RESUMEN
In vitro tests of cancer cell invasion are the "first line" tools of preclinical researchers for screening the multitude of chemical compounds or cell perturbations that may aid in halting or treating cancer malignancy. In order to have predictive value or to contribute to designing personalized treatment regimes, these tests need to take into account the cancer cell environment and measure effects on invasion in sufficient detail. The in vitro invasion assays presented here are a trade-off between feasibility in a multisample format and mimicking the complexity of the tumor microenvironment. They allow testing multiple samples and conditions in parallel using 3D-matrix-embedded cells and deal with the heterogeneous behavior of an invading cell population in time. We describe the steps to take, the technical problems to tackle and useful software tools for the entire workflow: from the experimental setup to the quantification of the invasive capacity of the cells. The protocol is intended to guide researchers to standardize experimental set-ups and to annotate their invasion experiments in sufficient detail. In addition, it provides options for image processing and a solution for storage, visualization, quantitative analysis, and multisample comparison of acquired cell invasion data.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Esferoides Celulares/citología , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Células MCF-7 , Microscopía de Contraste de Fase , Esferoides Celulares/metabolismoRESUMEN
Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.
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Citoesqueleto de Actina/genética , Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Fosfoproteínas/metabolismo , Proteína de Unión al GTP cdc42/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/genética , Embrión de Mamíferos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/fisiología , Unión Proteica , Multimerización de Proteína/genética , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The conserved alphaherpesviral serine/threonine kinase US3 causes dramatic actin rearrangements, associated with increased viral spread. Here, we show that US3 of pseudorabies virus (PRV) leads to activation (dephosphorylation) of the central actin regulator cofilin. A mutation that impairs US3 kinase activity and the group I p21-activated kinase inhibitor IPA-3 inhibited US3-mediated cofilin activation. Additionally, expression of phosphomimetic S3D cofilin significantly suppressed the ability of US3 to cause cell projections and cell rounding. In conclusion, the US3 kinase of PRV leads to activation (dephosphorylation) of cofilin, and cofilin contributes to US3-mediated actin rearrangements.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Herpesvirus Suido 1/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Internalización del Virus , Western Blotting , Forma de la Célula/fisiología , Inhibidores Enzimáticos/metabolismo , Microscopía Fluorescente , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Quinasas p21 Activadas/antagonistas & inhibidoresRESUMEN
We previously demonstrated that the Bcr-Abl oncogene, p210(bcr-abl), through its unique GEF domain, specifically activates RhoA and induces spontaneous amoeboid motility. We intend to study the pathways downstream RhoA controlling amoeboid motility. Mouse prolymphoblastic cells (Ba/F3 cell line) expressing different forms of Bcr-Abl were embedded in 3-dimensional (3D) Matrigel to study motility and explore the effects of inhibiting Rho pathway (inhibitors and siRNAs). The phosphorylation levels of cofilin-1 and destrin were analyzed by 2-dimensional electrophoresis. Composition of Bcr-Abl signalplex in different conditions was determined by coimmunoprecipitation. Ba/F3p190 and Ba/F3 expressing a mutant form of p210(bcr-abl) (unable to activate RhoA) cells presented a spontaneous motility, but not an amoeboid type. p210(bcr-abl)-induced amoeboid motility in a 3D matrix requires isoform-specific RhoA/ROCK-1/destrin signaling. Next to the conventional Rho/ROCK/MLC/myosin pathway, this pathway is a crucial determinant for amoeboid motility, specific for the destrin isoform (and not its coexpressed homologue cofilin-1). Also, the presence of destrin (and not cofilin-1) in the p210(bcr-abl) complex is dependent on ROCK1, and this signalplex is required for amoeboid motility. This underscores isoform-specific function within the ADF/cofilin family and provides new insight into Bcr-Abl signaling to amoeboid motility and possible impact on understanding chronic myeloid leukemia progression.
Asunto(s)
Amoeba/fisiología , Citocinas/metabolismo , Destrina/metabolismo , Proteínas de Fusión bcr-abl/fisiología , Proteínas de Neoplasias/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Ratones , Microscopía FluorescenteRESUMEN
Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic ß- and γ-actin. Because of the presence and localized translation of ß-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates ß-actin in gene regulation. Cell migration without ß-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking ß-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, ß-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of ß-actin knockout cells. This also explains why reintroducing ß-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in ß-actin knockout cells based on increased Rho-ROCK signaling and increased TGFß production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of ß-actin knockout cells indicating that other actins compensate for ß-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but ß-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/fisiología , Fibroblastos/metabolismo , Proteómica/métodos , Actinas/genética , Amidas/farmacología , Animales , Western Blotting , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Seudópodos/genética , Seudópodos/metabolismo , Seudópodos/fisiología , Piridinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: The Enabled/Vasodilator stimulated phosphoprotein (Ena/VASP) gene family comprises three genes in vertebrates: Vasp, Enabled homologue (Enah) and Ena-VASP like (Evl). Enah has the most complex gene structure. It has extra alternatively included exons compared to Vasp and Evl, and possibly one alternatively excluded intron S. The aim of this mapping study was to probe the occurrence of combinations of exon usage in Enah thereby identifying possible vertebrate ENAH splice variants. We investigated this via an in silico analysis and by performing a reverse transcription-polymerase chain reaction (RT-PCR) screen on mouse samples. We further probed the expression pattern of mouse Enah splice variants during development and in a selection of mouse adult tissues and mouse cell lines. RESULTS: In silico analysis of the vertebrate Ena/VASP gene family reveals that birds do not have Vasp, while fish have two Evl genes. Analysis of expressed sequence tags of vertebrate Enah splice variants confirms that an Enah transcript without alternative exons is ubiquitously expressed, but yields only limited information about the existence of other possible alternatively spliced Enah transcripts. Via a RT-PCR screen, we provide evidence that during mouse development and in adult mice at least eight and maximally sixteen different Enah transcripts are expressed. We also show that tissues and cell lines display specific expression profiles of these different transcripts. Exons previously associated with neuronal expression of Enah splice variants are also present in other tissues, in particular in heart. CONCLUSIONS: We propose a more uniform nomenclature for alternative exons in Enah. We provide an overview of distinct expression profiles of mouse Enah splice variants during mouse development, in adult mouse tissues and in a subset of mouse cell lines.
Asunto(s)
Empalme Alternativo , Proteínas del Citoesqueleto/genética , Animales , Moléculas de Adhesión Celular/clasificación , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Proteínas del Citoesqueleto/clasificación , Proteínas del Citoesqueleto/metabolismo , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Exones , Perfilación de la Expresión Génica , Intrones , Ratones , Proteínas de Microfilamentos/clasificación , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/clasificación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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/metabolismoRESUMEN
Nemaline myopathy is a neuromuscular disorder, characterized by muscle weakness and hypotonia and is, in 20% of the cases, caused by mutations in the gene encoding alpha-skeletal muscle actin, ACTA1. It is a heterogeneous disease with various clinical phenotypes and severities. In patients the ultrastructure of muscle cells is often disturbed by nemaline rods and it is thought this is the cause for muscle weakness. To search for possible defects during muscle cell differentiation we expressed alpha-actin mutants in myoblasts and allowed these cells to differentiate into myotubes. Surprisingly, we observed two striking new phenotypes in differentiating myoblasts: rounding up of cells and bleb formation, two features reminiscent of apoptosis. Indeed expression of these mutants induced cell death with apoptotic features in muscle cell culture, using AIF and endonuclease G, in a caspase-independent but calpain-dependent pathway. This is the first report on a common cellular defect induced by NM causing actin mutants, independent of their biochemical phenotypes or rod and aggregate formation capacity. These data suggest that lack of type II fibers or atrophy observed in nemaline myopathy patients may be also due to an increased number of dying muscle cells.
Asunto(s)
Actinas/fisiología , Apoptosis , Músculo Esquelético/metabolismo , Mutación/genética , Mioblastos/metabolismo , Miopatías Nemalínicas/patología , Animales , Animales Recién Nacidos , Factor Inductor de la Apoptosis/metabolismo , Calpaína/metabolismo , Caspasas/metabolismo , Diferenciación Celular , Membrana Celular/metabolismo , Células Cultivadas , Endodesoxirribonucleasas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Mutagénesis Sitio-Dirigida , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Fenotipo , Conformación Proteica , RatasRESUMEN
BACKGROUND: Nemaline myopathy is a neuromuscular disorder characterized by the presence of nemaline bodies in patient muscles. 20% of the cases are associated with alpha-skeletal muscle actin mutations. We previously showed that actin mutations can cause four different biochemical phenotypes and that expression of NM associated actin mutants in fibroblasts, myoblasts and myotubes induces a range of cellular defects. FINDINGS: We conducted the same biochemical experiments for twelve new actin mutants associated with nemaline myopathy. We observed folding and polymerization defects. Immunostainings of these and eight other mutants in transfected cells revealed typical cellular defects such as nemaline rods or aggregates, decreased incorporation in F-actin structures, membrane blebbing, the formation of thickened actin fibres and cell membrane blebbing in myotubes. CONCLUSION: Our results confirm that NM associated alpha-actin mutations induce a range of defects at the biochemical level as well as in cultured fibroblasts and muscle cells.
RESUMEN
Central core disease (CCD), congenital fibre type disproportion (CFTD), and nemaline myopathy (NM) are earlyonset clinically heterogeneous congenital myopathies, characterized by generalized muscle weakness and hypotonia. All three diseases are associated with alpha-skeletal muscle actin mutations. We biochemically characterized the CCD and CFTD causing actin mutants and show that all mutants fold correctly and are stable. Expression studies in fibroblasts, myoblasts, and myotubes show that these mutants incorporate in filamentous structures. However they do not intercalate between the nascent z-lines in differentiating muscle cell cultures. We also show that the distribution of mitochondria and of the ryanodine receptors, and calcium release properties from ryanodine receptors, are unchanged in myotubes expressing the CCD causing mutants. CFTD causing mutants induce partly similar phenotypes as NM associated ones, such as rods and thickened actin fibers in cell culture. Our results suggest that molecular mechanisms behind CFTD and NM may be partly related.
Asunto(s)
Actinas/genética , Miopatías Estructurales Congénitas/genética , Miopatía del Núcleo Central/genética , Actinas/deficiencia , Actinas/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Fibroblastos/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Modelos Moleculares , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Mioblastos/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Miopatías Estructurales Congénitas/metabolismo , Miopatía del Núcleo Central/metabolismo , Células 3T3 NIH , Conformación Proteica , Pliegue de Proteína , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
We showed earlier that human beta-thymosin 15 (Tb15) is up-regulated in prostate cancer, confirming studies from others that propagated Tb15 as a prostate cancer biomarker. In this first report on mouse Tb15, we show that, unlike in humans, four Tb15-like isoforms are present in mouse. We used phylogenetic analysis of deuterostome beta-thymosins to show that these four new isoforms cluster within the vertebrate Tb15-clade. Intriguingly, one of these mouse beta-thymosins, Tb15r, consists of two beta-thymosin domains. The existence of such a repeat beta-thymosin is so far unique in vertebrates, though common in lower eukaryotes. Biochemical data indicate that Tb15r potently sequesters actin. In a cellular context, Tb15r behaves as a bona fide beta-thymosin, lowering central stress fibre content. We reveal that a complex genomic organization underlies Tb15r expression: Tb15r results from read-through transcription and alternative splicing of two tandem duplicated mouse Tb15 genes. Transcript profiling of all mouse beta-thymosin isoforms (Tb15s, Tb4 and Tb10) reveals that two isoform switches occur between embryonic and adult tissues, and indicates Tb15r as the major mouse Tb15 isoform in adult cells. Tb15r is present also in mouse prostate cancer cell lines. This insight into the mouse Tb15 family is fundamental for future studies on Tb15 in mouse (prostate) cancer models.
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Timosina/química , Timosina/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Mapeo Cromosómico , Cartilla de ADN/genética , Duplicación de Gen , Humanos , Técnicas In Vitro , Masculino , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Células 3T3 NIH , Filogenia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Conejos , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Timosina/metabolismoRESUMEN
The chaperones prefoldin and the cytosolic chaperonin CCT-containing TCP-1 (CCT) guide the cytoskeletal protein actin to its native conformation. Performing an alanine scan of actin, we identified discrete recognition determinants for CCT interaction. Interestingly, one of these is similar and functional in the non-homologous protein Cdc20, suggesting that some of the binding information in the CCT target proteins is shared. The information in actin for recognition by CCT and for folding is different, as all but one of the mutants in the recognition determinants are folding-competent. In addition, some other actin mutants remain CCT-arrested and are not released in a native conformation, whereas others do fold but remain bound to CAP. Kinetic experiments provide evidence that CCT-mediated folding of non-native actin occurs in at least two steps, in which initially the recognition determinant 245-249 contacts CCT and the other determinants interact at later stages. Actin mutants that are CCT-arrested demonstrate that some regions neighbouring the recognition determinants are involved in modulating the correct folding transitions of actin on CCT, or its release from this chaperonin. Further, we found that the ATP binding of actin is not a prerequisite for its release, and we suggest that CAP may be involved in charging the nucleotide. Based on the kinetics of CCT binding and folding of actin and actin mutants, we propose a multi-step recognition-transition-release model. This also implies that the currently accepted notion of CCT-mediated actin folding is probably more complex.
Asunto(s)
Actinas/química , Chaperoninas/fisiología , Modelos Moleculares , Pliegue de Proteína , Actinas/genética , Secuencia de Aminoácidos , Sitios de Unión , Chaperonina con TCP-1 , Humanos , Cinética , Mutación Missense , Unión ProteicaRESUMEN
Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three beta-actin mutants that have been associated with diseases.
RESUMEN
Mutations in the gene encoding alpha-skeletal-muscle actin, ACTA1, cause congenital myopathies of various phenotypes that have been studied since their discovery in 1999. Although much is now known about the clinical aspects of myopathies resulting from over 60 different ACTA1 mutations, we have very little evidence for how mutations alter the behavior of the actin protein and thus lead to disease. We used a combination of biochemical and cell biological analysis to classify 19 myopathy mutants and found a range of defects in the actin. Using in vitro expression systems, we probed actin folding and actin's capacity to interact with actin-binding proteins and polymerization. Only two mutants failed to fold; these represent recessive alleles, causing severe myopathy, indicating that patients produce nonfunctional actin. Four other mutants bound tightly to cyclase-associated protein, indicating a possible instability in the nucleotide-binding pocket, and formed rods and aggregates in cells. Eleven mutants showed defects in the ability to co-polymerize with wild-type actin. Some of these could incorporate into normal actin structures in NIH 3T3 fibroblasts, but two of the three tested also formed aggregates. Four mutants showed no defect in vitro but two of these formed aggregates in cells, indicating functional defects that we have not yet tested for. Overall, we found a range of defects and behaviors of the mutants in vitro and in cultured cells, paralleling the complexity of actin-based muscle myopathy phenotypes.
Asunto(s)
Actinas/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Mutación , Actinas/química , Alelos , Animales , Fibroblastos/metabolismo , Ratones , Microscopía Fluorescente , Modelos Moleculares , Enfermedades Musculares/patología , Células 3T3 NIH , Fenotipo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , TransfecciónRESUMEN
Actin is one of the most conserved and versatile proteins capable of forming homopolymers and interacting with numerous other proteins in the cell. We performed an alanine mutagenesis scan covering the entire beta-actin molecule. Somewhat surprisingly, the majority of the mutants were capable of reaching a stable conformation. We tested the ability of these mutants to bind to various actin binding proteins, thereby mapping different interfaces with actin. Additionally, we tested their ability to copolymerize with alpha-actin in order to localize regions in actin that contact neighboring protomers in the filament. Hereby, we could discriminate between two existing models for filamentous actin and our data strongly support the right-handed double-stranded helix model. We present data corroborating this model in vivo. Mutants defective in copolymerization do not colocalize with the actin cytoskeleton and some impair its normal function, thereby disturbing cell shape.