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1.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34047769

RESUMO

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.


Assuntos
Nadadeiras de Animais/metabolismo , Movimento Celular , Inflamação/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Cicatrização , Infecção dos Ferimentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/lesões , Nadadeiras de Animais/microbiologia , Nadadeiras de Animais/patologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Microscopia de Fluorescência , Neutrófilos/microbiologia , Comunicação Parácrina , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , Fatores de Tempo , Infecção dos Ferimentos/genética , Infecção dos Ferimentos/microbiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
2.
Trends Cell Biol ; 31(2): 86-94, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33281034

RESUMO

The directed migration of leukocytes to sites of damage or infection is necessary for a productive immune response. There is substantial evidence supporting a key role for chemoattractants in directed migration, however, less is known about how cell-cell contacts affect the migratory behavior of leukocytes in innate immunity. Here, we explore how cell-cell contacts can affect the directed migration of innate immune cells, including their role in attracting, repelling, or stopping cell motility. Further investigation of cell contact dynamics as guidance cues may yield new insights into the regulation of innate immunity.

3.
Cell Commun Signal ; 18(1): 144, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900380

RESUMO

BACKGROUND: Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549. METHODS: We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation. RESULTS: We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation. CONCLUSION: In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases. Video abstract.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Pulmonares/metabolismo , Podossomos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Células A549 , Adenocarcinoma de Pulmão/patologia , Movimento Celular , Humanos , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/patologia , Podossomos/patologia
4.
Methods Mol Biol ; 2108: 281-293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939189

RESUMO

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.


Assuntos
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
5.
Elife ; 82019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31259685

RESUMO

Wound repair is controlled temporally and spatially to restore tissue homeostasis. Previously we reported that thermal damage of the larval zebrafish fin disrupts collagen organization and wound healing compared to tail transection (LeBert et al., 2018). Here we characterize different injury models in larval zebrafish to dissect temporal and spatial dynamics of repair in complex damage. We found that each damage model triggers distinct inflammatory and tissue responses, with Stat3 and TGFß playing key roles in the regulation of mesenchymal cells during simple repair. While thermal injury disrupts collagen fibers initially, healing is recovered as inflammation resolves, and mesenchymal cells and collagen fibers align. By contrast, infected wounds lead to persistent inflammation and loss of mesenchymal cells, resulting in minimal tissue repair. These wound models have broad physiological relevance, thereby providing a valuable advance in our toolkit to probe the dynamics of inflammation and wound repair in complex tissue damage.


Assuntos
Inflamação/patologia , Inflamação/fisiopatologia , Cicatrização , Ferimentos e Lesões/patologia , Animais , Peixe-Zebra
6.
J Hepatol ; 70(4): 710-721, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30572006

RESUMO

BACKGROUND & AIMS: Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) is an increasing clinical problem associated with progression to hepatocellular carcinoma (HCC). The effect of a high-fat diet on the early immune response in HCC is poorly understood, while the role of metformin in treating NAFLD and HCC remains controversial. Herein, we visualized the early immune responses in the liver and the effect of metformin on progression of HCC using optically transparent zebrafish. METHODS: We used live imaging to visualize liver inflammation and disease progression in a NAFLD/NASH-HCC zebrafish model. We combined a high-fat diet with a transgenic zebrafish HCC model induced by hepatocyte-specific activated beta-catenin and assessed liver size, angiogenesis, micronuclei formation and inflammation in the liver. In addition, we probed the effects of metformin on immune cell composition and early HCC progression. RESULTS: We found that a high-fat diet induced an increase in liver size, enhanced angiogenesis, micronuclei formation and neutrophil infiltration in the liver. Although macrophage number was not affected by diet, a high-fat diet induced changes in macrophage morphology and polarization with an increase in liver associated TNFα-positive macrophages. Treatment with metformin altered macrophage polarization, reduced liver size and reduced micronuclei formation in NAFLD/NASH-associated HCC larvae. Moreover, a high-fat diet reduced T cell density in the liver, which was reversed by treatment with metformin. CONCLUSIONS: These findings suggest that diet alters macrophage polarization and exacerbates the liver inflammatory microenvironment and cancer progression in a zebrafish model of NAFLD/NASH-associated HCC. Metformin specifically affects the progression induced by diet and modulates the immune response by affecting macrophage polarization and T cell infiltration, suggesting possible effects of metformin on tumor surveillance. LAY SUMMARY: This paper reports a new zebrafish model that can be used to study the effects of diet on liver cancer. We found that a high-fat diet promotes non-resolving inflammation in the liver and enhances cancer progression. In addition, we found that metformin, a drug used to treat diabetes, inhibits high-fat diet-induced cancer progression in this model, by reducing diet-induced non-resolving inflammation and potentially restoring tumor surveillance.


Assuntos
Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/tratamento farmacológico , Progressão da Doença , Imunidade Inata/efeitos dos fármacos , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/tratamento farmacológico , Metformina/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Polaridade Celular/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Inflamação/tratamento farmacológico , Inflamação/etiologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Metformina/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Peixe-Zebra
7.
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
8.
Sci Rep ; 7(1): 8547, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819224

RESUMO

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.


Assuntos
Actinas/metabolismo , Extensões da Superfície Celular/metabolismo , Macrófagos/metabolismo , Polimerização , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Comunicação Celular , Linhagem Celular , Humanos , Macrófagos/citologia , Camundongos , Transdução de Sinais , Imagem com Lapso de Tempo/métodos , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
9.
Methods Mol Biol ; 1519: 125-143, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27815877

RESUMO

The p21-family members of Rho GTPases are important for the control of actin cytoskeleton dynamics, and are critical regulators of phagocytosis. The three-dimensional structure of phagosomes and the highly compartmentalized nature of the signaling mechanisms during phagocytosis require high-resolution imaging using ratiometric biosensors to decipher Rho GTPase activities regulating phagosome formation and function. Here we describe methods for the expression and ratiometric imaging of FRET-based Rho GTPase biosensors in macrophages during phagocytosis. As an example, we show Cdc42 activity at the phagosome over Z-serial planes. In addition, we demonstrate the usage of a new, fast, and user-friendly deconvolution package that delivers significant improvements in the attainable details of Rho GTPase activity in phagosome structures.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Fagocitose , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática , Imageamento Tridimensional , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Estatística como Assunto , Proteína cdc42 de Ligação ao GTP/metabolismo
10.
Nature ; 539(7630): 575-578, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27828948

RESUMO

Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair, as well as homeostatic mechanisms that counteract ageing and neurodegeneration, cardiovascular disease, and cancer. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular 'brake' on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1KO) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1KO cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease, this Fat1 function may have importance in other settings of altered cell growth and metabolism.


Assuntos
Artérias/citologia , Artérias/metabolismo , Caderinas/metabolismo , Respiração Celular , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta/citologia , Aorta/lesões , Aorta/metabolismo , Artérias/lesões , Ácido Aspártico/metabolismo , Caderinas/química , Caderinas/deficiência , Proliferação de Células , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Mitocôndrias/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Neointima/metabolismo , Oxigênio/metabolismo , Consumo de Oxigênio
11.
J Immunol ; 196(8): 3479-93, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26951800

RESUMO

Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play nonredundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells, we developed a genetically encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, Rac1, and Cdc42 have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics, yet they had distinct spatial distributions in response to the exogenous stimulus, fMLF. Active Rac1 was mainly localized to the cell periphery, whereas active Rac2 was distributed throughout the cell, with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2, and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Neuropeptídeos/genética , Isoformas de Proteínas/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Linhagem Celular , Extensões da Superfície Celular/fisiologia , Células HEK293 , Humanos , Macrófagos/imunologia , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética
12.
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
14.
Methods Mol Biol ; 1172: 173-84, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908304

RESUMO

Cytokine stimulations of leukocytes many times result in transient activation of the p21 Rho family of small GTPases. The role of these molecules during cell migration and chemotaxis is well established. The traditional approach to study the activation dynamics of these proteins involves affinity pull-downs that are often cumbersome and prone to errors. Here, we describe a reagent and a method of simple "mix-and-measure" approach useful for determining the activation status of endogenous Cdc42 GTPase from cell lysates.


Assuntos
Bioensaio , Quimiocina CX3CL1/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Coloração e Rotulagem/métodos , Proteína cdc42 de Ligação ao GTP/genética , Animais , Extratos Celulares/química , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
15.
Methods Mol Biol ; 1172: 263-70, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908313

RESUMO

Immunofluorescence is an important technique required to observe expression, localization and colocalization of proteins within the cell. Here we describe the immunofluorescence and subsequent confocal microscopy technique of tumor necrosis factor-α (TNF) in human neutrophils (polymorphonuclear leukocytes; PMN). The qualitative technique can be used to observe the expression pattern changes from resting to stimulated leukocytes. Colocalization with other cytokines, proteins, or organelles can be observed. This immunofluorescence technique can be done in 1-2 days.


Assuntos
Citoplasma/química , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análise , Anticorpos/química , Núcleo Celular/química , Núcleo Celular/imunologia , Células Cultivadas , Citoplasma/imunologia , Imunofluorescência , Humanos , Lipopolissacarídeos/farmacologia , Microscopia Confocal , Neutrófilos/citologia , Neutrófilos/imunologia , Fixação de Tecidos , Fator de Necrose Tumoral alfa/biossíntese
16.
Methods Mol Biol ; 1172: 285-93, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908315

RESUMO

Most cytokines are stored in the cytoplasm until their release into the extracellular environment; however, some cytokines have been reported to localize in the nucleus. Traditional whole cell extract preparation does not provide information about the intracellular localization of cytokines. Here, we describe how to prepare cytoplasmic and nuclear extracts that can be analyzed by immunoblotting. While in this chapter we use this method to analyze intracellular localization of interleukin-8 (IL-8) in human mononuclear leukocytes, this protocol is adaptable to any cell type or protein of interest.


Assuntos
Núcleo Celular/química , Citoplasma/química , Interleucina-8/isolamento & purificação , Leucócitos Mononucleares/metabolismo , Western Blotting , Fracionamento Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Centrifugação com Gradiente de Concentração , Misturas Complexas/química , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Ficoll , Expressão Gênica , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia
17.
J Cell Biol ; 205(5): 737-51, 2014 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-24891603

RESUMO

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin-NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Talina/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto , Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Metástase Neoplásica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Trocador 1 de Sódio-Hidrogênio
18.
PLoS One ; 9(5): e96469, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24798463

RESUMO

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.


Assuntos
Técnicas Biossensoriais , Proteína cdc42 de Ligação ao GTP/análise , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Fluorometria , Camundongos , Células NIH 3T3
19.
Nat Cell Biol ; 16(6): 574-86, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24859002

RESUMO

Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, we developed a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. We describe here an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.


Assuntos
Neoplasias da Mama/enzimologia , Movimento Celular , Extensões da Superfície Celular/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Técnicas Biossensoriais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/patologia , Cortactina/metabolismo , Matriz Extracelular/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Ratos , Fatores de Tempo , Transfecção , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genética
20.
Immunol Rev ; 256(1): 222-39, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24117824

RESUMO

Macrophages are best known for their protective search and destroy functions against invading microorganisms. These processes are commonly known as chemotaxis and phagocytosis. Both of these processes require actin cytoskeletal remodeling to produce distinct F-actin-rich membrane structures called lamellipodia and phagocytic cups. This review will focus on the mechanisms by which macrophages regulate actin polymerization through initial receptor signaling and subsequent Arp2/3 activation by nucleation-promoting factors like the WASP/WAVE family, followed by remodeling of actin networks to produce these very distinct structures.


Assuntos
Membrana Celular/metabolismo , Quimiotaxia/fisiologia , Macrófagos/fisiologia , Fagocitose/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Humanos , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/metabolismo , Receptores de IgG/metabolismo
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