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2.
Commun Biol ; 4(1): 1091, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531530

RESUMO

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.

3.
Nat Commun ; 11(1): 605, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001718

RESUMO

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.


Assuntos
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
4.
Methods Mol Biol ; 1821: 87-106, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30062407

RESUMO

Genetically encoded FRET-based biosensors are increasingly popular and useful tools for examining signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic techniques used to characterize and to validate single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensors of the Rho GTPase-family proteins. Methods described here are generally applicable to other genetically encoded FRET-based biosensors by modifying the tested conditions to include additional/different regulators and inhibitors, as appropriate for the specific protein of interest.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Camundongos , Células RAW 264.7
5.
J Cell Biol ; 216(12): 4331-4349, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29061650

RESUMO

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and ßPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Integrina beta1/genética , Neoplasias Mamárias Animais/genética , Proteínas rac de Ligação ao GTP/genética , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Células HEK293 , Humanos , Integrina beta1/metabolismo , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/deficiência
6.
Nature ; 540(7631): 74-79, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27775718

RESUMO

Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.


Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , GTP Fosfo-Hidrolases/genética , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/química , Permeabilidade , Conformação Proteica/efeitos dos fármacos
7.
Genes Dev ; 29(8): 876-86, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25877922

RESUMO

Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.


Assuntos
Expressão Gênica , Técnicas Genéticas , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Sequência de Bases/genética , Proteínas do Capsídeo/genética , Linhagem Celular , Células Cultivadas , Códon/genética , Humanos , Levivirus/genética , Camundongos , Motivos de Nucleotídeos , Saccharomyces cerevisiae/genética
8.
Cell Adh Migr ; 8(6): 526-34, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25482645

RESUMO

The Rho-family of p21 small GTPases are directly linked to the regulation of actin-based motile machinery and play a key role in the control of cell migration. Aside from the original and most well-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerous isoforms of these key proteins have been identified and shown to have specific roles in regulating various cellular motility processes. The major difficulty in addressing these isoform-specific effects is that isoforms typically contain highly similar primary amino acid sequences and thus are able to interact with the same upstream regulators and the downstream effector targets. Here, we will introduce the major members of each GTPase subfamily and discuss recent advances in the design and application of fluorescent resonance energy transfer-based probes, which are at the forefront of the technologies available to directly probe the differential, spatiotemporal activation dynamics of these proteins in live single cells. Currently, it is possible to specifically detect the activation status of RhoA vs. RhoC isoforms, as well as Cdc42 vs. TC-10 isoforms in living cells. Clearly, additional efforts are still required to produce biosensor systems capable of detecting other isoforms of Rho GTPases including RhoB, Rac2/3, RhoG, etc. Through such efforts, we will uncover the isoform-specific roles of these near-identical proteins in living cells, clearly an important area of the Rho GTPase biology that is not yet fully appreciated.


Assuntos
Movimento Celular/fisiologia , Transferência Ressonante de Energia de Fluorescência , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Técnicas Biossensoriais , Humanos , Isoenzimas/metabolismo , Proteínas rho de Ligação ao GTP/química
9.
J Cell Sci ; 127(Pt 3): 673-85, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24284073

RESUMO

Vaccinia virus enhances its cell-to-cell spread by inducing Arp2/3-dependent actin polymerisation. This process is initiated by Src- and Abl-mediated phosphorylation of the viral transmembrane protein A36, leading to recruitment of a signalling network consisting of Grb2, Nck, WIP and N-WASP. Nck is a potent activator of N-WASP-Arp2/3-dependent actin polymerisation. However, recent observations demonstrate that an interaction between Nck and N-WASP is not required for vaccinia actin tail formation. We found that Cdc42 cooperates with Nck to promote actin tail formation by stabilising N-WASP beneath the virus. Cdc42 activation is mediated by the Rho guanine-nucleotide-exchange factor (GEF) intersectin-1 (ITSN1), which is recruited to the virus prior to its actin-based motility. Moreover, Cdc42, ITSN1 and N-WASP function collaboratively in a feed-forward loop to promote vaccinia-induced actin polymerisation. Outside the context of infection, we demonstrate that ITSN1 also functions together with Cdc42, Nck and N-WASP during phagocytosis mediated by the Fc gamma receptor. Our observations suggest that ITSN1 is an important general regulator of Cdc42-, Nck- and N-WASP-dependent actin polymerisation.


Assuntos
Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Oncogênicas/metabolismo , Vírus Vaccinia/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/ultraestrutura , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linhagem Celular , Proteína Adaptadora GRB2/genética , Humanos , Fosforilação , Transdução de Sinais/genética , Vírus Vaccinia/patogenicidade , Vírus Vaccinia/ultraestrutura , Proteína cdc42 de Ligação ao GTP/genética
10.
Curr Biol ; 23(11): 999-1006, 2013 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-23707428

RESUMO

Nck links phosphotyrosine-based signaling to Arp2/3-dependent actin polymerization during many different cellular processes as well as actin-based motility of enteropathogenic Escherichia coli (EPEC), vaccinia, and other vertebrate poxviruses by interacting with N-WASP/WASP. Nck also binds WASP-interacting protein (WIP), which inhibits the ability of N-WASP to activate the Arp2/3 complex until it receives an appropriate signaling input. Using mouse embryonic fibroblasts (MEFs) lacking Nck, WIP, or N-WASP, we have investigated whether an interaction of Nck with both WIP and N-WASP is required for their recruitment to vaccinia during Arp2/3-dependent actin assembly. We find that WIP or its homolog WIRE is required for N-WASP recruitment and actin-based motility of the virus. WIP contains two Nck-binding sites and is recruited to the virus, bound to N-WASP, by interacting with the second SH3 domain of Nck. N-WASP also contains two Nck-binding sites, but its recruitment is dependent on its interaction with WIP rather than Nck. The first and third SH3 domains of Nck are not required to recruit the WIP:N-WASP complex but are essential to stimulate actin assembly. We have established that WIP acts as an essential link between Nck and N-WASP. Our observations provide important insights into the hierarchy and connections in one of the major cellular signaling networks stimulating Arp2/3 complex-dependent actin polymerization.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Oncogênicas/metabolismo , Vírus Vaccinia/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Proteínas do Citoesqueleto , Fibroblastos , Camundongos , Fosfotirosina/metabolismo , Polimerização , Ligação Proteica , Transdução de Sinais , Domínios de Homologia de src
11.
J Cell Sci ; 125(Pt 12): 2825-30, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22454526

RESUMO

The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcγ receptor (FcγR)- but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FcγR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAM motif in the cytoplasmic tail of the receptor. In the absence of FcγR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FcγR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FcγR-mediated phagocytosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Oncogênicas/metabolismo , Fagocitose , Receptores de IgG/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genética , Ligação Proteica , Receptores de IgG/química , Receptores de IgG/genética , Transdução de Sinais , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/fisiopatologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética
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