RESUMEN
Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.
Asunto(s)
Citoesqueleto de Actina , Actinas , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , Citoesqueleto de Actina/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Actinas/metabolismo , Dominios PDZ , Unión Proteica , Citoesqueleto/metabolismo , Animales , Células HEK293RESUMEN
PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.
Asunto(s)
Calcio , Ácido Egtácico/análogos & derivados , Uniones Intercelulares , Calcio/metabolismo , Ionomicina , Uniones Intercelulares/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
Cell-cell junction formation requires actin cytoskeletal remodeling. Here, we show that PLEKHG4B, a Rho-guanine nucleotide exchange factor (Rho-GEF), plays a crucial role in epithelial cell-cell junction formation. Knockdown of PLEKHG4B decreased Cdc42 activity and tended to increase RhoA activity in A549 cells. A549 monolayer cells showed 'closed junctions' with closely packed actin bundles along the cell-cell contacts, but PLEKHG4B knockdown suppressed closed junction formation, and PLEKHG4B-knockdown cells exhibited 'open junctions' with split actin bundles located away from the cell-cell boundary. In Ca2+-switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and ß-catenin accumulation at cell-cell junctions. Furthermore, PLEKHG4B knockdown abrogated the reduction in myosin activity normally seen in the later stage of junction formation. The aberrant myosin activation and impairments in closed junction formation in PLEKHG4B-knockdown cells were reverted by ROCK inhibition or LARG/PDZ-RhoGEF knockdown. These results suggest that PLEKHG4B enables actin remodeling during epithelial cell-cell junction maturation, probably by reducing myosin activity in the later stage of junction formation, through suppressing LARG/PDZ-RhoGEF and RhoA-ROCK pathway activities. We also showed that annexin A2 participates in PLEKHG4B localization to cell-cell junctions.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Actinas , Uniones Intercelulares , Actinas/genética , Actinas/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Uniones Intercelulares/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (CEP104) localizes to the tip of the elongating axoneme, and CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus, CEP104 has been implicated in ciliogenesis. However, the mechanism by which CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the SXIP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end-binding protein, EB1, through the SXIP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation-associated ciliopathies.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Cerebelo/anomalías , Cerebelo/metabolismo , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Células HEK293 , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutación , Fosfoproteínas/metabolismo , Dominios Proteicos , Retina/anomalías , Retina/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
The Hippo signaling pathway suppresses cell proliferation and tumorigenesis. In the canonical Hippo pathway, large tumor suppressor kinases 1/2 (LATS1/2) phosphorylate the transcriptional coactivator yes-associated protein (YAP) and thereby suppress its nuclear localization and co-transcriptional activity. Nuclear Dbf2-related kinases 1/2 (NDR1/2), which are closely related to LATS1/2, also phosphorylate and inactivate YAP by suppressing its nuclear localization. Furry (FRY) is a cytoplasmic protein that associates with NDR1/2 and activates them, but its role in the nuclear/cytoplasmic localization of YAP remains unknown. Here, we constructed FRY-knockout cell lines to examine the role of FRY in YAP's cytoplasmic localization. FRY depletion markedly increased YAP nuclear localization and decreased NDR1/2 kinase activity and YAP phosphorylation levels, but did not affect LATS1/2 kinase activity. This indicated that FRY suppresses YAP's nuclear localization by promoting its phosphorylation via NDR1/2 activation. NDR1/2 depletion also promoted YAP nuclear localization, but depletion of both FRY and NDR1/2 increased the number of cells with YAP nuclear localization more strongly than did depletion of NDR1/2 alone, suggesting that FRY suppresses YAP nuclear localization by a mechanism in addition to NDR1/2 activation. Co-precipitation assays revealed that Fry uses its N-terminal 1-2400-amino-acid-long region to bind to YAP. Expression of full-length FRY or its 1-2400 N-terminal fragment restored YAP cytoplasmic localization in FRY-knockout cells. Taken together, these results suggest that FRY plays a crucial role in YAP cytoplasmic retention by promoting YAP phosphorylation via NDR1/2 kinase activation and by binding to YAP, leading to its cytoplasmic sequestration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Edición Génica , Células HEK293 , Humanos , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Señalizadoras YAPRESUMEN
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
Primary cilia are antenna-like sensory organelles that transmit various extracellular signals. Ciliogenesis requires the removal of CP110 and its interactor CEP97 from the mother centriole for initiating ciliary axoneme extension, but the underlying mechanism remains unknown. Here we show that, upon serum starvation, CEP97 is partially degraded by the ubiquitin-proteasome system. CEP97 was polyubiquitylated in serum-starved cells, and overexpression of a non-ubiquitylatable CEP97 mutant effectively blocked CP110 removal and ciliogenesis induced by serum-starvation. Through several screening steps, we identified the cullin-3-RBX1-KCTD10 complex as the E3 ligase that mediates CEP97 degradation and removal from the mother centriole. Depletion of each component of this E3 complex caused aberrant accumulation of CEP97 on the centrosome, suppressed the removal of CEP97 and CP110 from the mother centriole, and impaired ciliogenesis. Moreover, KCTD10 was specifically localized to the mother centriole. These results suggest that CEP97 degradation by the cullin-3-RBX1-KCTD10 complex plays a crucial role in serum-starvation-induced CP110 removal and ciliogenesis.
Asunto(s)
Centrosoma/metabolismo , Proteínas Cullin/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Axonema/metabolismo , Línea Celular , Centriolos/metabolismo , Humanos , Ubiquitina/metabolismoRESUMEN
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
Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F-actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil-like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma.
Asunto(s)
Actinas/metabolismo , Infiltración Neutrófila/inmunología , Proteína Quinasa C/metabolismo , Choque Hemorrágico/inmunología , Migración Transendotelial y Transepitelial/inmunología , Animales , Línea Celular , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Polimerizacion , Proteína Quinasa C/inmunología , Transducción de Señal/inmunología , PorcinosRESUMEN
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
Primary cilia are antenna-like sensory organelles extending from the surface of many cell types that play critical roles in tissue development and homeostasis. Here, we examined the effect of nutrient status on primary cilium formation. Glucose deprivation significantly increased the number of ciliated cells under both serum-fed and -starved conditions. Glucose deprivation-induced ciliogenesis was suppressed by overexpression of Rheb, an activator of the mammalian target of rapamycin complex-1 (mTORC1). Inactivating mTORC1 by rapamycin treatment or Raptor knockdown significantly promoted ciliogenesis. These results indicate that glucose deprivation promotes primary cilium formation through mTORC1 inactivation. Rapamycin treatment did not promote autophagy or degradation of OFD1, a negative regulator of ciliogenesis. In contrast, rapamycin treatment increased the level of the p27KIP1 (also known as CDKN1B) cyclin-dependent kinase inhibitor, and rapamycin-induced ciliogenesis was abrogated in p27KIP1-depleted cells. These results indicate that mTORC1 inactivation induces ciliogenesis through p27KIP1 upregulation, but not through autophagy. By contrast, glucose deprivation or rapamycin treatment shortened the cilium length. Thus, glucose deprivation and subsequent inactivation of mTORC1 play dual roles in ciliogenesis: triggering primary cilium formation and shortening cilium length.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Cilios/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Glucosa/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular , Cilios/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Homeostasis , Humanos , Proteínas/efectos de los fármacos , Proteínas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (Gas6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which Gas6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that Gas6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated Gas6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, Gas6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated Gas6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound Gas6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by Gas6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.
RESUMEN
Primary cilia are non-motile cilia that serve as cellular antennae for sensing and transducing extracellular signals. In general, primary cilia are generated by cell quiescence signals. Recent studies have shown that manipulations to increase actin assembly suppress quiescence-induced ciliogenesis. To further examine the role of actin dynamics in ciliogenesis, we analyzed the effect of jasplakinolide (Jasp), a potent inducer of actin polymerization, on ciliogenesis. Unexpectedly, Jasp treatment induced ciliogenesis in serum-fed cells cultured at low density. In contrast, Jasp had no apparent effect on ciliogenesis in cells cultured at higher densities. Jasp-induced ciliogenesis was correlated with a change in cell morphology from a flat and adherent shape to a round and weakly adherent one. Jasp treatment also induced the phosphorylation and cytoplasmic localization of the YAP transcriptional co-activator and suppressed cell proliferation in low density-cultured cells. Overexpression of an active form of YAP suppressed Jasp-induced ciliogenesis. These results suggest that Jasp induces ciliogenesis through cell rounding and cytoplasmic localization and inactivation of YAP. Knockdown of LATS1/2 only faintly suppressed Jasp-induced YAP phosphorylation, indicating that LATS1/2 are not primarily responsible for Jasp-induced YAP phosphorylation. Furthermore, overexpression of active Src kinase suppressed Jasp-induced cytoplasmic localization of YAP and ciliogenesis, suggesting that down-regulation of Src activity is involved in Jasp-induced YAP inactivation and ciliogenesis. Our data suggest that actin polymerization does not suppress ciliogenesis per se but rather that cell rounding and reduced cell adhesion are more crucially involved in Jasp-induced ciliogenesis.
Asunto(s)
Actinas/metabolismo , Cilios/efectos de los fármacos , Depsipéptidos/farmacología , Proteínas Nucleares/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Cilios/metabolismo , Cilios/ultraestructura , Humanos , Proteínas Nucleares/análisis , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Epitelio Pigmentado de la Retina/metabolismo , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/genéticaRESUMEN
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
Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor Pex5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.
Asunto(s)
Catalasa/metabolismo , Cilios/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Peroxisomas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Células Cultivadas , Citoplasma/metabolismo , Células HEK293 , Humanos , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Epitelio Pigmentado de la Retina/citología , Transducción de SeñalRESUMEN
Slingshot-1 (SSH1) is a protein phosphatase that specifically dephosphorylates and activates cofilin, an F-actin-severing protein. SSH1 binds to and co-localizes with F-actin, and the cofilin-phosphatase activity of SSH1 is markedly increased by binding to F-actin. In this study, we performed a secondary structure analysis of SSH1, which predicted the existence of a pleckstrin homology (PH)-like domain in the N-terminal region of SSH1. SSH1 also contains a DEK-C domain in the N-terminal region. The N-terminal fragment of SSH1 bound to and co-localized with F-actin, but mutation at Arg-96 or a Leu-His-Lys (LHK) motif in the PH-like domain reduced this activity. Furthermore, mutation at Arg-96 abrogated the cofilin-phosphatase activity of SSH1 in the presence of F-actin. These results suggest that the N-terminal PH-like domain plays a critical role in F-actin binding and F-actin-mediated activation of the cofilin-phosphatase activity of SSH1.
Asunto(s)
Factores Despolimerizantes de la Actina/química , Actinas/química , Mutación , Fosfoproteínas Fosfatasas/química , Secuencias de Aminoácidos , Animales , Dominio Catalítico , Dicroismo Circular , Células HEK293 , Células HeLa , Histidina/química , Humanos , Leucina/química , Lisina/química , Músculo Esquelético/metabolismo , Plásmidos/metabolismo , Dominios Homólogos a Pleckstrina , Unión Proteica , ConejosRESUMEN
The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses.
Asunto(s)
Epitelio/crecimiento & desarrollo , Odontogénesis/fisiología , Diente/crecimiento & desarrollo , Animales , División Celular/fisiología , Movimiento Celular/fisiología , Forma de la Célula/fisiología , Ratones , Microscopía Confocal/métodos , Modelos BiológicosRESUMEN
Centrosome amplification is a hallmark of virtually all types of cancers, including solid tumors and hematologic malignancies. Cancer cells with extra centrosomes use centrosome clustering (CC) to allow for successful division. Because normal cells do not rely on this mechanism, CC is regarded as a promising target to selectively eradicate cells harboring supernumerary centrosomes. To identify novel inhibitors of CC, we developed a cell-based high-throughput screen that reports differential drug cytotoxicity for isogenic cell populations with different centrosome contents. We identified CP-673451 and crenolanib, two chemically related compounds originally developed for the inhibition of platelet-derived growth factor receptor ß (PDGFR-ß), as robust inhibitors of CC with selective cytotoxicity for cells with extra centrosomes. We demonstrate that these compounds induce mitotic spindle multipolarity by activation of the actin-severing protein cofilin, leading to destabilization of the cortical actin network, and provide evidence that this activation is dependent on slingshot phosphatases 1 and 2 but unrelated to PDGFR-ß inhibition. More specifically, we found that although both compounds attenuated PDGF-BB-induced signaling, they significantly enhanced the phosphorylation of PDGFR-ß downstream effectors, Akt and MEK, in almost all tested cancer cell lines under physiologic conditions. In summary, our data reveal a novel mechanism of CC inhibition depending on cofilin-mediated cortical actin destabilization and identify two clinically relevant compounds interfering with this tumor cell-specific target. Cancer Res; 76(22); 6690-700. ©2016 AACR.
Asunto(s)
Actinas/metabolismo , Centrosoma/metabolismo , Cofilina 1/metabolismo , Línea Celular Tumoral , Humanos , Transducción de SeñalRESUMEN
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.