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1.
Cell ; 187(19): 5376-5392.e17, 2024 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-39197448

RESUMO

Animals defend a target level for their fundamental needs, including food, water, and sleep. Deviation from the target range, or "setpoint," triggers motivated behaviors to eliminate that difference. Whether and how the setpoint itself is encoded remains enigmatic for all motivated behaviors. Employing a high-throughput feeding assay in Drosophila, we demonstrate that the protein intake setpoint is set to different values in male, virgin female, and mated female flies to meet their varying protein demands. Leveraging this setpoint variability, we found, remarkably, that the information on the intake setpoint is stored within the protein hunger neurons as the resting membrane potential. Two RFamide G protein-coupled receptor (GPCR) pathways, by tuning the resting membrane potential in opposite directions, coordinately program and adjust the protein intake setpoint. Together, our studies map the protein intake setpoint to a single trackable physiological parameter and elucidate the cellular and molecular mechanisms underlying setpoint determination and modulation.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Neurônios , Receptores Acoplados a Proteínas G , Transdução de Sinais , Animais , Proteínas de Drosophila/metabolismo , Feminino , Masculino , Drosophila melanogaster/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Comportamento Alimentar
2.
Mol Cell ; 83(15): 2624-2640, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37419111

RESUMO

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.


Assuntos
Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo
3.
Cell ; 151(2): 304-19, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-23063122

RESUMO

Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.


Assuntos
Proteínas E4 de Adenovirus/química , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Infecções por Adenovirus Humanos/virologia , Linhagem Celular , Células Cultivadas , Cristalografia por Raios X , Humanos , Células Vegetais/virologia , Dobramento de Proteína , Nicotiana/virologia
4.
Proc Natl Acad Sci U S A ; 121(7): e2310430121, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38315854

RESUMO

Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.


Assuntos
Separação de Fases , Transcriptoma , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Oncogenes
5.
Mol Cell ; 69(2): 334-346.e4, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29307513

RESUMO

Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.


Assuntos
Imagem Óptica/métodos , Fosfotransferases/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Drosophila , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Fosfotransferases/metabolismo
6.
Nat Chem Biol ; 19(12): 1458-1468, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37349581

RESUMO

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.


Assuntos
Adesões Focais , Animais , Adesões Focais/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Fosforilação , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Movimento Celular/fisiologia , Adesão Celular/fisiologia
7.
PLoS Pathog ; 17(9): e1009898, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34478458

RESUMO

The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 µM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Etacridina/farmacologia , Inibidores de Proteases/farmacologia , Ativação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Proteases 3C de Coronavírus/antagonistas & inibidores , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Células Vero , Vírion/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
8.
Photochem Photobiol Sci ; 21(9): 1545-1555, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35041199

RESUMO

miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.


Assuntos
Mononucleotídeo de Flavina , Oxigênio Singlete , Mononucleotídeo de Flavina/química , Flavoproteínas/química , Oxigênio/química , Fosfatos , Riboflavina , Oxigênio Singlete/química
9.
Trends Biochem Sci ; 42(2): 111-129, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27814948

RESUMO

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.


Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Humanos , Fitocromo/química , Engenharia de Proteínas
11.
Nat Chem Biol ; 14(9): 870-875, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30061717

RESUMO

Amyloids adopt 'cross-ß' structures composed of long, twisted fibrils with ß-strands running perpendicular to the fibril axis. Recently, a toxic peptide was proposed to form amyloid-like cross-α structures in solution, with a planar bilayer-like assembly observed in the crystal structure. Here we crystallographically characterize designed peptides that assemble into spiraling cross-α amyloid-like structures, which resemble twisted ß-amyloid fibrils. The peptides form helical dimers, stabilized by packing of small and apolar residues, and the dimers further assemble into cross-α amyloid-like fibrils with superhelical pitches ranging from 170 Å to 200 Å. When a small residue that appeared critical for packing was converted to leucine, it resulted in structural rearrangement to a helical polymer. Fluorescently tagged versions of the designed peptides form puncta in mammalian cells, which recover from photobleaching with markedly different kinetics. These structural folds could be potentially useful for directing in vivo protein assemblies with predetermined spacing and stabilities.


Assuntos
Amiloide/química , Peptídeos/química , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Peptídeos/síntese química , Conformação Proteica
12.
J Am Chem Soc ; 141(11): 4526-4530, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30821975

RESUMO

A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.


Assuntos
Apoptose , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Imagem Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Conformação Proteica em Folha beta
13.
Nat Methods ; 13(9): 763-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27479328

RESUMO

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.


Assuntos
Técnicas Biossensoriais , Proteínas Luminescentes/isolamento & purificação , Ficocianina/química , Trichodesmium/metabolismo , Biliverdina/química , Ciclo Celular/fisiologia , Escherichia coli/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/efeitos da radiação , Mutação , Ficocianina/metabolismo , Conformação Proteica , Estabilidade Proteica , Subunidades Proteicas , Proteína Vermelha Fluorescente
14.
Anal Chem ; 90(24): 14287-14293, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30431263

RESUMO

Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein-protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.


Assuntos
Fator de Transcrição Ikaros/metabolismo , Peptídeo Hidrolases/metabolismo , Mapas de Interação de Proteínas , Bibliotecas de Moléculas Pequenas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Fator de Transcrição Ikaros/química , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Microscopia Confocal , Proteínas Nucleares/genética , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Talidomida/análogos & derivados , Talidomida/química , Talidomida/metabolismo , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases , Proteína Supressora de Tumor Von Hippel-Lindau/genética
15.
Nat Methods ; 12(8): 763-5, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26098020

RESUMO

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.


Assuntos
Proteínas de Fluorescência Verde/química , Raios Infravermelhos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , DNA/química , Biologia do Desenvolvimento , Drosophila melanogaster , Corantes Fluorescentes/química , Células HeLa , Histidina/química , Humanos , Proteínas Luminescentes/química , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Plasmídeos/metabolismo , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Transfecção , Peixe-Zebra
16.
Proc Natl Acad Sci U S A ; 112(11): 3338-43, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25733847

RESUMO

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.


Assuntos
Apoptose , Drosophila melanogaster/citologia , Corantes Fluorescentes/metabolismo , Genes Reporter , Animais , Carcinogênese/patologia , Caspases/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Células HEK293 , Humanos , Raios Infravermelhos , Peptídeo Hidrolases/metabolismo , Fatores de Tempo
17.
Biochemistry ; 55(31): 4263-74, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27471775

RESUMO

Using X-ray crystallography, continuum electrostatic calculations, and molecular dynamics simulations, we have studied the structure, protonation behavior, and dynamics of the biliverdin chromophore and its molecular environment in a series of genetically engineered infrared fluorescent proteins (IFPs) based on the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. Our study suggests that the experimentally observed enhancement of fluorescent properties results from the improved rigidity and planarity of the biliverdin chromophore, in particular of the first two pyrrole rings neighboring the covalent linkage to the protein. We propose that the increases in the levels of both motion and bending of the chromophore out of planarity favor the decrease in fluorescence. The chromophore-binding pocket in some of the studied proteins, in particular the weakly fluorescent parent protein, is shown to be readily accessible to water molecules from the solvent. These waters entering the chromophore region form hydrogen bond networks that affect the otherwise planar conformation of the first three rings of the chromophore. On the basis of our simulations, the enhancement of fluorescence in IFPs can be achieved either by reducing the mobility of water molecules in the vicinity of the chromophore or by limiting the interactions of the nearby protein residues with the chromophore. Finally, simulations performed at both low and neutral pH values highlight differences in the dynamics of the chromophore and shed light on the mechanism of fluorescence loss at low pH.


Assuntos
Proteínas de Bactérias/química , Proteínas Luminescentes/química , Proteínas de Bactérias/genética , Biliverdina/química , Cristalografia por Raios X , Deinococcus/química , Deinococcus/genética , Fluorescência , Raios Infravermelhos , Proteínas Luminescentes/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fitocromo/química , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade Estática
18.
Bioorg Med Chem Lett ; 26(14): 3359-3363, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27220724

RESUMO

Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling.


Assuntos
Fármacos Fotossensibilizantes/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Oxigênio Singlete/metabolismo , Modelos Moleculares , Estrutura Molecular , Processos Fotoquímicos , Fármacos Fotossensibilizantes/química , Proteínas Quinases Associadas a Fase S/química
19.
Proc Natl Acad Sci U S A ; 109(19): 7499-504, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22532663

RESUMO

We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Flavoproteínas/metabolismo , Proteínas Luminescentes/metabolismo , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Caspases/genética , Caspases/metabolismo , Morte Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Flavoproteínas/genética , Interneurônios/citologia , Interneurônios/metabolismo , Interneurônios/efeitos da radiação , Luz , Proteínas Luminescentes/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios Motores/efeitos da radiação , Neurônios/citologia , Neurônios/efeitos da radiação , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Oxigênio Singlete/metabolismo , Fatores de Tempo
20.
PLoS Biol ; 9(4): e1001041, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21483721

RESUMO

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.


Assuntos
Coloração e Rotulagem , 3,3'-Diaminobenzidina/análise , Estruturas Animais , Animais , Encéfalo/ultraestrutura , Caenorhabditis elegans/química , Células , Corantes Fluorescentes/análise , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Fototropinas/análise , Proteínas/análise
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