Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 63
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
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.

2.
Methods Mol Biol ; 2350: 43-68, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331278

RESUMO

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.


Assuntos
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
3.
Cell Signal ; 77: 109827, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33161094

RESUMO

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn2+/Ca2+, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.

4.
J Cell Biol ; 219(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33007084

RESUMO

In neurons, dendrites form the major sites of information receipt and integration. It is thus vital that, during development, the dendritic arbor is adequately formed to enable proper neural circuit formation and function. While several known processes shape the arbor, little is known of those that govern dendrite branching versus extension. Here, we report a new mechanism instructing dendrites to branch versus extend. In it, glutamate signaling activates mGluR5 receptors to promote Ckd5-mediated phosphorylation of the C-terminal PDZ-binding motif of delta-catenin. The phosphorylation state of this motif determines delta-catenin's ability to bind either Pdlim5 or Magi1. Whereas the delta:Pdlim5 complex enhances dendrite branching at the expense of elongation, the delta:Magi1 complex instead promotes lengthening. Our data suggest that these complexes affect dendrite development by differentially regulating the small-GTPase RhoA and actin-associated protein Cortactin. We thus reveal a "phospho-switch" within delta-catenin, subject to a glutamate-mediated signaling pathway, that assists in balancing the branching versus extension of dendrites during neural development.


Assuntos
Cateninas/metabolismo , Dendritos/fisiologia , Guanilato Quinases/metabolismo , Hipocampo/citologia , Proteínas com Domínio LIM/metabolismo , Neurogênese , Neurônios/citologia , Animais , Cateninas/genética , Guanilato Quinases/genética , Células HEK293 , Hipocampo/metabolismo , Humanos , Proteínas com Domínio LIM/genética , Neurônios/metabolismo , Fosforilação , Ratos , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
5.
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.

6.
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
7.
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
8.
J Mol Endocrinol ; 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30407917

RESUMO

Key features for progression to pancreatic ß-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in ß-cells, we manipulated their Rac1-specific GEF1 domain activity by using small molecule inhibitors and by genetically ablating Kalirin. We examined age related secretory granule behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-hour old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured ß-cell lines, demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin's participation to stimulatory glucose signaling.

9.
Nat Commun ; 9(1): 4144, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30297715

RESUMO

Studies of cancer cell migration have found two modes: one that is protease-independent, requiring micron-sized pores or channels for cells to squeeze through, and one that is protease-dependent, relevant for confining nanoporous matrices such as basement membranes (BMs). However, many extracellular matrices exhibit viscoelasticity and mechanical plasticity, irreversibly deforming in response to force, so that pore size may be malleable. Here we report the impact of matrix plasticity on migration. We develop nanoporous and BM ligand-presenting interpenetrating network (IPN) hydrogels in which plasticity could be modulated independent of stiffness. Strikingly, cells in high plasticity IPNs carry out protease-independent migration through the IPNs. Mechanistically, cells in high plasticity IPNs extend invadopodia protrusions to mechanically and plastically open up micron-sized channels and then migrate through them. These findings uncover a new mode of protease-independent migration, in which cells can migrate through confining matrix if it exhibits sufficient mechanical plasticity.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Hidrogéis/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Matriz Extracelular/química , Feminino , Humanos , Hidrogéis/química , Fenômenos Mecânicos , Camundongos Nus , Transplante Heterólogo
10.
J Cell Biol ; 217(11): 3873-3885, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30150290

RESUMO

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.


Assuntos
Actinas/metabolismo , Estruturas da Membrana Celular/metabolismo , Macrófagos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/genética , Animais , Sistemas CRISPR-Cas , Estruturas da Membrana Celular/genética , Deleção de Genes , Lipopolissacarídeos/farmacologia , Camundongos , Células RAW 264.7 , Proteínas rab de Ligação ao GTP/genética
11.
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
12.
Nat Chem Biol ; 14(9): 902, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29728601

RESUMO

In the version of this article originally published, the values for time shown on the x axis of Figure 5c were incorrect. The error has been corrected in all versions of the paper.

13.
Nat Chem Biol ; 14(6): 591-600, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29686359

RESUMO

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas rho de Ligação ao GTP/química , Animais , Técnicas Biossensoriais , Bradyrhizobium , Células HEK293 , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Optogenética , Plasmídeos , Ligação Proteica , Transdução de Sinais , Espectroscopia de Luz Próxima ao Infravermelho , Proteína cdc42 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/química
14.
Nat Commun ; 8(1): 1185, 2017 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084958

RESUMO

Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its fluctuating activity at the front edge. This differential RHOA activity determines polarized NC morphology and motility, and is regulated by the asymmetrically localized RhoGAP Deleted in liver cancer (DLC1) in the cytoplasm at the cell front. Importantly, the association of DLC1 with NEDD9 is crucial for its asymmetric localization and differential RHOA activity. Moreover, NC specifiers, SOX9 and SOX10, regulate NEDD9 and DLC1 expression, respectively. These results present a SOX9/SOX10-NEDD9/DLC1-RHOA regulatory axis to govern NCC migratory polarization.


Assuntos
Movimento Celular , Polaridade Celular , Proteínas Ativadoras de GTPase/metabolismo , Crista Neural/embriologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Técnicas Biossensoriais , Embrião de Galinha , Transferência Ressonante de Energia de Fluorescência , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/metabolismo , Fatores de Transcrição SOX9/metabolismo
15.
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
16.
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
17.
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
18.
Dev Cell ; 39(6): 708-723, 2016 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-27939686

RESUMO

Microtubules regulate signaling, trafficking, and cell mechanics, but the respective contribution of these functions to cell morphogenesis and migration in 3D matrices is unclear. Here, we report that the microtubule plus-end tracking protein (+TIP) SLAIN2, which suppresses catastrophes, is not required for 2D cell migration but is essential for mesenchymal cell invasion in 3D culture and in a mouse cancer model. We show that SLAIN2 inactivation does not affect Rho GTPase activity, trafficking, and focal adhesion formation. However, SLAIN2-dependent catastrophe inhibition determines microtubule resistance to compression and pseudopod elongation. Another +TIP, CLASP1, is also needed to form invasive pseudopods because it prevents catastrophes specifically at their tips. When microtubule growth persistence is reduced, inhibition of depolymerization is sufficient for pseudopod maintenance but not remodeling. We propose that catastrophe inhibition by SLAIN2 and CLASP1 supports mesenchymal cell shape in soft 3D matrices by enabling microtubules to perform a load-bearing function.


Assuntos
Mesoderma/metabolismo , Mesoderma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colágeno/metabolismo , Exocitose , Feminino , Adesões Focais/metabolismo , Células HEK293 , Humanos , Interfase , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Polimerização , Pseudópodes/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
19.
Sci Rep ; 6: 37874, 2016 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-27901093

RESUMO

The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/MenaINV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell - macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.


Assuntos
Macrófagos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Podossomos/metabolismo , Receptor Notch1/metabolismo , Migração Transendotelial e Transepitelial/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Humanos , Camundongos , Camundongos SCID , Invasividade Neoplásica/patologia , Podossomos/fisiologia , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Regulação para Cima/fisiologia
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...