RESUMEN
Keratin intermediate filament (IF) proteins constitute the major cytoskeletal components in epithelial cells. Missense mutations in keratin 5 (K5; also known as KRT5) or keratin 14 (K14; also known as KRT14), highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS). EBS-associated mutations disrupt keratin networks and change keratinocyte mechanics; however, molecular mechanisms by which mutations shape EBS pathology remain incompletely understood. Here, we demonstrate that, in contrast to keratin-deficient keratinocytes, cells expressing K14R125C, a mutation that causes severe EBS, generate lower traction forces, accompanied by immature focal adhesions with an altered cellular distribution. Furthermore, mutant keratinocytes display reduced directionality during collective migration. Notably, RhoA activity is downregulated in human EBS keratinocytes, and Rho activation rescues stiffness-dependent cell-extracellular matrix (ECM) adhesion formation of EBS keratinocytes. Collectively, our results strongly suggest that intact keratin IF networks regulate mechanotransduction through a Rho signaling pathway upstream of cell-ECM adhesion formation and organized cell migration. Our findings provide insights into the underlying pathophysiology of EBS.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Epidermólisis Ampollosa Simple , Queratinas , Citoesqueleto/metabolismo , Epidermólisis Ampollosa Simple/genética , Humanos , Queratinas/genética , Queratinas/metabolismo , Mecanotransducción Celular , Mutación/genética , TracciónRESUMEN
Epithelial to mesenchymal transition (EMT) is a fundamental biological process that occurs during development and tumorigenesis. The Rho family of GTPases (Rho-family) is a well-characterized regulator of actin cytoskeleton that gives rise to EMT-associated cell activities. Meanwhile, there are in total at least 66 different Rho-GTPase-activating proteins (Rho-GAPs), which, as an upstream regulator, inactivate specific members of the Rho-family in a cell context-dependent manner. However, molecular roles of individual Rho-GAPs are poorly understood, particularly regarding their involvements in EMT. Here, based on comprehensive screening on the whole Rho-GAP family, we identified specific Rho-GAPs that are responsible for the maintenance of epithelial cell phenotypes, suppressing EMT in human mammary epithelial cells. Specifically, we revealed that at least two Rho-GAPs, that is, ARHGAP4 and SH3BP1, critically regulate the cell morphology. Among them, we focused on ARHGAP4 and demonstrated with multidisciplinary approaches that this specific Rho-GAP regulates epithelial/mesenchymal-selective marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber-driven physical force generation in a manner reminiscent of the EMT process. Furthermore, we identified Septin9 with proteomic analyses as a negative regulator of ARHGAP4, which promotes the occurrence of EMT by activation of the FAK/Src signaling pathway. These findings shed light on the novel Rho-GAP-associated pathway in the EMT process under development and tumorigenesis.
Asunto(s)
Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Septinas/metabolismo , Citoesqueleto de Actina/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Adhesiones Focales/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Células MCF-7 , Morfogénesis/fisiología , Proteómica/métodos , Transducción de Señal/fisiologíaRESUMEN
Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Queratinas/metabolismo , Mecanotransducción Celular , Animales , Sitios de Unión , Perros , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , Mutación , Unión ProteicaRESUMEN
Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.Key words: epithelial tubulogenesis, Solo, keratin, Rho-GEF, myosin.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Animales , Técnicas de Cultivo de Célula , Colágeno/química , Citoesqueleto/metabolismo , Perros , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Filamentos Intermedios/metabolismo , Queratina-18/antagonistas & inhibidores , Queratina-18/genética , Queratina-8/genética , Células de Riñón Canino Madin Darby , Microscopía Fluorescente , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Cyclic stretch is an artificial model of mechanical force loading, which induces the reorientation of vascular endothelial cells and their stress fibers in a direction perpendicular to the stretch axis. Rho family GTPases are crucial for cyclic-stretch-induced endothelial cell reorientation; however, the mechanism underlying stretch-induced activation of Rho family GTPases is unknown. A screen of short hairpin RNAs targeting 63 Rho guanine nucleotide exchange factors (Rho-GEFs) revealed that at least 11 Rho-GEFs Abr, alsin, ARHGEF10, Bcr, GEF-H1 (also known as ARHGEF2), LARG (also known as ARHGEF12), p190RhoGEF (also known as ARHGEF28), PLEKHG1, P-REX2, Solo (also known as ARHGEF40) and α-PIX (also known as ARHGEF6) which specifically or broadly target RhoA, Rac1 and/or Cdc42, are involved in cyclic-stretch-induced perpendicular reorientation of endothelial cells. Overexpression of Solo induced RhoA activation and F-actin accumulation at cell-cell and cell-substrate adhesion sites. Knockdown of Solo suppressed cyclic-stretch- or tensile-force-induced RhoA activation. Moreover, knockdown of Solo significantly reduced cyclic-stretch-induced perpendicular reorientation of endothelial cells when cells were cultured at high density, but not when they were cultured at low density or pretreated with EGTA or VE-cadherin-targeting small interfering RNAs. These results suggest that Solo is involved in cell-cell-adhesion-mediated mechanical signal transduction during cyclic-stretch-induced endothelial cell reorientation.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Estrés Mecánico , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Recuento de Células , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , ARN Interferente Pequeño/metabolismo , Fibras de Estrés/efectos de los fármacos , Fibras de Estrés/metabolismo , Resistencia a la Tracción/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions fracture when claudin/JAM-A-deficient cells undergo spontaneous cell stretching. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells.
Asunto(s)
Proteínas de Uniones Estrechas , Uniones Estrechas , Actinas/genética , Actinas/metabolismo , Claudinas/metabolismo , Células Epiteliales/metabolismo , Uniones Intercelulares/genética , Uniones Intercelulares/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Células de Riñón Canino Madin Darby , Animales , PerrosRESUMEN
Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow, and lamellipodium extension using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin-stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization, and in MCF-7 cells endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.
Asunto(s)
Actinas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Quinasas Lim/metabolismo , Seudópodos/enzimología , Actinas/genética , Animales , Línea Celular Tumoral , Humanos , Quinasas Lim/genética , Ratones , Fosforilación/fisiología , Fotoblanqueo , Seudópodos/genéticaRESUMEN
Claudin-based tight junctions (TJs) are formed at the most apical part of cell-cell contacts in epithelial cells. Previous studies suggest that scaffolding proteins ZO-1 and ZO-2 (ZO proteins) determine the location of TJs by interacting with claudins, but this idea is not conclusive. To address the role of the ZO proteins binding to claudins at TJs, a COOH-terminal PDZ domain binding motif-deleted claudin-3 mutant, which lacks the ZO protein binding, was stably expressed in claudin-deficient MDCK cells. The COOH-terminus-deleted claudin-3 was localized at the apicolateral region similar to full-length claudin-3. Consistently, freeze-fracture electron microscopy revealed that the COOH-terminus-deleted claudin-3-expressing cells reconstituted belts of TJs at the most apical region of the lateral membrane and restored functional epithelial barriers. These results suggest that the interaction of claudins with ZO proteins is not a prerequisite for TJ formation at the most apical part of cell-cell contacts.
Asunto(s)
Claudinas , Uniones Estrechas , Línea Celular , Claudina-1/metabolismo , Claudina-3/genética , Claudina-3/metabolismo , Claudina-5/metabolismo , Claudinas/genética , Claudinas/metabolismo , Humanos , Dominios PDZ , Unión Proteica , Uniones Estrechas/metabolismoRESUMEN
Aggregation, fibril formation, and deposition of amyloid ß (Aß) protein are believed to be the central pathogeneses of Alzheimer's disease (AD). Numerous studies have shown that fibril formation is promoted by preformed seeds at the beginning of the aggregation process. Therefore, aggregated molecules that promote fibrillization of Aß protein as seeds could affect the pathology. We recently found that approximately 40 amino acid hydrophobic peptides, BBF2H7-derived small peptide (BSP) fragments, are generated via intramembranous cleavage under endoplasmic reticulum (ER) stress conditions. Interestingly, similar to Aß protein, the fragments exhibit a high aggregation propensity and form fibril structures. It has been noted that ER stress is involved in the pathogenesis of AD. In this study, we examined the effect of BSP fragments on aggregation and cytotoxicity of Aß1-40 protein, which is generated as a major species of Aß protein, but has a lower aggregative property than Aß1-42 protein. We demonstrated that BSP fragments promote aggregation of Aß1-40 protein. Aggregates of Aß1-40 protein mediated by BSP fragments also exhibited potent neurotoxicity. Our findings suggest the possibility that BSP fragments affect accumulation of Aß proteins and are involved in the pathogenesis of AD.
Asunto(s)
Amiloide/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Neuronas/metabolismo , Péptidos beta-Amiloides/metabolismo , Línea Celular , Humanos , Fragmentos de Péptidos/metabolismoRESUMEN
Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force-induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell-cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell-cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell-cell junctions related to K8/K18 filaments and plakoglobin.
Asunto(s)
Movimiento Celular/fisiología , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/fisiología , Quinasas Asociadas a rho/fisiología , Amidas/farmacología , Animales , Polaridad Celular , Colágeno , Citoesqueleto/fisiología , Desmosomas/fisiología , Perros , Geles , Técnicas de Silenciamiento del Gen , Queratina-18/antagonistas & inhibidores , Queratina-18/genética , Queratina-18/fisiología , Queratina-8/antagonistas & inhibidores , Queratina-8/genética , Queratina-8/fisiología , Células de Riñón Canino Madin Darby , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Estrés Mecánico , Imagen de Lapso de Tiempo , gamma Catenina/antagonistas & inhibidores , gamma Catenina/genética , gamma Catenina/fisiología , Proteína de Unión al GTP rac1/fisiología , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, ß4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of ß4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.
Asunto(s)
Células Acinares/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemidesmosomas/metabolismo , Animales , Línea Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Integrina beta4/química , Integrina beta4/metabolismo , Queratina-18/genética , Queratina-18/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
All cells sense and respond to various mechanical forces in and mechanical properties of their environment. To respond appropriately, cells must be able to sense the location, direction, strength and duration of these forces. Recent progress in mechanobiology has provided a better understanding of the mechanisms of mechanoresponses underlying many cellular and developmental processes. Various roles of mechanoresponses in development and tissue homeostasis have been elucidated, and many molecules involved in mechanotransduction have been identified. However, the whole picture of the functions and molecular mechanisms of mechanotransduction remains to be understood. Recently, novel mechanisms for sensing and transducing mechanical stresses via the cytoskeleton, cell-substrate and cell-cell adhesions and related proteins have been identified. In this review, we outline the roles of the cytoskeleton, cell-substrate and cell-cell adhesions, and related proteins in mechanosensing and mechanotransduction. We also describe the roles and regulation of Rho-family GTPases in mechanoresponses.
Asunto(s)
Citoesqueleto/metabolismo , Mecanotransducción Celular , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Adhesión Celular , HumanosRESUMEN
Mechanical force-induced cytoskeletal reorganization is essential for cell and tissue remodeling and homeostasis; however, the underlying cellular mechanisms remain elusive. Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor (GEF) involved in cyclical stretch-induced human endothelial cell reorientation and convergent extension cell movement in zebrafish gastrula. In this study, we show that Solo binds to keratin-8/keratin-18 (K8/K18) intermediate filaments through multiple sites. Solo overexpression promotes the formation of thick actin stress fibers and keratin bundles, whereas knockdown of Solo, expression of a GEF-inactive mutant of Solo, or inhibition of ROCK suppresses stress fiber formation and leads to disorganized keratin networks, indicating that the Solo-RhoA-ROCK pathway serves to precisely organize keratin networks, as well as to promote stress fibers. Of importance, knockdown of Solo or K18 or overexpression of GEF-inactive or deletion mutants of Solo suppresses tensile force-induced stress fiber reinforcement. Furthermore, knockdown of Solo or K18 suppresses tensile force-induced RhoA activation. These results strongly suggest that the interplay between Solo and K8/K18 filaments plays a crucial role in tensile force-induced RhoA activation and consequent actin cytoskeletal reinforcement.