitors reveal varying patterns of inheritance and post-formation introgression. We discern regions with signatures of selective sweeps and detect 8,187 differentially expressed genes with implications for B. napus diversification. This study highlights the complex origin and evolution of B. napus providing insights that can further facilitate B. napus breeding and germplasm preservation.


Assuntos
Brassica napus/genética , Brassica napus/metabolismo , Ploidias , Regulação da Expressão Gênica de Plantas , Genômica , Organelas , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Tubérculos , Polimorfismo de Nucleotídeo Único , RNA de Plantas/genética , Análise de Sequência de RNA , Transcriptoma
9.
Nat Neurosci ; 22(7): 1043-1045, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235905
10.
Nat Chem Biol ; 15(6): 589-597, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086330

RESUMO

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.


Assuntos
Luz , Engenharia Metabólica , Redes e Vias Metabólicas/efeitos da radiação , Optogenética/métodos , Organelas/metabolismo , Organelas/efeitos da radiação , Biologia Sintética , Indóis/metabolismo , Organelas/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Synechocystis/efeitos da radiação
11.
Plant Cell Rep ; 38(7): 803-818, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079194

RESUMO

Plant cells are characterized by a unique group of interconvertible organelles called plastids, which are descended from prokaryotic endosymbionts. The most studied plastid type is the chloroplast, which carries out the ancestral plastid function of photosynthesis. During the course of evolution, plastid activities were increasingly integrated with cellular metabolism and functions, and plant developmental processes, and this led to the creation of new types of non-photosynthetic plastids. These include the chromoplast, a carotenoid-rich organelle typically found in flowers and fruits. Here, we provide an introduction to non-photosynthetic plastids, and then review the structures and functions of chromoplasts in detail. The role of chromoplast differentiation in fruit ripening in particular is explored, and the factors that govern plastid development are examined, including hormonal regulation, gene expression, and plastid protein import. In the latter process, nucleus-encoded preproteins must pass through two successive protein translocons in the outer and inner envelope membranes of the plastid; these are known as TOC and TIC (translocon at the outer/inner chloroplast envelope), respectively. The discovery of SP1 (suppressor of ppi1 locus1), which encodes a RING-type ubiquitin E3 ligase localized in the plastid outer envelope membrane, revealed that plastid protein import is regulated through the selective targeting of TOC complexes for degradation by the ubiquitin-proteasome system. This suggests the possibility of engineering plastid protein import in novel crop improvement strategies.


Assuntos
Cloroplastos/metabolismo , Plastídeos/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/genética , Organelas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmídeos/genética , Plastídeos/genética , Transporte Proteico
12.
PLoS Pathog ; 15(5): e1007670, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31121005

RESUMO

To elicit effective invasion and egress from infected cells, obligate intracellular parasites of the phylum Apicomplexa rely on the timely and spatially controlled exocytosis of specialized secretory organelles termed the micronemes. The effector molecules and signaling events underpinning this process are intricate; however, recent advances within the field of Toxoplasma gondii research have facilitated a broader understanding as well as a more integrated view of this complex cascade of events and have unraveled the importance of phosphatidic acid (PA) as a lipid mediator at multiple steps in this process.


Assuntos
Cálcio/metabolismo , GMP Cíclico/metabolismo , Exocitose/fisiologia , Organelas/metabolismo , Ácidos Fosfatídicos/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Animais , Interações Hospedeiro-Parasita , Humanos , Organelas/parasitologia , Transporte Proteico , Proteínas de Protozoários/metabolismo , Transdução de Sinais
13.
Anal Chim Acta ; 1068: 60-69, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31072478

RESUMO

Three hydrogen sulfide (H2S) probes based on an azonia-cyanine skeleton were successfully designed and prepared. Probe 1a, containing 4-chloro-7-nitro-1,2,3-benzoxadiazole connected to the cyanine dye, had an emission at 660 nm that was enhanced 4.5-fold by the reduced photoinduced electron transfer process when reacting with H2S. Probes 1b and 1c were constructed from cyanine dyes with electron withdrawing 2,4-dinitrophenyl and 7-nitrobenzo[c] [1,2,5]oxadiazol-4-yl groups, respectively. Probes 1b and 1c gave off-on type responses with 169- and 17-fold fluorescent enhancements at 639 nm with H2S. Their emission properties were influenced by intramolecular hydrogen bonds and intramolecular charge transfer processes. The detection limits of probes 1a-1c were calculated at 178, 121, and 9.6 nM, respectively. The intracellular imaging experiments with HeLa cells indicated probe 1a was a mitochondria-targeting H2S probe, while probes 1b and 1c were lysosome-targeting H2S probes.


Assuntos
Carbocianinas/química , Corantes Fluorescentes/química , Sulfeto de Hidrogênio/análise , Imagem Óptica , Organelas/química , Carbocianinas/síntese química , Teoria da Densidade Funcional , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura Molecular , Células Tumorais Cultivadas
14.
J Plant Res ; 132(3): 431-438, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30980216

RESUMO

Euglena gracilis has an organelle resembling hematochrome, with an appearance similar to the eyespot and the absorption band spectrally overlapped with that of the carotenoid. To discriminate the hematochrome-like granules and eyespot, scan-free, non-invasive, absorbance spectral imaging A(x, y, λ) microscopy of single live cells, where A(x, y, λ) means absorbance at a position (x, y) on a two-dimensional image at a specific wavelength λ was applied. This technique was demonstrated to be a powerful tool for basic research on intracellular structural analysis. By this method, characteristic absorption spectra specific to the hematochrome-like granule or eyespot were identified among a variety of spectra observed depending on the location inside the organelles. The hematochrome-like granule was dark orange and deep green in its outline and had a characteristic absorption peak at 620 nm as well as at 676 to 698 nm, suggesting that its origin is a component of chloroplast including chlorophyll a. Furthermore, the representative spectra of these organelles were derived by principal component analysis of the absorbance and its position in absorbance image, indicating that they can be distinguished from each other and other regions. It was also confirmed that even in areas where these organelles and chloroplasts overlap, one can distinguish them from each other. The present research clarified the absorption spectra of the eyespot with 1 × 1 µm spatial resolution and those unpublished of hematochrome-like granules of E. gracilis, and indicated that one can statistically distinguish these organelles by this method.


Assuntos
Euglena gracilis/metabolismo , Organelas/metabolismo , Animais , Euglena gracilis/fisiologia , Microscopia Intravital , Microespectrofotometria , Organelas/fisiologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/fisiologia
15.
Cell Mol Life Sci ; 76(20): 4117-4130, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31028425

RESUMO

Intracellular traffic amongst organelles represents a key feature for eukaryotes and is orchestrated principally by members of Rab family, the largest within Ras superfamily. Given that variations in Rab repertoire have been fundamental in animal diversification, we provided the most exhaustive survey regarding the Rab toolkit of chordates. Our findings reveal the existence of 42 metazoan conserved subfamilies exhibiting a univocal intron/exon structure preserved from cnidarians to vertebrates. Since the current view does not capture the Rab complexity, we propose a new Rab family classification in three distinct monophyletic clades. The Rab complement of chordates shows a dramatic diversification due to genome duplications and independent gene duplications and losses with sharp differences amongst cephalochordates, tunicates and gnathostome vertebrates. Strikingly, the analysis of the domain architecture of this family highlighted the existence of chimeric calcium-binding Rabs, which are animal novelties characterized by a complex evolutionary history in gnathostomes and whose role in cellular metabolism is obscure. This work provides novel insights in the knowledge of Rab family: our hypothesis is that chordates represent a hotspot of Rab variability, with many events of gene gains and losses impacting intracellular traffic capabilities. Our results help to elucidate the role of Rab members in the transport amongst endomembranes and shed light on intracellular traffic routes in vertebrates. Then, since the predominant role of Rabs in the molecular communication between different cellular districts, this study paves to way to comprehend inherited or acquired human disorders provoked by dysfunctions in Rab genes.


Assuntos
Evolução Biológica , Cordados/genética , Genoma , Família Multigênica , Filogenia , Proteínas rab de Ligação ao GTP/genética , Animais , Transporte Biológico , Cordados/classificação , Bases de Dados Genéticas , Éxons , Duplicação Gênica , Variação Genética , Humanos , Íntrons , Organelas/genética , Organelas/metabolismo , Domínios Proteicos , Sintenia , Proteínas rab de Ligação ao GTP/classificação , Proteínas rab de Ligação ao GTP/metabolismo
16.
Cell Biol Int ; 43(7): 728-738, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30969020

RESUMO

This review summarizes original and literature data on changes in the ultrastructure of major cell organelles during apoptosis obtained by transmission electron microscopy. Organelles that make the most crucial contribution to the initiation of apoptosis: plasma membrane, mitochondria, proteasomes, Golgi apparatus, and endoplasmic reticulum, were of our prime attention. The nucleus and cytoskeleton that undergo essential changes, were considered as well. Special attention was paid to the data on ultrastructural changes in the cell organelles observed recently by electron microscopic tomography and correlative microscopy, in particular, to remodeling of mitochondrial crista junctions and microtubules during the execution phase of apoptosis.


Assuntos
Apoptose , Organelas/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Células U937
17.
Methods Mol Biol ; 1945: 251-264, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945250

RESUMO

This chapter describes the procedures necessary to create generative models of the spatial organization of cells directly from microscope images and use them to automatically provide geometries for spatial simulations of cell processes and behaviors. Such models capture the statistical variation in the overall cell architecture as well as the number, shape, size, and spatial distribution of organelles and other structures. The different steps described include preparing images, learning models, evaluating model quality, creating sampled cell geometries by various methods, and combining those geometries with biochemical model specifications to enable simulations.


Assuntos
Células/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Simulação por Computador , Humanos , Modelos Biológicos , Organelas/ultraestrutura
18.
Proc Natl Acad Sci U S A ; 116(15): 7343-7352, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918125

RESUMO

Mechanoreceptive organelles (MOs) are specialized subcellular entities in mechanoreceptors that transform extracellular mechanical stimuli into intracellular signals. Their ultrastructures are key to understanding the molecular nature and mechanics of mechanotransduction. Campaniform sensilla detect cuticular strain caused by muscular activities or external stimuli in Drosophila Each campaniform sensillum has an MO located at the distal tip of its dendrite. Here we analyzed the molecular architecture of the MOs in fly campaniform mechanoreceptors using electron microscopic tomography. We focused on the ultrastructural organization of NompC (a force-sensitive channel) that is linked to the array of microtubules in these MOs via membrane-microtubule connectors (MMCs). We found that NompC channels are arranged in a regular pattern, with their number increasing from the distal to the proximal end of the MO. Double-length MMCs in nompC 29+29ARs confirm the ankyrin-repeat domain of NompC (NompC-AR) as a structural component of MMCs. The unexpected finding of regularly spaced NompC-independent linkers in nompC 3 suggests that MMCs may contain non-NompC components. Localized laser ablation experiments on mechanoreceptor arrays in halteres suggest that MMCs bear tension, providing a possible mechanism for why the MMCs are longer when NompC-AR is duplicated or absent in mutants. Finally, mechanical modeling shows that upon cuticular deformation, sensillar architecture imposes a rotational activating force, with the proximal end of the MO, where more NOMPC channels are located, being subject to larger forces than the distal end. Our analysis reveals an ultrastructural pattern of NompC that is structurally and mechanically optimized for the sensory functions of campaniform mechanoreceptors.


Assuntos
Proteínas de Drosophila , Mecanorreceptores , Mecanotransdução Celular , Organelas , Canais de Receptores Transientes de Potencial , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Mecanorreceptores/química , Mecanorreceptores/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Organelas/química , Organelas/genética , Organelas/metabolismo , Canais de Receptores Transientes de Potencial/química , Canais de Receptores Transientes de Potencial/genética , Canais de Receptores Transientes de Potencial/metabolismo
20.
J Biotechnol ; 297: 19-27, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-30902643

RESUMO

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Vetores Genéticos/metabolismo , Proteínas Luminescentes/metabolismo , Organelas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas
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