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1.
Nat Commun ; 10(1): 5725, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844058

RESUMO

Many traits vary among isogenic individuals in homogeneous environments. In microbes, plants and animals, variation in the protein chaperone system affects many such traits. In the animal model C. elegans, the expression level of hsp-16.2 chaperone biomarkers correlates with or predicts the penetrance of mutations and lifespan after heat shock. But the physiological mechanisms causing cells to express different amounts of the biomarker were unknown. Here, we used an in vivo microscopy approach to dissect different contributions to cell-to-cell variation in hsp-16.2 expression in the intestines of young adult animals, which generate the most lifespan predicting signal. While we detected both cell autonomous intrinsic noise and signaling noise, we found both contributions were relatively unimportant. The major contributor to cell-to-cell variation in biomarker expression was general differences in protein dosage. The hsp-16.2 biomarker reveals states of high or low effective dosage for many genes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Dosagem de Genes , Proteínas de Choque Térmico/genética , Longevidade/genética , Penetrância , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Genes Reporter/genética , Proteínas de Choque Térmico/metabolismo , Microscopia Intravital/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Modelos Animais , Imagem Molecular , Transdução de Sinais/genética
2.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
3.
Emerg Microbes Infect ; 8(1): 1574-1583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682177

RESUMO

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has become an important re-emerging pathogen with its rapid spread to many non-endemic areas. The lack of effective vaccines and antiviral agents is largely attributed to the elusive infection and dissemination dynamics in vivo. In this study, we designed and developed a novel, replication-competent, CHIKV reporter virus (CHIKV-iRFP) encoding a near infrared fluorescent protein (iRFP). In vitro and in vivo characterization demonstrated that CHIKV-iRFP retained similar replication and virulence phenotypes to its parental virus. Neonatal BABL/c mice and IFNAR-/- A129 mice were highly susceptible to CHIKV-iRFP infection. Following intracranial (i.c.) inoculation, CHIKV-iRFP efficiently replicated and disseminated into whole body, resulting in rapid death in an age-dependent manner. Remarkably, upon footpad injection, CHIKV-iRFP readily disseminated from footpad to head and whole skeleton, with a specific tropism for bone marrow. Taken together, this novel reporter virus provides a powerful tool to track real time CHIKV replication and to test the in vivo efficacy of vaccines and antiviral therapeutics.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Animais , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Feminino , Fluorescência , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Virulência , Replicação Viral
4.
Nat Cell Biol ; 21(11): 1382-1392, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685990

RESUMO

In the unicellular eukaryote Saccharomyces cerevisiae, Cln3-cyclin-dependent kinase activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in their protein production rate. Rather than scaling with cell size dynamics, these pulses follow the intrinsic metabolic dynamics, peaking around Start. Using a viral-based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population levels, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells across growth conditions. Thus, uncoupling between two fundamental physiological parameters drives cell cycle commitment.


Assuntos
Ciclinas/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Divisão Celular , Ciclinas/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteômica/métodos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única , Transcrição Genética
5.
Nat Cell Biol ; 21(11): 1370-1381, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685997

RESUMO

Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence.


Assuntos
Actinas/genética , Movimento Celular/genética , Drosophila melanogaster/embriologia , Mecanotransdução Celular , Peixe-Zebra/embriologia , Actinas/metabolismo , Animais , Polaridade Celular , Rastreamento de Células , Cofilina 1/genética , Cofilina 1/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemócitos/citologia , Hemócitos/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Miosinas/genética , Miosinas/metabolismo , Cultura Primária de Células , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
6.
BMC Cancer ; 19(1): 934, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590660

RESUMO

BACKGROUND: Leukemia is a cancer of blood and bone marrow cells, causing about 300,000 deaths worldwide. Photodynamic therapy (PDT) is a promising alternative for the treatment of malignant tumors. KillerRed is a genetically encoded red fluorescent protein photosensitizer (PS). In this study, we aimed to investigate the effects of KillerRed-mediated PDT on chronic myelogenous leukemia K562 cells, acute monocytic leukemia NB4 cells, and acute monocytic leukemia THP1 cells. METHODS: KillerRed was expressed in Escherichia coli cells, purified by Q-Sepharose column, and confirmed by western-blotting. The PDT effect on cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). Cell apoptosis was determined by PE Annexin V/7-AAD staining and flow cytometry. The distribution of KillerRed in leukemia cells was detected by confocal laser scanning microscopy (CLSM) and western-blotting. The ROS generation was measured by flow cytometry. RESULTS: Pure KillerRed was obtained with a yield of about 37 mg per liter of bacterial cells. KillerRed photodynamic inactivated the leukemia cells in a concentration-dependent manner, but exhibited no obvious dark toxicity. PDT mediated by KillerRed could also induce apoptotic response (mainly early apoptosis) in the three cell lines. The CLSM imaging indicated that KillerRed was distributed within the cytoplasm and nuclei of leukemia cells, causing damages to the cytoplasm and leaving the nuclear envelope intact during light irradiation. KillerRed distributed both in the cytosol and nuclei was confirmed by western blotting, and ROS significantly increased in PDT treated cells compared to the cells treated with KillerRed alone. CONCLUSIONS: Our studies demonstrated that KillerRed-mediated PDT could effectively inactivate K562, NB4, and THP1 leukemia cells and trigger cell apoptosis, and it has potential to be used individually or complementally, in the treatment of leukemia.


Assuntos
Leucemia/tratamento farmacológico , Proteínas Luminescentes , Fotoquimioterapia , Fármacos Fotossensibilizantes , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia/metabolismo , Proteínas Luminescentes/isolamento & purificação , Proteínas Luminescentes/metabolismo , Fármacos Fotossensibilizantes/isolamento & purificação , Fármacos Fotossensibilizantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Nat Commun ; 10(1): 4550, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591396

RESUMO

It is believed that long-term memory (LTM) cannot be formed immediately because it must go through a protein synthesis-dependent consolidation process. However, the current study uses Drosophila aversive olfactory conditioning to show that such processes are dispensable for context-dependent LTM (cLTM). Single-trial conditioning yields cLTM that is formed immediately in a protein-synthesis independent manner and is sustained over 14 days without decay. Unlike retrieval of traditional LTM, which requires only the conditioned odour and is mediated by mushroom-body neurons, cLTM recall requires both the conditioned odour and reinstatement of the training-environmental context. It is mediated through lateral-horn neurons that connect to multiple sensory brain regions. The cLTM cannot be retrieved if synaptic transmission from any one of these centres is blocked, with effects similar to those of altered encoding context during retrieval. The present study provides strong evidence that long-term memory can be formed easily without the need for consolidation.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Proteínas de Drosophila/biossíntese , Memória de Longo Prazo/fisiologia , Corpos Pedunculados/fisiologia , Animais , Animais Geneticamente Modificados , Neurônios Dopaminérgicos/metabolismo , Proteínas de Drosophila/genética , Feminino , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Corpos Pedunculados/citologia , Corpos Pedunculados/metabolismo , Vias Neurais/fisiologia , Odorantes , Transmissão Sináptica/fisiologia
8.
Nat Commun ; 10(1): 4481, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578371

RESUMO

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Trimetoprima/farmacologia , Animais , Anti-Infecciosos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Microscopia Confocal
9.
PLoS Biol ; 17(10): e3000492, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626642

RESUMO

Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network.


Assuntos
Astrócitos/citologia , Morte Celular/genética , Microglia/citologia , Retina/citologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatologia , Comunicação Celular , Contagem de Células , Toxina Diftérica/toxicidade , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Hemorragia Retiniana/genética , Hemorragia Retiniana/metabolismo , Hemorragia Retiniana/fisiopatologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Elife ; 82019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31612858

RESUMO

In response to proteotoxic stress, chloroplasts communicate with the nuclear gene expression system through a chloroplast unfolded protein response (cpUPR). We isolated Chlamydomonas reinhardtii mutants that disrupt cpUPR signaling and identified a gene encoding a previously uncharacterized cytoplasmic protein kinase, termed Mars1-for mutant affected in chloroplast-to-nucleus retrograde signaling-as the first known component in cpUPR signal transmission. Lack of cpUPR induction in MARS1 mutant cells impaired their ability to cope with chloroplast stress, including exposure to excessive light. Conversely, transgenic activation of cpUPR signaling conferred an advantage to cells undergoing photooxidative stress. Our results indicate that the cpUPR mitigates chloroplast photodamage and that manipulation of this pathway is a potential avenue for engineering photosynthetic organisms with increased tolerance to chloroplast stress.


Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Testes Genéticos , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Oxirredução , Estresse Oxidativo , Fotossíntese/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
11.
Mar Biotechnol (NY) ; 21(5): 671-682, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31502176

RESUMO

Primordial germ cells (PGCs) as the precursors of germ cells are responsible for transmitting genetic information to the next generation. Visualization of teleost PGCs in vivo is essential to research the origination and development of germ cells and facilitate further manipulation on PGCs isolation, cryopreservation, and surrogate breeding. In this study, artificially synthesized mRNAs constructed by fusing fluorescent protein coding region to the 3' untranslated region (3'UTR) of nanos3 or vasa (mCherry-Smnanos3 3'UTR or mCherry-Smvasa 3'UTR mRNA) were injected into turbot (Scophthalmus maximus) fertilized eggs for tracing PGCs. The results demonstrated that the fluorescent PGCs differentiated from somatic cells and aligned on both sides of the trunk at the early segmentation period, then migrated and located at the dorsal part of the gut where the gonad would form. In the same way, we also found that the zebrafish (Danio rerio) vasa 3'UTR could trace turbot PGCs, while the vasa 3'UTR s of marine medaka (Oryzias melastigma) and red seabream (Pagrus major) failed, although they could label the marine medaka PGCs. In addition, through comparative analysis, we discovered that some potential sequence elements in the3 'UTRs of nanos3 and vasa, such as GCACs, 62-bp U-rich regions and nucleotide 187-218 regions might be involved in PGCs stabilization. The results of this study provided an efficient, rapid, and specific non-transgenic approach for visualizing PGCs of economical marine fish in vivo.


Assuntos
Rastreamento de Células/métodos , Proteínas de Peixes/genética , Linguados/genética , Células Germinativas/metabolismo , Proteínas Recombinantes de Fusão/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas de Peixes/metabolismo , Linguados/crescimento & desenvolvimento , Linguados/metabolismo , Genes Reporter , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microinjeções , Conformação de Ácido Nucleico , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Oryzias/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto
12.
Nat Commun ; 10(1): 4384, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558717

RESUMO

Protein phosphatases are involved in embryonic development, metabolic homeostasis, stress response, cell cycle transitions, and many other essential biological mechanisms. Unlike kinases, protein phosphatases remain understudied and less characterized. Traditional genetic and biochemical methods have contributed significantly to our understanding; however, these methodologies lack precise and acute spatiotemporal control. Here, we report the development of a light-activated protein phosphatase, the dual specificity phosphatase 6 (DUSP6 or MKP3). Through genetic code expansion, MKP3 is placed under optical control via two different approaches: (i) incorporation of a caged cysteine into the active site for controlling catalytic activity and (ii) incorporation of a caged lysine into the kinase interaction motif for controlling the protein-protein interaction between the phosphatase and its substrate. Both strategies are expected to be applicable to the engineering of a wide range of light-activated phosphatases. Applying the optogenetically controlled MKP3 in conjunction with live cell reporters, we discover that ERK nuclear translocation is regulated in a graded manner in response to increasing MKP3 activity.


Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , Ativação Enzimática/efeitos da radiação , Proteínas Luminescentes/metabolismo , Optogenética/métodos , Raios Ultravioleta , Fosfatase 6 de Especificidade Dupla/química , Fosfatase 6 de Especificidade Dupla/genética , Células HEK293 , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Mutação , Imagem com Lapso de Tempo/métodos
13.
Nat Methods ; 16(9): 862-865, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471614

RESUMO

Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA-protein complexes that enable live imaging of mRNA in living cells.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , RNA Mensageiro/análise , Aptâmeros de Nucleotídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo
14.
Nat Methods ; 16(9): 815-829, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471616

RESUMO

The applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be.


Assuntos
Pesquisa Biomédica , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Animais , Humanos
15.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509958

RESUMO

The current paper reviews the applications of luminescence bioassays for monitoring the results of low-intensity exposures which produce a stimulative effect. The impacts of radioactivity of different types (alpha, beta, and gamma) and bioactive compounds (humic substances and fullerenols) are under consideration. Bioassays based on luminous marine bacteria, their enzymes, and fluorescent coelenteramide-containing proteins were used to compare the results of the low-intensity exposures at the cellular, biochemical, and physicochemical levels, respectively. High rates of luminescence response can provide (1) a proper number of experimental results under comparable conditions and, therefore, proper statistical processing, with this being highly important for "noisy" low-intensity exposures; and (2) non-genetic, i.e., biochemical and physicochemical mechanisms of cellular response for short-term exposures. The results of cellular exposures were discussed in terms of the hormesis concept, which implies low-dose stimulation and high-dose inhibition of physiological functions. Dependencies of the luminescence response on the exposure time or intensity (radionuclide concentration/gamma radiation dose rate, concentration of the bioactive compounds) were analyzed and compared for bioassays of different organization levels.


Assuntos
Exposição Ambiental/análise , Monitoramento Ambiental/métodos , Luminescência , Medições Luminescentes/métodos , Bactérias/metabolismo , Bactérias/efeitos da radiação , Fulerenos/metabolismo , Fulerenos/efeitos da radiação , Substâncias Húmicas/efeitos da radiação , Proteínas Luminescentes/metabolismo , Radiação Ionizante , Espectrometria de Fluorescência/métodos
16.
Nat Commun ; 10(1): 3552, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391532

RESUMO

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Microscopia/métodos , Plasmídeos/genética , Imagem Individual de Molécula/métodos , Proteína 9 Associada à CRISPR/genética , Dosagem de Genes , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia/instrumentação , Modelos Genéticos , Método de Monte Carlo , Motivos de Nucleotídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula/instrumentação , Fatores de Tempo
17.
J Microbiol Biotechnol ; 29(9): 1488-1493, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31387342

RESUMO

The rising cases of multidrug-resistant Acinetobacter baumannii (Ab) and the lack of effective drugs call for quick attention. Here, based on a Tn7 transposon and Xer/dif system, we constructed a stable, selectable marker-free autoluminescent Ab capable of producing visible light without extra substrates. Utilization of this autoluminescent reporter strain has the potential to reduce the time, effort and costs required for the evaluation of activities of anti-Ab drug candidates in vitro.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas Luminescentes/genética , Testes de Sensibilidade Microbiana/métodos , Acinetobacter baumannii/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Contagem de Colônia Microbiana , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Engenharia Genética , Genoma Bacteriano/genética , Proteínas Luminescentes/metabolismo , Mutagênese Insercional , Deleção de Sequência
18.
Cell Prolif ; 52(5): e12668, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31379046

RESUMO

OBJECTIVES: Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells. MATERIALS AND METHODS: Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. RESULTS: The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. CONCLUSIONS: Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.


Assuntos
Técnicas de Cultura de Células/métodos , Derme/citologia , Fibroblastos/citologia , Queratinócitos/citologia , Células Cultivadas , Derme/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrogéis/química , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal
19.
Sensors (Basel) ; 19(16)2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31405152

RESUMO

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL-ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.


Assuntos
Trifosfato de Adenosina/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Medições Luminescentes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Engenharia de Proteínas , Análise de Célula Única
20.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366154

RESUMO

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.


Assuntos
Grafite/química , Proteínas de Fluorescência Verde/química , Hidrogéis/química , Proteínas Imobilizadas/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Alginatos/química , Técnicas Biossensoriais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metacrilatos/química , Nanopartículas/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química
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