Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 52
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
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
2.
Int J Ment Health Syst ; 14: 24, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32211054

RESUMO

Background: The increasing number of people who experience mental disorders is a global problem. People with mental disorders have high rates of co-morbidity and significantly poorer oral health outcomes than the general public. However, their oral health remains largely a hidden and neglected issue. A complex range of factors impact the oral health of this group. These include anxiety and dental phobia, dietary habits, including the heavy consumption of sugary drinks, substance misuse of tobacco, alcohol, and/or psychostimulants, the adverse orofacial side effects of anti-psychotic and anti-depression medications, and financial, geographic, and social barriers to accessing oral health care. Methods: The aim of this realist systematic review is to (a) identify and synthesise evidence that explores oral health interventions for people living with mental disorders; (b) explore the context and mechanisms that have contributed to the success of interventions or the barriers and challenges; (c) produce program theories on causal, contextual and mechanistic factors to facilitate outcomes and (d) produce recommendations and guidelines to guide future oral health interventions for people with mental disorders at both the policy and practice level. Using a five-step process, that incorporates primary data collection from key stakeholders, a beginning theoretical framework will be developed to describe contextual and mechanistic factors and how they might impact on the success or failure of oral health interventions for people with mental disorders. Key database searches will be conducted, with data extraction focused on the factors that might have impacted on intervention implementation and outcomes. Quality appraisal of studies will occur, and the theoretical framework will be populated with extracted data. Stakeholder input will support the development and refinement of a theory on oral health interventions for people with mental disorders. Discussion: This will be the first review to take a realist approach to explore the broad scope of causal factors that impact on the success or failure of oral health interventions for people with mental disorders. The approach includes extensive stakeholder engagement and will advance realist systematic review methodology. Review outcomes will be important in guiding policy and practice to ensure oral health interventions better meet the needs of people with mental disorders.Systematic review registration This review protocol is registered with PROSPERO (Number) 155969.

3.
Invest Ophthalmol Vis Sci ; 61(2): 30, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084266

RESUMO

Purpose: Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. Methods: Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. Results: Adult neural retina released EVs at a rate of 1.42 +/- 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs. Conclusions: The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.


Assuntos
Vesículas Extracelulares/metabolismo , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Transporte Proteico/fisiologia , Transporte de RNA/fisiologia , Retina/metabolismo , Animais , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/metabolismo
4.
Methods Mol Biol ; 2108: 273-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939188

RESUMO

Macrophages are known to play multiple roles in the breast cancer microenvironment including the promotion of tumor cell invasion that is dependent on soluble factors or through direct contact. Macrophages can also enhance the production of Tunneling Nanotubes (TNTs) in tumor cells which can be mimicked using macrophage-conditioned medium. TNTs are long thin F-actin structures that connect two or more cells together that have been found in many different cell types including macrophages and tumor cells and have been implicated in enhancing tumor cells functions, such as invasion. Here we describe basic procedures used to stimulate tumor cell TNT formation through macrophage-conditioned medium along with methods for quantifying TNTs.


Assuntos
Biomarcadores , Macrófagos/metabolismo , Macrófagos/patologia , Microscopia , Citoesqueleto de Actina , Actinas/metabolismo , Animais , Transporte Biológico , Comunicação Celular , Meios de Cultivo Condicionados/metabolismo , Imunofluorescência , Camundongos , Microscopia/métodos , Células RAW 264.7 , Microambiente Tumoral
5.
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
6.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30659112

RESUMO

The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study, we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs), that occurs between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long-distance tumor cell streaming in vitro Using an in vivo zebrafish model that recreates macrophage-mediated tumor cell invasion, we observed TNT-mediated macrophage-dependent tumor cell invasion, distant metastatic foci and areas of metastatic spread. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that leads to tumor progression and metastasis.


Assuntos
Neoplasias da Mama/genética , Comunicação Celular/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Macrófagos/metabolismo , Neoplasias Mamárias Animais/genética , Animais , Transporte Biológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Embrião não Mamífero , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Macrófagos/ultraestrutura , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Cultura Primária de Células , Células RAW 264.7 , Ratos , Transdução de Sinais , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismo , Peixe-Zebra
7.
Cancer Rep (Hoboken) ; 2(6): e1213, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-32467880

RESUMO

Background: Metastasis is the cause of most cancer-related deaths. It is known that breast cancer cells in proximity to macrophages become more invasive in an Epidermal Growth Factor (EGF) dependent manner. Tunneling nanotubes (TNTs) are thin, F-actin containing, cellular protrusions that mediate intercellular communication and have been identified in many tumors. The mechanism of TNT formation varies between different cell types. M-Sec (TNFAIP2) has been demonstrated to be involved in TNT formation in some cell types including macrophages. Yet, the requirement of M-Sec in tumor cell TNT formation in response to macrophages has not been explored. Aim: The aim of this study was to determine whether EGF was required for macrophage induced tumor cell TNTs in an M-Sec dependent manner and what possible roles tumor cell TNTs play in tumor cell migration and invasion. Methods and Results: Macrophage Conditioned Media (CM) was used to induce an increase in TNTs in a number of breast cancer cell lines as measured by live cell microscopy. Tumor cell TNT formation by CM was dependent on the presence of EGF which was sufficient to induce TNT formation. CM treatment enhanced the level of M-Sec identified using western blot analysis. Reduction of endogenous M-Sec levels via shRNA in MTLn3 mammary adenocarcinoma cells inhibited the formation of TNTs. The role of tumor cell TNTs in cell behavior was tested using in vitro transwell and 3D invasion assays. No effect on chemotaxis was detected but 3D invasion was reduced following the knockdown of M-Sec in tumor cell TNTs. Conclusions: Our results show that EGF was necessary and sufficient for tumor cell TNT formation which was dependent on cellular M-Sec levels. While tumor cell TNTs may not play a role in individual cell behaviors like chemotaxis, they may be important in more complex tumor cell behaviors such as 3D invasion.

8.
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
9.
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
10.
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
11.
J Clin Invest ; 126(10): 3837-3851, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27599296

RESUMO

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Movimento Celular , Proteínas do Citoesqueleto , Células HEK293 , Humanos , Sinapses Imunológicas/metabolismo , Linfonodos/citologia , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Mapas de Interação de Proteínas , Multimerização Proteica , Transporte Proteico , Linfócitos T/fisiologia
12.
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
13.
Int J Biochem Cell Biol ; 71: 44-54, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26704468

RESUMO

Cell-cell communication is critical to coordinate the activity and behavior of a multicellular organism. The cells of the immune system not only must communicate with similar cells, but also with many other cell types in the body. Therefore, the cells of the immune system have evolved multiple ways to communicate. Exosomes and tunneling nanotubes (TNTs) are two means of communication used by immune cells that contribute to immune functions. Exosomes are small membrane vesicles secreted by most cell types that can mediate intercellular communication and in the immune system they are proposed to play a role in antigen presentation and modulation of gene expression. TNTs are membranous structures that mediate direct cell-cell contact over several cell diameters in length (and possibly longer) and facilitate the interaction and/or the transfer of signals, material and other cellular organelles between connected cells. Recent studies have revealed additional, but sometimes conflicting, structural and functional features of both exosomes and TNTs. Despite the new and exciting information in exosome and TNT composition, origin and in vitro function, biologically significant functions are still being investigated and determined. In this review, we discuss the current field regarding exosomes and TNTs in immune cells providing evaluation and perspectives of the current literature.


Assuntos
Comunicação Celular/imunologia , Exossomos/metabolismo , Humanos
14.
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
15.
Sci Signal ; 7(353): ra112, 2014 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-25429076

RESUMO

Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)-responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding Mena(INV), an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down Mena(INV) or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that Mena(INV) and TMEM frequency are correlated prognostic markers and CSF-1 and Mena(INV) may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Macrófagos/fisiologia , Proteínas dos Microfilamentos/análise , Invasividade Neoplásica/fisiopatologia , Proteínas de Neoplasias/análise , Migração Transendotelial e Transepitelial/fisiologia , Processamento Alternativo , Animais , Comunicação Autócrina , Biomarcadores Tumorais/genética , Biópsia por Agulha Fina , Neoplasias da Mama/química , Neoplasias da Mama/genética , Caderinas/biossíntese , Caderinas/genética , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/classificação , Carcinoma Ductal de Mama/genética , Técnicas de Cocultura , Células Endoteliais/citologia , Éxons , Feminino , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Terapia de Alvo Molecular , Gradação de Tumores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Comunicação Parácrina , Prognóstico , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente Tumoral
16.
Cytoskeleton (Hoboken) ; 71(9): 542-54, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25186818

RESUMO

The ability of macrophages to migrate to sites of infection and inflammation is critical for their role in the innate immune response. Macrophage cell lines have made it possible to study the roles of individual proteins responsible for migration using molecular biology, but it has not been possible to reliably elicit the motility of macrophage cell lines in two dimensions. In the past, measurements of the motility of macrophage cell lines have been largely limited to transwell assays which provide limited quantitative information on motility and limited ability to visualize cell morphology. We used microcontact printing to create polydimethylsiloxane (PDMS) surfaces functionalized with fibronectin that otherwise support little macrophage adhesion. We used these surfaces to measure macrophage migration in two dimensions and found that these cells migrate efficiently in a uniform field of colony-stimulating factor-1, CSF-1. Knockdown of Cdc42 led to a nonstatistically significant reduction in motility, whereas chemical inhibition of PI3K activity led to a complete loss of motility. Inhibition of the RhoA kinase, ROCK, did not abolish the motility of these cells but caused a quantitative change in motility, reducing motility significantly on high concentrations of fibronectin but not on low concentrations. This study illustrates the importance of studying cell motility on well controlled materials to better understand the exact roles of specific proteins on cell migration. © 2014 Wiley Periodicals, Inc.


Assuntos
Quimiotaxia/fisiologia , Técnicas In Vitro/métodos , Macrófagos/citologia , Linhagem Celular , Fibronectinas , Humanos
17.
Methods Mol Biol ; 1172: 115-23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908299

RESUMO

Growth factor-dependent pairing and motility between tumor cells and tumor-associated macrophages on extracellular matrix (ECM) fibers of the tumor microenvironment have been shown to enhance intravasation and metastatic spread of breast carcinomas. We describe an in vitro motility assay that combines time-lapse wide-field microscopy and micro-patterned linear adhesive substrates to reconstitute the in vivo behavior between macrophages, tumor cells, and ECM fibers in orthotopic rodent tumor models observed by intravital imaging. Commercially available linear stripes of 650 nm dye-labeled fibronectin microlithographed onto glass cover slips are sequentially plated with fluorescently labeled MTLn3 tumor cells and bone marrow-derived macrophages and time-lapse imaged for up to 8 h. Incubation with pharmacological inhibitors during the assay can identify important paracrine or autocrine signaling pathways involved in the macrophage-tumor cell interaction. This high-resolution motility assay will lead to a more detailed description of immune cell-tumor cell behavior as well as interrogating additional cell types within the tumor microenvironment which use cytokine/growth factor paracrine signaling interactions to facilitate intravasation and metastasis.


Assuntos
Bioensaio/métodos , Comunicação Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Macrófagos/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Fibronectinas/química , Fibronectinas/metabolismo , Gefitinibe , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Camundongos , Imagem Molecular , Quinazolinas/farmacologia , Transdução de Sinais , Imagem com Lapso de Tempo , Tirfostinas/farmacologia
18.
Methods Mol Biol ; 1172: 125-35, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908300

RESUMO

Macrophages, which are organized throughout every tissue, represent a key component of the immune system and the recruitment of macrophages to specific sites is important in normal host defense. However, when inappropriately recruited macrophages may damage or destroy healthy tissue; this is seen in several autoimmune diseases such as arthritis. Many cytokines, including CSF-1 and chemokines, are often upregulated in inflamed tissues and can induce the directional migration of macrophages towards the highest concentration of the cytokine in a process called chemotaxis. Chemokines were first described as chemoattractant cytokines synthesized at sites of inflammation that stimulate the directional migration of leukocytes and mediate inflammation. Whereas specific receptors for chemoattractants reside over the entire cell surface, macrophages can detect very shallow chemotactic gradients leading to spatially defined responses to the chemoattractant such as the extension of directed protrusions leading to cell migration. In this chapter we describe a method for the localized delivery of chemoattractants via a micropipette needle to macrophages in culture followed by methods for imaging and an outline of quantifying macrophage responses.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Análise de Célula Única/métodos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Rastreamento de Células/métodos , Células Cultivadas , Quimiocina CX3CL1/farmacologia , Fatores Quimiotáticos/farmacologia , Corantes Fluorescentes , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Microinjeções , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Análise de Célula Única/estatística & dados numéricos
19.
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
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...