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1.
Nat Rev Mol Cell Biol ; 24(2): 86-106, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36104625

RESUMEN

Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called 'invadosomes', are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.


Asunto(s)
Podosomas , Podosomas/metabolismo , Actinas/metabolismo , Matriz Extracelular/metabolismo , Microtúbulos/metabolismo , Proteolisis
2.
Nat Commun ; 12(1): 7300, 2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34911937

RESUMEN

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.


Asunto(s)
Neoplasias de la Mama/inmunología , Macrófagos/inmunología , Células Madre Neoplásicas/citología , Animales , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Microscopía Intravital , Ratones , Ratones SCID , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/inmunología , Células Madre Neoplásicas/inmunología , Receptores Notch/genética , Receptores Notch/inmunología , Transducción de Señal , Microambiente Tumoral/inmunología
3.
Cells ; 10(6)2021 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-34205257

RESUMEN

Aligned collagen fibers provide topography for the rapid migration of single tumor cells (streaming migration) to invade the surrounding stroma, move within tumor nests towards blood vessels to intravasate and form distant metastases. Mechanisms of tumor cell motility have been studied extensively in the 2D context, but the mechanistic understanding of rapid single tumor cell motility in the in vivo context is still lacking. Here, we show that streaming tumor cells in vivo use collagen fibers with diameters below 3 µm. Employing 1D migration assays with matching in vivo fiber dimensions, we found a dependence of tumor cell motility on 1D substrate width, with cells moving the fastest and the most persistently on the narrowest 1D fibers (700 nm-2.5 µm). Interestingly, we also observed nuclear deformation in the absence of restricting extracellular matrix pores during high speed carcinoma cell migration in 1D, similar to the nuclear deformation observed in tumor cells in vivo. Further, we found that actomyosin machinery is aligned along the 1D axis and actomyosin contractility synchronously regulates cell motility and nuclear deformation. To further investigate the link between cell speed and nuclear deformation, we focused on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex proteins and SRF-MKL1 signaling, key regulators of mechanotransduction, actomyosin contractility and actin-based cell motility. Analysis of The Cancer Genome Atlas dataset showed a dramatic decrease in the LINC complex proteins SUN1 and SUN2 in primary tumor compared to the normal tissue. Disruption of LINC complex by SUN1 + 2 KD led to multi-lobular elongated nuclei, increased tumor cell motility and concomitant increase in F-actin, without affecting Lamin proteins. Mechanistically, we found that MKL1, an effector of changes in cellular G-actin to F-actin ratio, is required for increased 1D motility seen in SUN1 + 2 KD cells. Thus, we demonstrate a previously unrecognized crosstalk between SUN proteins and MKL1 transcription factor in modulating nuclear shape and carcinoma cell motility in an in vivo relevant 1D microenvironment.


Asunto(s)
Movimiento Celular , Núcleo Celular/metabolismo , Matriz Extracelular/metabolismo , Neoplasias Mamarias Animales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismo , Microambiente Tumoral , Animales , Línea Celular Tumoral , Núcleo Celular/patología , Matriz Extracelular/patología , Femenino , Neoplasias Mamarias Animales/patología , Ratones SCID , Ratas
4.
Nat Chem Biol ; 16(9): 1034, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32694868

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

5.
Nat Chem Biol ; 15(12): 1183-1190, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31740825

RESUMEN

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.


Asunto(s)
Acetilesterasa/química , Factores Despolimerizantes de la Actina/química , Optogenética , Tubulina (Proteína)/química , Acetilación
6.
Biotechniques ; 66(3): 113-119, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30869550

RESUMEN

We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time. Use of actin monoclonal antibodies revealed significant limitations under a variety of fixation-permeabilization conditions. Evaluation of commonly used live-cell probes provides evidence for actin filament bias, with TagRFP-Lifeact excluded from lamellipodia, but not mEGFP-Lifeact or F-tractin-EGFP.


Asunto(s)
Citoesqueleto de Actina/genética , Actinas/aislamiento & purificación , Colorantes Fluorescentes/química , Citoesqueleto de Actina/química , Actinas/química , Anticuerpos/química , Anticuerpos/farmacología , Colorantes Fluorescentes/farmacología , Maleimidas/química , Maleimidas/farmacología , Faloidina/química , Faloidina/farmacología , Seudópodos/química , Seudópodos/genética
7.
Trends Cell Biol ; 27(8): 595-607, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28412099

RESUMEN

Invadopodia are a subset of invadosomes that are implicated in the integration of signals from the tumor microenvironment to support tumor cell invasion and dissemination. Recent progress has begun to define how tumor cells regulate the plasticity necessary for invadopodia to assemble and function efficiently in the different microenvironments encountered during dissemination in vivo. Exquisite mapping by many laboratories of the pathways involved in integrating diverse invadopodium initiation signals, from growth factors, to extracellular matrix (ECM) and cell-cell contact in the tumor microenvironment, has led to insight into the molecular basis of this plasticity. Here, we integrate this new information to discuss how the invadopodium is an important conductor that orchestrates tumor cell dissemination during metastasis.


Asunto(s)
Movimiento Celular , Neoplasias/metabolismo , Podosomas/metabolismo , Microambiente Tumoral , Animales , Comunicación Celular , Matriz Extracelular/metabolismo , Humanos , Invasividad Neoplásica , Neoplasias/patología
8.
Sci Rep ; 6: 37874, 2016 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-27901093

RESUMEN

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.


Asunto(s)
Macrófagos/metabolismo , Proteínas de Microfilamentos/metabolismo , Podosomas/metabolismo , Receptor Notch1/metabolismo , Migración Transendotelial y Transepitelial/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Humanos , Ratones , Ratones SCID , Invasividad Neoplásica/patología , Podosomas/fisiología , Transducción de Señal/fisiología , Microambiente Tumoral/fisiología , Regulación hacia Arriba/fisiología
9.
Sci Rep ; 6: 36142, 2016 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-27824079

RESUMEN

Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B.


Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , Cortactina/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Podosomas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cortactina/genética , Femenino , Humanos , Ratones , Proteínas de Microfilamentos/genética , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Fosforilación/genética , Podosomas/genética , Podosomas/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo
10.
J Phys Chem A ; 120(12): 2041-8, 2016 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-26916368

RESUMEN

The gas-phase molecular structure of ketene has been determined using samples generated by the pyrolysis of acetic anhydride (giving acetic acid and ketene), using one permutation of the very-high-temperature (VHT) inlet nozzle system designed and constructed for the gas electron diffraction (GED) apparatus based at the University of Canterbury. The gas-phase structures of acetic anhydride, acetic acid, and ketene are presented and compared to previous electron diffraction and microwave spectroscopy data to show improvements in data extraction and manipulation with current methods. Acetic anhydride was modeled with two conformers, rather than a complex dynamic model as in the previous study, to allow for inclusion of multiple pyrolysis products. The redetermined gas-phase structure of acetic anhydride (obtained using the structure analysis restrained by ab initio calculations for electron diffraction method) was compared to that from the original study, providing an improvement on the description of the low vibrational torsions compared to the dynamic model. Parameters for ketene and acetic acid (both generated by the pyrolysis of acetic anhydride) were also refined with higher accuracy than previously reported in GED studies, with structural parameter comparisons being made to prior experimental and theoretical studies.

11.
Mol Biol Cell ; 26(21): 3867-78, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-26337385

RESUMEN

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.


Asunto(s)
Proteínas de Microfilamentos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Actinas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Proteínas del Citoesqueleto , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Femenino , Humanos , Metástasis de la Neoplasia , Fosforilación , Isoformas de Proteínas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos
12.
Methods Mol Biol ; 1172: 115-23, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24908299

RESUMEN

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.


Asunto(s)
Bioensayo/métodos , Comunicación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Macrófagos/efectos de los fármacos , Glándulas Mamarias Humanas/efectos de los fármacos , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/química , Fibronectinas/metabolismo , Gefitinib , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Ratones , Imagen Molecular , Quinazolinas/farmacología , Transducción de Señal , Imagen de Lapso de Tiempo , Tirfostinos/farmacología
13.
Curr Biol ; 23(21): 2079-89, 2013 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-24206842

RESUMEN

BACKGROUND: Tks5 regulates invadopodium formation, but the precise timing during invadopodium lifetime (initiation, stabilization, maturation) when Tks5 plays a role is not known. RESULTS: We report new findings based on high-resolution spatiotemporal live-cell imaging of invadopodium precursor assembly. Cortactin, N-WASP, cofilin, and actin arrive together to form the invadopodium precursor, followed by Tks5 recruitment. Tks5 is not required for precursor initiation but is needed for precursor stabilization, which requires the interaction of the phox homology (PX) domain of Tks5 with PI(3,4)P2. During precursor formation, PI(3,4)P2 is uniformly distributed but subsequently starts accumulating at the precursor core 3-4 min after core initiation, and conversely, PI(3,4,5)P3 gets enriched in a ring around the precursor core. SHIP2, a 5'-inositol phosphatase, localizes at the invadopodium core and regulates PI(3,4)P2 levels locally at the invadopodium. The timing of SHIP2 arrival at the invadopodium precursor coincides with the onset of PI(3,4)P2 accumulation. Consistent with its late arrival, we found that SHIP2 inhibition does not affect precursor formation but does cause decreases in mature invadopodia and matrix degradation, whereas SHIP2 overexpression increases matrix degradation. CONCLUSIONS: Together, these findings lead us to propose a new sequential model that provides novel insights into molecular mechanisms underlying invadopodium precursor initiation, stabilization, and maturation into a functional invadopodium.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Metástasis de la Neoplasia/patología , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Técnicas Biosensibles , Extensiones de la Superficie Celular/metabolismo , Femenino , Humanos , Inositol Polifosfato 5-Fosfatasas , Proteínas de Microfilamentos/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética
14.
Nat Rev Mol Cell Biol ; 14(7): 405-15, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23778968

RESUMEN

Recently, a consensus has emerged that cofilin severing activity can generate free actin filament ends that are accessible for F-actin polymerization and depolymerization without changing the rate of G-actin association and dissociation at either filament end. The structural basis of actin filament severing by cofilin is now better understood. These results have been integrated with recently discovered mechanisms for cofilin activation in migrating cells, which led to new models for cofilin function that provide insights into how cofilin regulation determines the temporal and spatial control of cell behaviour.


Asunto(s)
Factores Despolimerizantes de la Actina/fisiología , Movimiento Celular , Factores Despolimerizantes de la Actina/química , Actinas/metabolismo , Animales , Extensiones de la Superficie Celular/metabolismo , Humanos , Modelos Moleculares , Fosforilación , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas
15.
J Cell Sci ; 126(Pt 15): 3356-69, 2013 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-23704350

RESUMEN

Protrusion formation is the first step that precedes cell movement of motile cells. Spatial control of actin polymerization is necessary to achieve directional protrusion during cell migration. Here we show that the spatial coordinators p190RhoGEF and p190RhoGAP regulate actin polymerization during leading edge protrusions by regulating the actin barbed end distribution and amplitude. The distribution of RhoC activity and proper balance of cofilin activation achieved by p190RhoGEF and p190RhoGAP determines the direction of final protrusive activity. These findings provide a new insight into the dynamic plasticity in the amplitude and distribution of barbed ends, which can be modulated by fine-tuning RhoC activity by upstream GEFs and GAPs for directed cell motility.


Asunto(s)
Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Actinas/ultraestructura , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/patología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Quimiotaxis/fisiología , Neoplasias Mamarias Experimentales/patología , Ratas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transfección , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genética
16.
Mol Biol Cell ; 24(11): 1661-75, S1-11, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23552693

RESUMEN

ß1 integrin has been shown to promote metastasis in a number of tumor models, including breast, ovarian, pancreatic, and skin cancer; however, the mechanism by which it does so is poorly understood. Invasive membrane protrusions called invadopodia are believed to facilitate extracellular matrix degradation and intravasation during metastasis. Previous work showed that ß1 integrin localizes to invadopodia, but its role in regulating invadopodial function has not been well characterized. We find that ß1 integrin is required for the formation of mature, degradation-competent invadopodia in both two- and three-dimensional matrices but is dispensable for invadopodium precursor formation in metastatic human breast cancer cells. ß1 integrin is activated during invadopodium precursor maturation, and forced ß1 integrin activation enhances the rate of invadopodial matrix proteolysis. Furthermore, ß1 integrin interacts with the tyrosine kinase Arg and stimulates Arg-dependent phosphorylation of cortactin on tyrosine 421. Silencing ß1 integrin with small interfering RNA completely abrogates Arg-dependent cortactin phosphorylation and cofilin-dependent barbed-end formation at invadopodia, leading to a significant decrease in the number and stability of mature invadopodia. These results describe a fundamental role for ß1 integrin in controlling actin polymerization-dependent invadopodial maturation and matrix degradation in metastatic tumor cells.


Asunto(s)
Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Integrina beta1/genética , Proteínas Tirosina Quinasas/genética , Seudópodos/metabolismo , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Cortactina/genética , Cortactina/metabolismo , Humanos , Integrina beta1/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Proteínas Tirosina Quinasas/metabolismo , Seudópodos/genética , Seudópodos/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Tirosina/metabolismo
17.
Intravital ; 1(1): 77-85, 2012 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24634804

RESUMEN

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF) / colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as "streams." Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging.

18.
Curr Biol ; 21(8): 635-44, 2011 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-21474314

RESUMEN

BACKGROUND: RhoGTPases have been implicated in the regulation of cancer metastasis. Invasive carcinoma cells form invadopodia, F-actin-rich matrix-degrading protrusions that are thought to be important for tumor cell invasion and intravasation. Regulation of actin dynamics at invadopodial protrusions is crucial to drive invasion. This process requires the severing activity of cofilin to generate actin-free barbed ends. Previous work demonstrates that cofilin's severing activity is tightly regulated through multiple mechanisms, including regulation of cofilin serine phosphorylation by Rho GTPases. However, it is not known which Rho GTPase is involved in regulating cofilin's phosphorylation status at invadopodia. RESULTS: We show here, for the first time, how RhoC activation is controlled at invadopodia and how this activation regulates cofilin phosphorylation to control cofilin's generation of actin-free barbed ends. Live-cell imaging of fluorescent RhoC biosensor reveals that RhoC activity is spatially confined to areas surrounding invadopodia. This spatiotemporal restriction of RhoC activity is controlled by "spatially distinct regulatory elements" that confine RhoC activation within this compartment. p190RhoGEF localizes around invadopodia to activate RhoC, whereas p190RhoGAP localizes inside invadopodia to deactivate the GTPase within the structure. RhoC activation enhances cofilin phosphorylation outside invadopodia. CONCLUSION: These results show how RhoC activity is spatially regulated at invadopodia by p190RhoGEF and p190RhoGAP. RhoC activation in areas surrounding invadopodia restricts cofilin activity to within the invadopodium core, resulting in a focused invadopodial protrusion. This mechanism likely enhances tumor cell invasion during metastasis.


Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Adenocarcinoma/metabolismo , Movimiento Celular , Extensiones de la Superficie Celular/metabolismo , Neoplasias Mamarias Animales/metabolismo , Metástasis de la Neoplasia/patología , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Adenocarcinoma/patología , Animales , Técnicas Biosensibles , Neoplasias Mamarias Animales/patología , Fosforilación , Ratas , Serina/metabolismo , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genética , Proteína rhoC de Unión a GTP
19.
Mol Biol Cell ; 20(21): 4509-23, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19741095

RESUMEN

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.


Asunto(s)
Movimiento Celular/fisiología , Cofilina 1/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Forma de la Célula , Cofilina 1/genética , Humanos , Ratones , Células 3T3 NIH , Seudópodos/metabolismo , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
20.
Inorg Chem ; 48(17): 8603-12, 2009 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-19663455

RESUMEN

The molecular structures of allyl-, allenyl-, propargyl-, vinyl-, ethynyl-, phenyl-, benzyl-, and chloromethyl-phosphine have been determined from gas-phase electron diffraction data employing the SARACEN method. The experimental geometric parameters are compared with those obtained using ab initio calculations performed at the MP2 level using both Pople-type basis sets and the correlation-consistent basis sets of Dunning. The structure and conformational behavior of each molecule have been analyzed and, where possible, comparisons made to the analogous amine. For systems with multiple conformers, differences in the CCP bond angle of approximately 5 degrees between conformers are common. Trends in the key parameters are identified and compared with those found in similar systems.


Asunto(s)
Simulación por Computador , Electrones , Modelos Químicos , Fosfinas/química , Teoría Cuántica , Aminas/química , Gases/química , Modelos Moleculares , Estructura Molecular , Fosfinas/síntesis química
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