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Nat Commun ; 11(1): 605, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001718


Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.

Optogenética , Proteínas/metabolismo , Actinas/metabolismo , Movimento Celular/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Luz , Engenharia de Proteínas
Nat Protoc ; 13(5): 1121-1136, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29700485


Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.

Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética/métodos , Transporte Proteico/efeitos da radiação , Transcrição Genética/efeitos da radiação , Proteínas de Bactérias/metabolismo , Células HeLa , Humanos
Chembiochem ; 19(12): 1334-1340, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29465801


Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner, QPAS1, is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested the functionality of two BphP1-QPAS1-based optogenetic tools-an NIR- and blue-light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA)-in several cell types, including cortical neurons. We found that the performance of these optogenetic tools often relied on physiological properties of a specific cell type, such as nuclear transport, which could limit the applicability of the blue-light-sensitive component of iRIS. In contrast, the NIR-light-sensing component of iRIS performed well in all tested cell types. The TA system showed the best performance in cervical cancer (HeLa), bone cancer (U-2 OS), and human embryonic kidney (HEK-293) cells. The small size of the QPAS1 component allowed the design of adeno-associated virus (AAV) particles, which were applied to deliver the TA system to neurons.

Neurônios/metabolismo , Optogenética/métodos , Proteínas/genética , Ativação Transcricional/efeitos da radiação , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Expressão Gênica/efeitos da radiação , Células HEK293 , Células HeLa , Humanos , Raios Infravermelhos , Luz , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Fitocromo/análise , Fitocromo/genética , Engenharia de Proteínas/métodos , Proteínas/análise , Ratos
Chem Rev ; 117(9): 6423-6446, 2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28401765


Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore. Because biliverdin possesses the largest electron-conjugated chromophore system among linear tetrapyrroles, BphPs exhibit the most NIR-shifted spectra that reside within the NIR tissue transparency window. Here we analyze phytochrome structure and photochemistry to describe the molecular mechanisms by which they function. We then present strategies to engineer BphP-based NIR fluorescent proteins and review their properties and applications in modern imaging technologies. We next summarize designs of reporters and biosensors and describe their use in the detection of protein-protein interactions, proteolytic activities, and posttranslational modifications. Finally, we provide an overview of optogenetic tools developed from phytochromes and describe their use in light-controlled cell signaling, gene expression, and protein localization. Our review provides guidelines for the selection of NIR probes and tools for noninvasive imaging, sensing, and light-manipulation applications, specifically focusing on probes developed for use in mammalian cells and in vivo.

Técnicas Biossensoriais/métodos , Raios Infravermelhos , Proteínas Luminescentes/genética , Optogenética/métodos , Fitocromo/genética , Engenharia de Proteínas/métodos , Animais , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Fitocromo/química , Fitocromo/metabolismo
Nat Chem Biol ; 13(6): 633-639, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28346403


Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1-PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix-PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1-Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1-Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Luz , Complexos Multienzimáticos/química , Optogenética , Monoéster Fosfórico Hidrolases/química , Proteínas Quinases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Bioensaio , Epigênese Genética/genética , Citometria de Fluxo , Deleção de Genes , Células HeLa , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fitocromo/metabolismo , Engenharia de Proteínas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo