RESUMEN
At invasion, transformed mammary epithelial cells expand into the stroma through a disrupted myoepithelial (ME) cell layer and basement membrane (BM). The intact ME cell layer has thus been suggested to act as a barrier against invasion. Here, we investigate the mechanisms behind the disruption of ME cell layer. We show that the expression of basal/ME proteins CK5, CK14, and α-SMA altered along increasing grade of malignancy, and their loss affected the maintenance of organotypic 3D mammary architecture. Furthermore, our data suggests that loss of CK5 prior to invasive stage causes decreased levels of Zinc finger protein SNAI2 (SLUG), a key regulator of the mammary epithelial cell lineage determination. Consequently, a differentiation bias toward luminal epithelial cell type was detected with loss of mature, α-SMA-expressing ME cells and reduced deposition of basement membrane protein laminin-5. Therefore, our data discloses the central role of CK5 in mammary epithelial differentiation and maintenance of normal ME layer.
RESUMEN
Contractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Miosinas/metabolismo , Fibras de Estrés/metabolismoRESUMEN
Epithelial integrity is lost upon cancer progression as cancer cells detach from the primary tumor site and start to invade to the surrounding tissues. Invasive cancers of epithelial origin often express altered levels of TRP-family cation channels. Upregulation of TRPV6 Ca2+-channel has been associated with a number of human malignancies and its high expression in breast cancer has been linked to both proliferation and invasive disease. The mechanisms behind the potential of TRPV6 to induce invasive progression have, however, not been well elucidated. Here we show that TRPV6 is connected to both E-cadherin-based adherens junctions and intracellular cytoskeletal structures. Loss of TRPV6 from normal mammary epithelial cells led to disruption of epithelial integrity and abnormal 3D-mammo sphere morphology. Furthermore, expression level of TRPV6 was tightly linked to the levels of common EMT markers, suggesting that TRPV6 may have a role in the mesenchymal invasion of breast cancer cells. Thus, either too low or too high TRPV6 levels compromise homeostasis of the mammary epithelial sheets and may promote the progression of pathophysiological conditions.
Asunto(s)
Canales de Calcio/metabolismo , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Canales Catiónicos TRPV/metabolismo , Actomiosina/análisis , Actomiosina/metabolismo , Uniones Adherentes/metabolismo , Neoplasias de la Mama/metabolismo , Cadherinas/análisis , Cadherinas/metabolismo , Canales de Calcio/análisis , Línea Celular , Línea Celular Tumoral , Células Epiteliales/citología , Femenino , Humanos , Canales Catiónicos TRPV/análisisRESUMEN
Defects in the maintenance of intercellular junctions are associated with loss of epithelial barrier function and consequent pathological conditions, including invasive cancers. Epithelial integrity is dependent on actomyosin bundles at adherens junctions, but the origin of these junctional bundles is incompletely understood. Here we show that peripheral actomyosin bundles can be generated from a specific actin stress fiber subtype, transverse arcs, through their lateral fusion at cell-cell contacts. Importantly, we find that assembly and maintenance of peripheral actomyosin bundles are dependent on the mechanosensitive CaMKK2/AMPK signaling pathway and that inhibition of this route leads to disruption of tension-maintaining actomyosin bundles and re-growth of stress fiber precursors. This results in redistribution of cellular forces, defects in monolayer integrity, and loss of epithelial identity. These data provide evidence that the mechanosensitive CaMKK2/AMPK pathway is critical for the maintenance of peripheral actomyosin bundles and thus dictates cell-cell junctions through cellular force distribution.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Actomiosina/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Células Epiteliales/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Fenómenos Biomecánicos , Cadherinas/metabolismo , Moléculas de Adhesión Celular , Comunicación Celular , Línea Celular , Movimiento Celular , Polaridad Celular , Células Cultivadas , Perros , Activación Enzimática , Células Epiteliales/citología , Transición Epitelial-Mesenquimal , Femenino , Humanos , Proteínas de Microfilamentos , Modelos Biológicos , Fenotipo , Fosfoproteínas , Fibras de Estrés/metabolismo , Regulación hacia ArribaRESUMEN
Controlled transport of macromolecules between the cytoplasm and nucleus is essential for homeostatic regulation of cellular functions. For instance, gene expression entails coordinated nuclear import of transcriptional regulators to activate transcription and nuclear export of the resulting messenger RNAs for cytoplasmic translation. Here we link these two processes by reporting a novel role for the mRNA export factor Ddx19/Dbp5 in nuclear import of MKL1, the signal-responsive transcriptional activator of SRF. We show that Ddx19 is not a general nuclear import factor, and that its specific effect on MKL1 nuclear import is separate from its role in mRNA export. Both helicase and nuclear pore-binding activities of Ddx19 are dispensable for MKL1 nuclear import, but RNA binding is required. Mechanistically, Ddx19 operates by modulating the conformation of MKL1, which affects its interaction with Importin-ß for efficient nuclear import. Thus, Ddx19 participates in mRNA export, translation and nuclear import of a key transcriptional regulator.
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Transporte Activo de Núcleo Celular , ARN Helicasas DEAD-box/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transactivadores/metabolismo , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Homeostasis , Humanos , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Unión Proteica , Conformación Proteica , ARN/metabolismo , beta Carioferinas/metabolismoRESUMEN
Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina , Actinas/genética , Núcleo Celular/metabolismo , Expresión Génica , Vía de Señalización Hippo , Humanos , Proteínas de Microfilamentos/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/genéticaRESUMEN
Phactr proteins bind actin and protein phosphatase 1 (PP1), and are involved in processes ranging from angiogenesis to cell cycle regulation. Phactrs share a highly conserved RPEL domain with the myocardin-related transcription factor (MRTF) family, where actin binding to this domain regulates both the nuclear localization and the activity of these transcription coactivators. We show here that in contrast to MRTF-A, the RPEL domain is dispensable for the subcellular localization of Phactr4. Instead, we find the domain facilitating competitive binding of monomeric actin and PP1 to Phactr4. Binding of actin to Phactr4 influences the activity of PP1 and the phosphorylation status of one of its downstream targets, cofilin. Consequently, at low actin monomer levels, Phactr4 guides PP1 to dephosphorylate cofilin. This active form of cofilin is then able to sever and depolymerize actin filaments and thus restore the actin monomer pool. Accordingly, our data discloses the central role of Phactr4 in a feedback loop, where actin monomers regulate their own number via the activation of a key regulator of actin dynamics. Depending on the protein context, the RPEL domain can thus elicit mechanistically different responses to maintain the cellular actin balance.
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Actinas/metabolismo , Cofilina 1/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 1/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas del Citoesqueleto , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Proteínas Nucleares/genéticaRESUMEN
Besides its essential and well established role as a component of the cytoskeleton, actin is also present in the cell nucleus, where it has been linked to many processes that control gene expression. For example, nuclear actin regulates the activity of specific transcription factors, associates with all three RNA polymerases, and is a component of many chromatin remodelling complexes. Despite the fact that two export receptors, Crm1 and exportin 6, have been linked to nuclear export of actin, the mechanism by which actin enters the nucleus to elicit these essential functions has not been determined. It is also unclear whether actin is actively exchanged between the nucleus and the cytoplasm, and whether this connection has any functional significance for the cell. By applying a variety of live-cell imaging techniques we revealed that actin constantly shuttles in and out of the nucleus. The fast transport rates, which depend on the availability of actin monomers, suggest an active transport mechanism in both directions. Importantly, we identified importin 9 as the nuclear import factor for actin. Furthermore, our RNAi experiments showed that the active maintenance of nuclear actin levels by importin 9 is required for maximal transcriptional activity. Measurements of nuclear export rates and depletion studies also clarified that nuclear export of actin is mediated by exportin 6, and not by Crm1. These results demonstrate that cytoplasmic and nuclear actin pools are dynamically connected and identify the nuclear import and export mechanisms of actin.
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Actinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Carioferinas/fisiología , Transcripción Genética/fisiología , beta Carioferinas/fisiología , Factores Despolimerizantes de la Actina/fisiología , Actinas/genética , Animales , Línea Celular , Citoplasma/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/citología , Genes Reporteros , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/análisis , Humanos , Carioferinas/antagonistas & inhibidores , Ratones , Microscopía Confocal , Células 3T3 NIH , Fotoblanqueo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP ran/fisiología , Proteína Exportina 1RESUMEN
Myocardin-related transcription factors (MRTFs) are actin-regulated transcriptional coactivators, which bind G-actin through their N-terminal RPEL domains. In response to signal-induced actin polymerisation and concomitant G-actin depletion, MRTFs accumulate in the nucleus and activate target gene transcription through their partner protein SRF. Nuclear accumulation of MRTFs in response to signal is inhibited by increased G-actin level. Here, we study the mechanism by which MRTF-A enters the nucleus. We show that MRTF-A contains an unusually long bipartite nuclear localisation signal (NLS), comprising two basic elements separated by 30 residues, embedded within the RPEL domain. Using siRNA-mediated protein depletion in vivo, and nuclear import assays in vitro, we show that the MRTF-A extended bipartite NLS uses the importin (Imp)α/ß-dependent import pathway, and that import is inhibited by G-actin. Interaction of the NLS with the Impα-Impß heterodimer requires both NLS basic elements, and is dependent on the Impα major and minor binding pockets. Binding of the Impα-Impß heterodimer to the intact MRTF-A RPEL domain occurs competitively with G-actin. Thus, MRTF-A contains an actin-sensitive nuclear import signal.