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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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
Biol Cell ; 111(8): 199-212, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30905068

RESUMO

Motile cilia of epithelial multiciliated cells transport vital fluids along organ lumens to promote essential respiratory, reproductive and brain functions. Progenitors of multiciliated cells undergo massive and coordinated organelle remodelling during their differentiation for subsequent motile ciliogenesis. Defects in multiciliated cell differentiation lead to severe cilia-related diseases by perturbing cilia-based flows. Recent work designated the machinery of mitosis as the orchestrator of the orderly progression of differentiation associated with multiple motile cilia formation. By examining the events leading to motile ciliogenesis with a methodological prism of mitosis, we contextualise and discuss the recent findings to broaden the spectrum of questions related to the differentiation of mammalian multiciliated cells.


Assuntos
Centríolos/metabolismo , Cílios/fisiologia , Células Epiteliais , Mitose/fisiologia , Organelas/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Transformação Celular Neoplásica , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Camundongos , Leveduras/metabolismo
11.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923194

RESUMO

Nature regulates interference between cellular processes-allowing more complexity of life-by confining specific functions to organelles. Inspired by this concept, we designed an artificial organelle dedicated to protein engineering. We generated a membraneless organelle to translate only one type of messenger RNA-by recruiting an RNA-targeting system, stop codon-suppression machinery, and ribosomes-by means of phase separation and spatial targeting. This enables site-specific protein engineering with a tailored noncanonical function in response to one specific codon in the entire genome only in the protein of choice. Our results demonstrate a simple yet effective approach to the generation of artificial organelles that provides a route toward customized orthogonal translation and protein engineering in semisynthetic eukaryotic cells.


Assuntos
Códon/genética , Código Genético , Organelas/metabolismo , Organelas/ultraestrutura , Biossíntese de Proteínas/genética , Engenharia de Proteínas/métodos , RNA Mensageiro/genética , Animais , Células COS , Caenorhabditis elegans/genética , Membrana Celular , Cercopithecus aethiops , Células HEK293 , Humanos , Lisina/análogos & derivados , Lisina/genética , Methanosarcina , Organelas/química , RNA de Transferência/química , Ribossomos/química , Biologia Sintética
12.
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
13.
Nat Commun ; 10(1): 1287, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894536

RESUMO

Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.


Assuntos
Membrana Celular/metabolismo , Células Eucarióticas/metabolismo , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Terminologia como Assunto , Animais , Fracionamento Celular/métodos , Membrana Celular/ultraestrutura , Células Eucarióticas/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Microscopia/instrumentação , Microscopia/métodos , Organelas/ultraestrutura , Proteínas/genética , Proteínas/metabolismo , Coloração e Rotulagem/métodos
14.
Nat Commun ; 10(1): 1325, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902985

RESUMO

Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.


Assuntos
Células Artificiais/metabolismo , Fotossíntese , Biossíntese de Proteínas , Trifosfato de Adenosina/metabolismo , Células Artificiais/efeitos dos fármacos , Metabolismo Energético/efeitos da radiação , Luz , Organelas/metabolismo , Organelas/efeitos da radiação , Fotossíntese/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Lipossomas Unilamelares/metabolismo
15.
Opt Lett ; 44(6): 1359-1362, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874650

RESUMO

The health of a eukaryotic cell depends on the proper functioning of its cell organelles. Characterizing these nanometer- to micrometer-scaled specialized subunits without disturbing the cell is challenging but can also provide valuable insights regarding the state of a cell. We show that objective-based scanning surface plasmon resonance microscopy can be used to analyze the refractive index of cell organelles quantitatively in a noninvasive and label-free manner with a lateral resolution at the diffraction limit.


Assuntos
Microscopia/métodos , Organelas/metabolismo , Refratometria/métodos , Ressonância de Plasmônio de Superfície , Animais , Neurônios/citologia , Ratos
16.
Photochem Photobiol Sci ; 18(5): 1212-1217, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30834414

RESUMO

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.


Assuntos
Técnicas Biossensoriais , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Organelas/metabolismo , Animais , Células COS , Cercopithecus aethiops , Vaga-Lumes , Concentração de Íons de Hidrogênio , Organelas/química
17.
Methods Mol Biol ; 1920: 265-275, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737696

RESUMO

The Balbiani body (Bb) is a large membrane-less organelle, densely packed with mitochondria, endoplasmic reticulum, proteins, and RNA. The Bb is present in many vertebrate female gametes. In frogs, the Bb is established early during oogenesis and operates as a maternal inherited embryonic determinant that specifies germline identity through the formation of germplasm. We describe here two techniques to isolate the Bb/germplasm from Xenopus laevis primary oocytes.


Assuntos
Fracionamento Celular , Oócitos/metabolismo , Oogênese , Organelas/metabolismo , Xenopus laevis , Animais , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração , Células Germinativas/metabolismo , Mitocôndrias/metabolismo , Oogênese/genética
18.
Methods Mol Biol ; 1920: 295-302, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737698

RESUMO

Proteomic characterization of isolated organelles can provide insight into the functional components of the structure and novel targets for further testing. Germplasm in developing oocytes is difficult to isolate for protein identification because not all types of germplasm are stable outside of the cytoplasm. In zebrafish, the Balbiani body forms a proteinaceous aggregate that contains the germplasm and we found is stable outside of the oocyte. Here we present a manual isolation protocol that collects intact Balbiani bodies from stage I zebrafish oocytes. We lysed oocytes by passing them through a syringe, and then used a fine injection needle to wick up Balbiani bodies by capillary action with minimal buffer solution. Using this protocol we collected sufficient material for proteomic analysis of the zebrafish Balbiani body.


Assuntos
Fracionamento Celular , Oócitos/metabolismo , Organelas/metabolismo , Proteoma , Proteômica , Peixe-Zebra/metabolismo , Animais , Biomarcadores , Fracionamento Celular/métodos , Imunofluorescência/métodos , Oogênese
19.
Int J Mol Sci ; 20(3)2019 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-30744118

RESUMO

Ion channels are transmembrane proteins that conduct specific ions across biological membranes. Ion channels are present at the onset of many cellular processes, and their malfunction triggers severe pathologies. Potassium channels (KChs) share a highly conserved signature that is necessary to conduct K⁺ through the pore region. To be functional, KChs require an exquisite regulation of their subcellular location and abundance. A wide repertoire of signatures facilitates the proper targeting of the channel, fine-tuning the balance that determines traffic and location. These signature motifs can be part of the secondary or tertiary structure of the protein and are spread throughout the entire sequence. Furthermore, the association of the pore-forming subunits with different ancillary proteins forms functional complexes. These partners can modulate traffic and activity by adding their own signatures as well as by exposing or masking the existing ones. Post-translational modifications (PTMs) add a further dimension to traffic regulation. Therefore, the fate of a KCh is not fully dependent on a gene sequence but on the balance of many other factors regulating traffic. In this review, we assemble recent evidence contributing to our understanding of the spatial expression of KChs in mammalian cells. We compile specific signatures, PTMs, and associations that govern the destination of a functional channel.


Assuntos
Ativação do Canal Iônico , Canais de Potássio/metabolismo , Animais , Transporte Biológico , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Organelas/metabolismo , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transdução de Sinais
20.
Methods Mol Biol ; 1941: 201-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707436

RESUMO

Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.


Assuntos
Biomarcadores/metabolismo , Encéfalo/metabolismo , Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Organelas/metabolismo , Receptores de Glutamato/metabolismo , Frações Subcelulares/metabolismo , Humanos
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