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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
J Cell Sci ; 132(8)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30890647


In addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here, we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID, to identify binding partners for nuclear actin. Common high-confidence interactions highlight the role of actin in chromatin-remodeling complexes and identify the histone-modifying complex human Ada-Two-A-containing (hATAC) as a novel actin-containing nuclear complex. Actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. Transient interactions detected through BioID link actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative splicing in minigene assays, likely by affecting the transcription elongation rate. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes.This article has an associated First Person interview with the first author of the paper.

Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/química , Citoesqueleto/metabolismo , Histona Acetiltransferases/metabolismo , Splicing de RNA , Proteínas Adaptadoras de Transdução de Sinal/genética , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Expressão Gênica , Células HeLa , Histona Acetiltransferases/genética , Humanos , Espectrometria de Massas , Ativação Transcricional
Sci Rep ; 7(1): 3692, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623258


Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.

Cromatina/genética , Cromatina/metabolismo , Fenômenos Fisiológicos Virais , Liberação de Vírus , Animais , Transporte Biológico , Biomarcadores , Linhagem Celular , Núcleo Celular , Feminino , Histonas/metabolismo , Camundongos , Microscopia Confocal
Curr Opin Cell Biol ; 46: 33-38, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28092729


In addition to its essential roles as part of the cytoskeleton, actin has also been linked to many processes in the nucleus. Recent data has demonstrated the presence of both monomeric and polymeric actin in the nucleus, and implied distinct functional roles for these actin pools. Monomeric actin seems to be involved in regulation of gene expression through transcription factors, chromatin regulating complexes and RNA polymerases. In addition to cytoplasmic actin regulators, nuclear proteins, such as emerin, can regulate actin polymerization properties specifically in this compartment. Besides of structural roles, nuclear actin filaments may be required for organizing the nuclear contents and for the maintenance of genomic integrity.

Actinas/metabolismo , Proteínas Nucleares/metabolismo , Actinas/química , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Citoplasma/metabolismo , Humanos , Proteínas Nucleares/química