RESUMEN
Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.[Figure: see text].
Asunto(s)
Proteogenómica , Humanos , Células Endoteliales de la Vena Umbilical Humana , Isoformas de Proteínas/genética , Empalme Alternativo , ARNRESUMEN
The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell-cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP-actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell-cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilin(S3A) inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA-GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Células Epiteliales/fisiología , Cinesinas/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Antígenos CD , Cadherinas/metabolismo , Adhesión Celular , Perros , Células Epiteliales/ultraestructura , Quinasas Lim/metabolismo , Células de Riñón Canino Madin Darby , Microtúbulos/metabolismo , Unión Proteica , Transporte de Proteínas , Transducción de Señal , Quinasas Asociadas a rho/metabolismoRESUMEN
Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.
Asunto(s)
Actomiosina/fisiología , Uniones Adherentes/fisiología , Células Epiteliales/fisiología , Mecanotransducción Celular/fisiología , Contracción Muscular/fisiología , Proteína de Unión al GTP rhoA/fisiología , Actomiosina/química , Uniones Adherentes/química , Animales , Simulación por Computador , Células Epiteliales/química , Humanos , Modelos Biológicos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/fisiología , Estrés Mecánico , Proteína de Unión al GTP rhoA/químicaRESUMEN
KIF17 is a kinesin-2 family motor that interacts with EB1 at microtubule (MT) plus-ends and contributes to MT stabilization in epithelial cells. The mechanism by which KIF17 affects MTs and how its activity is regulated are not yet known. Here, we show that EB1 and the KIF17 autoinhibitory tail domain (KIF17-Tail) interacted competitively with the KIF17 catalytic motor domain (K370). Both EB1 and KIF17-Tail decreased the K0.5MT of K370, with opposing effects on MT-stimulated ATPase activity. Importantly, K370 had independent effects on MT dynamic instability, resulting in formation of long MTs without affecting polymerization rate or total polymer mass. K370 also inhibited MT depolymerization induced by dilution in vitro and by nocodazole in cells, suggesting that it acts by protecting MT plus-ends. Interestingly, KIF17-Tail bound MTs and tubulin dimers, delaying initial MT polymerization in vitro and MT regrowth in cells. However, neither EB1 nor KIF17-Tail affected K370-mediated MT polymerization or stabilization significantly in vitro, and EB1 was dispensable for MT stabilization by K370 in cells. Thus, although EB1 and KIF17-Tail may coordinate KIF17 catalytic activity, our data reveal a novel and direct role for KIF17 in regulating MT dynamics.
Asunto(s)
Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Multimerización de Proteína/fisiología , Tubulina (Proteína)/metabolismo , Animales , Células CACO-2 , Catálisis , Bovinos , Humanos , Cinesinas/química , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/química , Microtúbulos/genética , Unión Proteica , Estructura Terciaria de Proteína , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.
Asunto(s)
Conexinas , Células Endoteliales , Células Endoteliales/metabolismo , Puntos de Control del Ciclo Celular/genética , Conexinas/genética , Conexinas/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Núcleo Celular/metabolismo , Proteína alfa-4 de Unión ComunicanteRESUMEN
The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the adult SVZ control NSC behavior are unclear. We found that Cx43 is highly expressed by SVZ NSCs and ECs, and its deletion in either leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by single-cell RNA sequencing and in vitro studies showing that ECs control NSC proliferation by regulating expression of genes associated with NSC quiescence and/or activation in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights inform adult NSC regulation and maintenance aimed at stem cell therapies for neurodegenerative disorders.
Asunto(s)
Conexina 43 , Ventrículos Laterales , Células Endoteliales/metabolismo , Encéfalo/metabolismo , Neurogénesis/fisiologíaRESUMEN
During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-ß signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-ß1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.
Asunto(s)
Células Endoteliales , Factor de Crecimiento Transformador beta1 , Animales , Arterias/metabolismo , Ciclo Celular , Células Endoteliales/metabolismo , Ratones , Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , VenasRESUMEN
Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC(50) of 16.4 ± 1.6 µM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G(2)/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.
Asunto(s)
Apoptosis , Autofagia , Microtúbulos/efectos de los fármacos , Naftoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 3/metabolismo , Ciclo Celular , Puntos de Control del Ciclo Celular , Línea Celular Tumoral/efectos de los fármacos , Supervivencia Celular , Sistema Libre de Células/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Inmunoprecipitación , Concentración 50 Inhibidora , Microscopía Confocal , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Índice Mitótico , Estructura Molecular , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Tubulina (Proteína)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Heterotypic cell lineages relentlessly exchange biomechanical signals among themselves in metazoan organs. Hence, cell-cell communications are pivotal for organ physiology and pathogenesis. Every cell lineage of an organ responds differently to a specific signal due to its unique receptibility and signal interpretation capacity. These distinct cellular responses generate a system-scale signaling network that helps in generating a specific organ phenotype. Although the reciprocal biochemical signal exchange between non-identical neighboring cells is known to be an essential factor for organ functioning, if, then how, mechanical cues incite these signals is not yet quite explored. Cells within organ tissues experience multiple mechanical forces, such as stretching, bending, compression, and shear stress. Forms and magnitudes of mechanical forces influence biochemical signaling in a cell-specific manner. Additionally, the biophysical state of acellular extracellular matrix (ECM) can transmit exclusive mechanical cues to specific cells of an organ. As it scaffolds heterotypic cells and tissues in close proximities, therefore, ECM can easily be contemplated as a mechanical conduit for signal exchange among them. However, force-stimulated signal transduction is not always physiological, aberrant force sensing by tissue-resident cells can transduce anomalous signals to each other, and potentially can promote pathological phenotypes. Herein, I attempt to put forward a perspective on how mechanical forces may influence signal transductions among heterotypic cell populations and how they feedback each other to achieve a transient or perpetual alteration in metazoan organs. A mechanistic insight of organ scale mechanotransduction can emanate the possibility of finding potential biomarkers and novel therapeutic strategies to deal with pathogenesis and organ regeneration.
Asunto(s)
Comunicación Celular , Mecanotransducción Celular , Animales , Matriz Extracelular , Fenómenos Mecánicos , Estrés MecánicoRESUMEN
Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton.
Asunto(s)
Caveolas/metabolismo , Células Epiteliales/metabolismo , Proteínas Oncogénicas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células CACO-2 , Caveolina 1/metabolismo , Células Epiteliales/ultraestructura , Forminas/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Proteínas Oncogénicas/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estrés MecánicoRESUMEN
Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Vitamina K 3/farmacología , Ciclo Celular , Línea Celular Tumoral , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Confocal , Microtúbulos/metabolismo , Unión Proteica , Espectrometría de Fluorescencia , Vitamina K 3/metabolismoRESUMEN
Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.
Asunto(s)
Microtúbulos/química , Microtúbulos/metabolismo , Naftoquinonas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Cabras , Humanos , Cinética , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microtúbulos/ultraestructura , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Unión Proteica , Espectrometría de Fluorescencia , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructuraRESUMEN
Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric Gα12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly.
Asunto(s)
Uniones Adherentes/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Estrés Mecánico , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Cadherinas/metabolismo , Humanos , Resistencia a la TracciónRESUMEN
We report an experimental approach to study the mechanosensitivity of cell-cell contact upon mechanical stimulation in suspended cell-doublets. The doublet is placed astride an hourglass aperture, and a hydrodynamic force is selectively exerted on only one of the cells. The geometry of the device concentrates the mechanical shear over the junction area. Together with mechanical shear, the system also allows confocal quantitative live imaging of the recruitment of junction proteins (e.g., E-cadherin, ZO-1, occludin, and actin). We observed the time sequence over which proteins were recruited to the stretched region of the contact. The compressed side of the contact showed no response. We demonstrated how this mechanism polarizes the stress-induced recruitment of junctional components within one single junction. Finally, we demonstrated that stabilizing the actin cortex dynamics abolishes the mechanosensitive response of the junction. Our experimental design provides an original approach to study the role of mechanical force at a cell-cell contact with unprecedented control over stress application and quantitative optical analysis.
RESUMEN
Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across αE-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Epitelio/metabolismo , Mamíferos/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animales , Antígenos CD , Células CACO-2 , Proteínas Fetales/metabolismo , Forminas , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Microfilamentos/metabolismo , Miosina Tipo II/metabolismo , Proteínas Nucleares/metabolismo , Estabilidad Proteica , Reproducibilidad de los Resultados , Estrés Fisiológico , Uniones Estrechas/metabolismo , alfa CateninaRESUMEN
Cellular contractility, driven by actomyosin networks coupled to cadherin cell-cell adhesion junctions, is a major determinant of cellular rearrangement during morphogenesis. It now emerges that contractility arises as the emergent property of a mechanochemical feedback system that encompasses the signals that regulate contractility and the elements of the actomyosin network itself.
Asunto(s)
Cadherinas/metabolismo , Adhesión Celular/fisiología , Uniones Intercelulares/fisiología , Mecanotransducción Celular/fisiología , Morfogénesis/fisiología , Contracción Muscular/fisiología , Actomiosina/metabolismo , Animales , HumanosRESUMEN
Morphogenesis requires dynamic coordination between cell-cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell-cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.
Asunto(s)
Uniones Adherentes/metabolismo , Proteínas de Microfilamentos/fisiología , Proteínas de la Zonula Occludens/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Adhesión Celular , Forma de la Célula , Citoesqueleto/metabolismo , Perros , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Técnicas de Silenciamiento del Gen , Células de Riñón Canino Madin Darby , Proteínas de Microfilamentos/metabolismo , Morfogénesis , Proteínas de la Zonula Occludens/genética , Proteínas de la Zonula Occludens/metabolismoRESUMEN
We have previously reported that RORγ expression was decreased in ER-ve breast cancer, and increased expression improves clinical outcomes. However, the underlying RORγ dependent mechanisms that repress breast carcinogenesis have not been elucidated. Here we report that RORγ negatively regulates the oncogenic TGF-ß/EMT and mammary stem cell (MaSC) pathways, whereas RORγ positively regulates DNA-repair. We demonstrate that RORγ expression is: (i) decreased in basal-like subtype cancers, and (ii) inversely correlated with histological grade and drivers of carcinogenesis in breast cancer cohorts. Furthermore, integration of RNA-seq and ChIP-chip data reveals that RORγ regulates the expression of many genes involved in TGF-ß/EMT-signaling, DNA-repair and MaSC pathways (including the non-coding RNA, LINC00511). In accordance, pharmacological studies demonstrate that an RORγ agonist suppresses breast cancer cell viability, migration, the EMT transition (microsphere outgrowth) and mammosphere-growth. In contrast, RNA-seq demonstrates an RORγ inverse agonist induces TGF-ß/EMT-signaling. These findings suggest pharmacological modulation of RORγ activity may have utility in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Reparación del ADN , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Factor de Crecimiento Transformador beta/genética , Benzamidas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Metástasis de la Neoplasia , Piperazinas/farmacología , Propanoles/farmacología , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.