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Commun Biol ; 4(1): 1091, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531530


During breast cancer metastasis, cancer cell invasion is driven by actin-rich protrusions called invadopodia, which mediate the extracellular matrix degradation required for the success of the invasive cascade. In this study, we demonstrate that TC10, a member of a Cdc42 subfamily of p21 small GTPases, regulates the membrane type 1 matrix metalloproteinase (MT1-MMP)-driven extracellular matrix degradation at invadopodia. We show that TC10 is required for the plasma membrane surface exposure of MT1-MMP at these structures. By utilizing our Förster resonance energy transfer (FRET) biosensor, we demonstrate the p190RhoGAP-dependent regulation of spatiotemporal TC10 activity at invadopodia. We identified a pathway that regulates invadopodia-associated TC10 activity and function through the activation of p190RhoGAP and the downstream interacting effector Exo70. Our findings reveal the role of a previously unknown regulator of vesicular fusion at invadopodia, TC10 GTPase, in breast cancer invasion and metastasis.

Methods Mol Biol ; 2350: 43-68, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331278


Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.

Técnicas Biossensoriais/métodos , Imunofluorescência/métodos , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte , Linhagem Celular , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência/métodos , Genes Reporter , Camundongos , Ligação Proteica , Proteínas rho de Ligação ao GTP/genética
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
Methods Mol Biol ; 2108: 281-293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939189


Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.

Luz , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Optogenética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Expressão Gênica , Genes Reporter , Camundongos , Microscopia de Fluorescência , Optogenética/métodos , Ligação Proteica , Células RAW 264.7 , Transfecção