RESUMEN
BACKGROUND: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear. METHODS: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo. RESULTS: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed iplectin (i = invadopodial). iPlectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed iplectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency. CONCLUSIONS: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and iplectin. CLINICAL TRIAL REGISTRATION NUMBER: NCT04608357.
Asunto(s)
Neoplasias de la Mama , Metaloproteinasa 14 de la Matriz , Invasividad Neoplásica , Podosomas , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Animales , Femenino , Embrión de Pollo , Línea Celular Tumoral , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Podosomas/metabolismo , Plectina/metabolismo , Plectina/genética , Membrana Corioalantoides/metabolismo , Movimiento Celular , ARN Interferente Pequeño/genéticaRESUMEN
Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Neoplasias de la Mama , Podosomas , Proteínas Qa-SNARE , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Invasividad Neoplásica , Podosomas/metabolismo , Transporte de Proteínas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Invasion of neighboring extracellular matrix (ECM) by malignant tumor cells is a hallmark of metastatic progression. This invasion can be mediated by subcellular structures known as invadopodia, the function of which depends upon soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated vesicular transport of cellular cargo. Recently, it has been shown the SNARE Syntaxin4 (Stx4) mediates trafficking of membrane type 1-matrix metalloproteinase (MT1-MMP) to invadopodia, and that Stx4 is regulated by Munc18c in this context. Here, it is observed that expression of a construct derived from the N-terminus of Stx4, which interferes with Stx4-Munc18c interaction, leads to perturbed trafficking of MT1-MMP, and reduced invadopodium-based invasion in vitro, in models of triple-negative breast cancer (TNBC). Expression of Stx4 N-terminus also led to increased survival and markedly reduced metastatic burden in multiple TNBC models in vivo. The findings are the first demonstration that disrupting Stx4-Munc18c interaction can dramatically alter metastatic progression in vivo, and suggest that this interaction warrants further investigation as a potential therapeutic target. IMPLICATIONS: Disrupting the interaction of Syntaxin4 and Munc18c may be a useful approach to perturb trafficking of MT1-MMP and reduce metastatic potential of breast cancers.
Asunto(s)
Neoplasias de la Mama , Podosomas , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/patología , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Invasividad Neoplásica/patología , Podosomas/metabolismo , Proteínas SNARE/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
During metastasis, cancer cells can invade extracellular matrix (ECM) through a process mediated by matrix-degrading protrusions of the plasma membrane, termed invadopodia. Formation of invadopodia correlates with cells' invasive and metastatic potential, and thus presents a potential target for therapeutic approaches to target metastatic progression. Invadopodia formation is dependent on the recruitment of proteins involved in intracellular signaling, actin cytoskeleton remodeling, and proteolytic matrix modification. The latter includes matrix degrading enzymes such as MT1-MMP, MMP2, and MMP9. These essential invadopodium-associated enzymes are required for localized matrix degradation, and their localization at invadopodia is central to invadopodium-based cancer cell invasion. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle traffic, including that involved in the transport of invadopodium-associated proteins, and in so doing promote modification of ECM and modulation of signaling pathways involved in the movement of cancer cells. Specific SNARE complexes have been found to support invadopodia formation, and these complexes are, in turn, regulated by associated proteins that interact specifically with SNAREs. Targeting SNARE regulatory proteins thus provides a possible approach to disrupt SNARE-dependent delivery of invadopodial proteins, including MT1-MMP, to sites of ECM modification. Here, we review recent studies of SNARE regulators that hold potential as targets for the development of anti-metastatic therapies for patients burdened with invadopodia-forming cancer types.
RESUMEN
Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of ß1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of ß1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and ß1 integrin were sequestered at the cell surface when ß1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of ß1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and ß1 integrin ultimately led to a loss of invasive behaviour.
Asunto(s)
Integrina beta1/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Integrina beta1/genética , Metaloproteinasa 14 de la Matriz/genética , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genéticaRESUMEN
Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Receptor TIE-2/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Secuencia de Aminoácidos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/genética , Glioma/patología , Células HEK293 , Humanos , Invasividad Neoplásica , TransfecciónRESUMEN
Malignant cancer cells can invade extracellular matrix (ECM) through the formation of F-actin-rich subcellular structures termed invadopodia. ECM degradation at invadopodia is mediated by matrix metalloproteinases (MMPs), and recent findings indicate that membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP, also known as MMP14) has a primary role in this process. Maintenance of an invasive phenotype is dependent on internalization of MT1-MMP from the plasma membrane and its recycling to sites of ECM remodeling. Internalization of MT1-MMP is dependent on its phosphorylation, and here we examine the role of ß1 integrin-mediated signaling in this process. Activation of ß1 integrin using the antibody P4G11 induced phosphorylation and internalization of MT1-MMP and resulted in increased cellular invasiveness and invadopodium formation in vitro We also observed phosphorylation of Src and epidermal growth factor receptor (EGFR) and an increase in their association in response to ß1 integrin activation, and determined that Src and EGFR promote phosphorylation of MT1-MMP on Thr567 These results suggest that MT1-MMP phosphorylation is regulated by a ß1 integrin-Src-EGFR signaling pathway that promotes recycling of MT1-MMP to sites of invadopodia formation during cancer cell invasion.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Integrina beta1 , Metaloproteinasa 14 de la Matriz , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Invasividad Neoplásica , Fosforilación , Transducción de SeñalRESUMEN
Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.
Asunto(s)
Membrana Celular/fisiología , Fusión de Membrana/fisiología , Cuerpos Multivesiculares/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Comunicación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Exocitosis/efectos de los fármacos , Células HCT116 , Células HeLa , Histamina/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fusión de Membrana/efectos de los fármacos , Cuerpos Multivesiculares/efectos de los fármacos , Fosforilación/efectos de los fármacos , Cloruro de Potasio/farmacología , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Histamínicos H1/efectos de los fármacos , Análisis de la Célula Individual , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMEN
Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.
Asunto(s)
Adenocarcinoma/metabolismo , Matriz Extracelular/metabolismo , Fibrosarcoma/metabolismo , Proteínas Munc18/metabolismo , Proteínas de Neoplasias/metabolismo , Podosomas/metabolismo , Proteínas Qa-SNARE/metabolismo , Adenocarcinoma/patología , Unión Competitiva , Línea Celular Tumoral , Receptores ErbB/metabolismo , Matriz Extracelular/patología , Fibrosarcoma/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas Munc18/antagonistas & inhibidores , Proteínas Munc18/química , Proteínas Munc18/genética , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Podosomas/patología , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/química , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/antagonistas & inhibidores , Proteína 2 de Membrana Asociada a Vesículas/química , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1-matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.
Asunto(s)
Extensiones de la Superficie Celular/enzimología , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Invasividad Neoplásica , Transporte de ProteínasRESUMEN
Acquisition of an invasive phenotype is prerequisite for tumor metastasis. Degradation of the extracellular matrix (ECM), and subsequent invasion by tumor cells, is mediated, in part, through subcellular structures called invadopodia. Src-dependent cytoskeletal rearrangements are required to form invadopodia, and here we identify an association between Src, epidermal growth factor receptor (EGFR), and ß1 integrin that facilitates invadopodia formation. The association of Src, EGFR and ß1 integrin is dependent upon membrane traffic that is mediated by syntaxin13 (officially known as STX12) and SNAP23; a similar dependence on these two SNARE proteins was observed for invadopodium-based matrix degradation and cell invasion. Inhibition of SNARE function impaired the delivery of Src and EGFR to developing invadopodia, as well as the ß1-integrin-dependent activation of Src and phosphorylation of EGFR on Tyr residue 845. We also identified an association between SNAP23 and ß1 integrin, and inhibition of ß1 integrin increased this association, whereas the interaction between syntaxin13 and SNAP23 was reduced. The results suggest that SNARE-dependent trafficking is regulated, in part, by ß1 integrin and is required for the delivery of Src and EGFR to sites of invadopodia formation in order to support tumor cell invasion.
Asunto(s)
Receptores ErbB/metabolismo , Integrina beta1/metabolismo , Proteínas Qa-SNARE/fisiología , Proteínas Qb-SNARE/fisiología , Proteínas Qc-SNARE/fisiología , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Humanos , Invasividad Neoplásica , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Seudópodos/metabolismoRESUMEN
In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.
Asunto(s)
Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas R-SNARE/metabolismo , Western Blotting , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Electroforesis en Gel de Poliacrilamida , Endosomas/genética , Endosomas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Metaloproteinasa 14 de la Matriz/genética , Microscopía Fluorescente , Mutación , Fosforilación/genética , Fosforilación/fisiología , Proteínas R-SNARE/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.
Asunto(s)
Membrana Celular/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Proteínas SNARE/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismo , Animales , Western Blotting , Células CHO , Adhesión Celular , Movimiento Celular , Células Cultivadas , Cricetinae , Cricetulus , Etilmaleimida/farmacología , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling. RESULTS: We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling. CONCLUSIONS: Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.
Asunto(s)
Transporte de Proteínas , Seudópodos/metabolismo , Proteínas SNARE/metabolismo , Animales , Células CHO , Adhesión Celular/genética , Movimiento Celular/genética , Cricetinae , Cricetulus , Endocitosis/genética , Matriz Extracelular/metabolismo , Células HeLa , Humanos , Ingeniería de Proteínas , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/genética , Seudópodos/genética , Proteínas SNARE/genética , Eliminación de Secuencia/genéticaRESUMEN
Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity (MIA), secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins α(4)ß(1) and α(5)ß(1), facilitating cell detachment and promoting formation of metastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I (COPI)- and coat protein complex II (COPII)-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca(2+) ions. Interestingly, the Ca(2+)-activated K(+)-channel, subfamily N, member 4 (KCa3.1), known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant-negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.
Asunto(s)
Movimiento Celular , Proteínas de la Matriz Extracelular/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Actinas/metabolismo , Calcio/metabolismo , Proteína Coat de Complejo I/metabolismo , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Pirazoles/farmacologíaRESUMEN
Cellular remodeling of the extracellular matrix (ECM), an essential component of many physiological and pathological processes, is dependent on the trafficking and secretion of matrix metalloproteinases (MMPs). Soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic has documented roles in cell-ECM interactions and the present study specifically examines SNARE function in the trafficking of MMPs during ECM degradation. Using the invasive human fibrosarcoma cell line HT-1080, we demonstrate that a plasma membrane SNARE, SNAP23, and an endosomal v-SNARE, VAMP3 (also known as cellubrevin), partly colocalize with MMP2 and MMP9, and that inhibition of these SNAREs using dominant-negative SNARE mutants impaired secretion of the MMPs. Inhibition of VAMP3, SNAP23 or syntaxin-13 using dominant-negative SNARES, RNA interference or tetanus toxin impaired trafficking of membrane type 1 MMP to the cell surface. Consistent with these observations, we found that blocking the function of these SNAREs reduced the ability of HT-1080 cells to degrade a gelatin substrate in situ and impaired invasion of HT-1080 cells in vitro. The results reveal the importance of VAMP3, syntaxin-13 and SNAP23 in the trafficking of MMP during degradation of ECM substrates and subsequent cellular invasion.
Asunto(s)
Movimiento Celular/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Transporte de Proteínas/fisiología , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteína 3 de Membrana Asociada a Vesículas/metabolismo , Línea Celular Tumoral , Gelatina/metabolismo , Humanos , Invasividad Neoplásica , Transporte de Proteínas/efectos de los fármacos , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Interferencia de ARN , Toxina Tetánica/farmacología , Proteína 3 de Membrana Asociada a Vesículas/genéticaRESUMEN
Membrane ruffles are actin-rich protrusions of the plasma membrane that can be observed on the surface of many cell types. Phase contrast and fluorescent microscopy are widely used in the analysis of ruffles, which are commonly identified in cells stained with fluorescently labeled phalloidin. Currently, comparison of cellular ruffle formation under different experimental conditions is generally qualitative or semiquantitative. Ruffle structures are often defined using manual tracing and thresholding methods. Here, we report the rapid and accurate segmentation of ruffles from two-dimensional confocal projections of cells using automated method based on well-established image processing and analysis methods. Line-shaped ruffles were detected using line detectors and were then separated from the filtered images. Automated categorizing of the segmented line structures enabled accurate quantification of the ruffles. This automated approach is efficient and reliable and hence can serve as a powerful tool in studies of the mechanism of ruffle formation.
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Actinas/química , Microscopía Confocal/métodos , Animales , Automatización , Células CHO , Cricetinae , Técnica del Anticuerpo FluorescenteRESUMEN
In the present study, we examined the role of soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic in the formation of focal adhesions during cell spreading. CHO-K1 cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (E329Q-NSF) were unable to spread as well as control cells and they formed focal adhesions (FAs) that were larger than those in control cells. FA formation was impaired in cells transfected with a dominant-negative form of RhoA, but, significantly, not in cells simultaneously expressing dominant-negative NSF. Treatment of E329Q-NSF-expressing cells with the ROCK inhibitor Y-27632 did inhibit FA formation. The results are consistent with a model of cell adhesion in which SNARE-mediated membrane traffic is required for both the elaboration of lamellipodia and the modulation of biochemical signals that control RhoA-mediated FA assembly.
Asunto(s)
Adhesiones Focales , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Proteínas SNARE/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Amidas/farmacología , Animales , Transporte Biológico , Células CHO , Membrana Celular/metabolismo , Movimiento Celular , Cricetinae , Cricetulus , Adhesiones Focales/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Sensibles a N-Etilmaleimida/antagonistas & inhibidores , Proteínas Sensibles a N-Etilmaleimida/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Piridinas/farmacología , Proteínas SNARE/genética , Fibras de Estrés/metabolismo , Toxina Tetánica/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rhoRESUMEN
In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of alpha5beta1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.