RESUMEN
ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.
Asunto(s)
Proteínas de Ciclo Celular , Centrosoma , Microtúbulos , Proteínas del Tejido Nervioso , Células-Madre Neurales , Neurogénesis , Animales , Microtúbulos/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neurogénesis/genética , Células-Madre Neurales/metabolismo , Centrosoma/metabolismo , Proliferación Celular , Movimiento Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Tubulina (Proteína)/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genéticaRESUMEN
The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine filamentous actin (F-actin) structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of myocardin-related transcription factor, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G protein-coupled receptor Smog, G protein αq subunit, Rho1 guanosine triphosphatase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand folded gastrulation to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, an NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.
Asunto(s)
Actinas , Astrocitos , Proteínas de Drosophila , Células-Madre Neurales , Receptores Acoplados a Proteínas G , Transducción de Señal , Animales , Actinas/metabolismo , Astrocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Citoesqueleto de Actina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.
RESUMEN
The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, a hallmark of qNSCs. Here, we have found that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus that acts as the major acentrosomal microtubule-organizing center in qNSCs. A Golgi-resident GTPase Arf1 and its guanine nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector mini spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. Our findings have established Drosophila qNSCs as a regeneration model and identified Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation.
RESUMEN
Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.
RESUMEN
Changes in mitochondrial dynamics (fusion and fission) are known to occur during stem cell differentiation; however, the role of this phenomenon in tissue aging remains unclear. Here, we report that mitochondrial dynamics are shifted toward fission during aging of Drosophila ovarian germline stem cells (GSCs), and this shift contributes to aging-related GSC loss. We found that as GSCs age, mitochondrial fragmentation and expression of the mitochondrial fission regulator, Dynamin-related protein (Drp1), are both increased, while mitochondrial membrane potential is reduced. Moreover, preventing mitochondrial fusion in GSCs results in highly fragmented depolarized mitochondria, decreased BMP stemness signaling, impaired fatty acid metabolism, and GSC loss. Conversely, forcing mitochondrial elongation promotes GSC attachment to the niche. Importantly, maintenance of aging GSCs can be enhanced by suppressing Drp1 expression to prevent mitochondrial fission or treating with rapamycin, which is known to promote autophagy via TOR inhibition. Overall, our results show that mitochondrial dynamics are altered during physiological aging, affecting stem cell homeostasis via coordinated changes in stemness signaling, niche contact, and cellular metabolism. Such effects may also be highly relevant to other stem cell types and aging-induced tissue degeneration.
Asunto(s)
Células Madre Germinales Adultas/metabolismo , Dinámicas Mitocondriales/genética , Células Madre/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Drosophila , Femenino , Masculino , Transducción de SeñalRESUMEN
Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade ß-catenin. Disruption of ß-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and ß-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.
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Proteínas Argonautas/metabolismo , Senescencia Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Germinativas/metabolismo , Animales , Proteínas Argonautas/genética , Cadherinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Ovario/citología , Ovario/metabolismo , Retroelementos/genética , Transducción de Señal , Nicho de Células Madre/fisiología , Células Madre/metabolismo , Receptores Toll-Like/metabolismo , beta Catenina/metabolismoRESUMEN
Insect oogenesis is greatly affected by nutrient availability. When nutrients are abundant, oocytes are rapidly generated, but the process is slowed to conserve energy under nutrient-deficient conditions. To properly allocate limited resources toward oogenesis, systemic factors coordinate the behavioral response of ovarian germline stem cells (GSCs) to nutritional inputs by acting on the GSC itself, GSC supporting cells (the niche), or the adipose tissue surrounding the ovary. In this review, we describe current knowledge of the Drosophila ovarian GSC-niche-adipocyte system and major nutrient sensing pathways (insulin/IGF signaling, TOR signaling, and GCN2-dependent amino acid sensing) that intrinsically or extrinsically regulate GSC responses to nutrient signals.
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Drosophila/fisiología , Células Madre Oogoniales/metabolismo , Transducción de Señal , Adipocitos/metabolismo , Animales , Femenino , Células Madre Oogoniales/fisiología , Nicho de Células Madre/fisiologíaRESUMEN
Tumor invasion and metastasis are the major causes of treatment failure and mortality in lung cancer patients. In this study, we identified a group of genes with differential expression in in situ and invasive lung adenocarcinoma tissues by expression profiling; among these genes we further characterized the association of the upregulation of PRNP, the gene encoding cellular Prion protein (PrPc), with lung adenocarcinoma invasiveness. Immunohistochemistry on clinical specimens showed an association of PrPc expression with invasive but not in situ lung adenocarcinoma. Consistently, the expression of PrPc was higher in the highly invasive than in the lowly invasive lung adenocarcinoma cell lines. Knockdown of PrPc expression in cultured lung adenocarcinoma cells decreased their lamellipodium formation, in vitro migration and invasion, and in vivo experimental lung metastasis. Phosphorylation of JNKs was found to correlate with PrPc expression and the inhibition of JNKs suppressed the PrPc-induced up-regulation of lamellipodium formation, cell migration, and invasion. Moreover, we identified the nuclear factor, interleukin 3 regulated (NFIL3) protein as a transcriptional activator of the PRNP promoter. Accordingly, NFIL3 promoted lung cancer cell migration and invasion in a PrPc-dependent manner. High NFIL3 expression in clinical specimens of lung adenocarcinoma was also associated with tumor invasiveness. Overall, our observations suggest that the NFIL3/PrPc axis, through regulating lamellipodium formation and cell mobility via JNK signaling, plays a critical role in lung cancer invasiveness and metastasis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Neoplasias Pulmonares/genética , Proteínas Priónicas/genética , Seudópodos/genética , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Hibridación in Situ , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Regiones Promotoras Genéticas/genética , Seudópodos/patologíaRESUMEN
In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. Here, we demonstrate that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. Our results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Células Germinativas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Animales , Diferenciación Celular , Drosophila/citología , Drosophila/crecimiento & desarrollo , Femenino , Células Germinativas/metabolismo , Masculino , Ovario/citología , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Nicho de Células MadreRESUMEN
Adult stem cells maintain tissue homeostasis. This unique capability largely depends on the stem cell niche, a specialized microenvironment, which preserves stem cell identity through physical contacts and secreted factors. In many cancers, latent tumor cell niches are thought to house stem cells and aid tumor initiation. However, in developing tissue and cancer it is unclear how the niche is established. The well-characterized germline stem cells (GSCs) and niches in the Drosophila melanogaster ovary provide an excellent model to address this fundamental issue. As such, we conducted a small-scale RNAi screen of 560 individually expressed UAS-RNAi lines with targets implicated in female fertility. RNAi was expressed in the soma of larval gonads, and screening for reduced egg production and abnormal ovarian morphology was performed in adults. Twenty candidates that affect ovarian development were identified and subsequently knocked down in the soma only during niche formation. Feminization factors (Transformer, Sex lethal, and Virilizer), a histone methyltransferase (Enhancer of Zeste), a transcriptional machinery component (Enhancer of yellow 1), a chromatin remodeling complex member (Enhancer of yellow 3) and a chromosome passenger complex constituent (Incenp) were identified as potentially functioning in the control of niche size. The identification of these molecules highlights specific molecular events that are critical for niche formation and will provide a basis for future studies to fully understand the mechanisms of GSC recruitment and maintenance.
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Drosophila/genética , Regulación de la Expresión Génica , Células Germinativas/metabolismo , Infertilidad Femenina/genética , Nicho de Células Madre , Animales , Diferenciación Celular , Femenino , Técnicas de Silenciamiento del Gen , Pruebas Genéticas/métodos , Masculino , Ovario/citología , Ovario/metabolismo , Fenotipo , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Diet is an important regulator of stem cell homeostasis; however, the underlying mechanisms of this regulation are not fully known. Here, we report that insulin signaling mediates dietary maintenance of Drosophila ovarian germline stem cells (GSCs) by promoting the extension of niche escort cell (EC) membranes to wrap around GSCs. This wrapping may facilitate the delivery of bone morphogenetic protein stemness factors from ECs in the niche to GSCs. In addition to the effects on GSCs, insulin signaling-mediated regulation of EC number and protrusions controls the division and growth of GSC progeny. The effects of insulin signaling on EC membrane extension are, at least in part, driven by enhanced translation of Failed axon connections (Fax) via Ribosomal protein S6 kinase. Fax is a membrane protein that may participate in Abelson tyrosine kinase-regulated cytoskeletal dynamics and is known to be involved in axon bundle formation. Therefore, we conclude that dietary cues stimulate insulin signaling in the niche to regulate EC cellular structure, probably via Fax-dependent cytoskeleton remodeling. This mechanism enhances intercellular contact and facilitates homeostatic interactions between somatic and germline cells in response to diet.
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Extensiones de la Superficie Celular/fisiología , Dieta , Células Germinativas/fisiología , Homeostasis/fisiología , Insulina/metabolismo , Nicho de Células Madre/fisiología , Animales , Western Blotting , Supervivencia Celular/fisiología , Señales (Psicología) , Drosophila/citología , Drosophila/metabolismo , Drosophila/fisiología , Proteínas de Drosophila/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Células Germinativas/citología , Células Germinativas/metabolismo , Ovario/metabolismo , Ovario/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. We report that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. We also report that Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell-cell adhesion by negative regulation of E-cadherin expression. Our results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation.
Asunto(s)
Adhesión Celular , Células Germinativas/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Nicho de Células Madre , Animales , Drosophila melanogaster , Femenino , Ovario/citología , Ovario/metabolismoRESUMEN
Wnt/ß-catenin signaling controls various cell fates in metazoan development, and its dysregulation is often associated with cancer formation. However, regulations of this signaling pathway are not completely understood. Here, we report that Lzap, a tumor suppressor, controls nuclear translocation of ß-catenin. In zebrafish embryos disruption of lzap increases the expression of chordin (chd), which encodes a bone morphogenetic protein (BMP) antagonist that is localized in prospective dorsal cells and promotes dorsal fates. Consistently, lzap-deficient embryos with attenuated BMP signaling are dorsalized, which can be rescued by overexpression of zebrafish lzap or bmp2b or human LZAP. The expansion of chd expression in embryos lacking lzap is due to the accumulation of nuclear ß-catenin in ventral cells, in which ß-catenin is usually degraded. Furthermore, the activity of GSK3, a master regulator of ß-catenin degradation, is suppressed in lzap-deficient embryos via inhibitory phosphorylation. Finally, we also report that a similar regulatory axis is also likely to be present in a human tongue carcinoma cell line, SAS. Our results reveal that Lzap is a novel regulator of GSK3 for the maintenance of ventral cell properties and may prevent carcinogenesis via the regulation of ß-catenin degradation.
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Genes Supresores de Tumor , Glucógeno Sintasa Quinasa 3/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/fisiología , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , beta Catenina/metabolismo , Animales , Linaje de la Célula , FosforilaciónRESUMEN
INTRODUCTION: Detection of epidermal growth factor receptor (EGFR) mutation has become the most critical molecular test in managing patients with advanced lung adenocarcinoma. Whether patients with discrepant EGFR mutation results determined by low- and high-sensitivity methods have different clinical outcomes with EGFR tyrosine kinase inhibitor (TKI) treatment needs to be further evaluated. METHODS: Genomic DNA from serial lung adenocarcinoma samples that were EGFR wild-type determined by direct sequencing (DS) were reanalyzed using Scorpion/Amplification Refractory Mutation System (ARMS). The outcomes with EGFR-TKI treatment among patients with discrepant EGFR mutation results between DS and Scorpion/ARMS versus patients with EGFR mutations detected by DS were studied. RESULTS: Of the 130 tumors studied, 28 (21.5%) were found to have EGFR mutations by Scorpion/ARMS. Discrepant EGFR mutation testing results were more common in samples from nonsmokers than in samples from smokers (30.7% versus 9.1%; p = 0.003) and in pleural than in nonpleural samples (62.5% versus 18.9%; p = 0.012). There was no significant difference in the abundance of cancer cells in region(s) selected for testing (26.2% in tumor cell percentage ≤50 versus 16.9% in tumor cell percentage >50; p = 0.201). During EGFR-TKI treatment, the progression-free survival in patients with discrepant EGFR mutation results was similar to those with EGFR mutations detected by DS (median, 13.4 versus 10.9 months; p = 0.225). CONCLUSIONS: DS overlooked EGFR mutation in a significant number of lung adenocarcinoma patients. These patients could have obtained the same benefit from EGFR-TKI when a high-sensitivity method such as Scorpion/ARMS was applied.
Asunto(s)
Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Resultado del TratamientoRESUMEN
BACKGROUND AND OBJECTIVE: Therapeutic responses of lung adenocarcinoma patients to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) are closely associated with activating mutations within the EGFR tyrosine kinase domain. Screening activating EGFR mutations prior to selection for therapeutic strategy has been considered extremely valuable for clinical management of lung adenocarcinoma patients in Asian countries including Taiwan, where the EGFR mutation rate is higher than in the rest of the world. Currently there is no consensus on the method of choice to assess EGFR mutations in tumour tissue. METHODS: We enrolled 445 lung adenocarcinoma patients for analysis of tumour EGFR mutations using polymerase chain reaction (PCR)-direct sequencing, scorpion/amplified refractory mutation system (ARMS) technology and immunohistochemistry with mutation-specific antibodies. RESULTS: Two hundred forty-five patients (245/445; 55%) were found to harbour activating EGFR mutations using PCR-direct sequencing method, with a majority of patients (233/245; 95%) carrying exon 19 deletion or p.L858R point mutations. One hundred three of 200 patients were negative for EGFR mutations from PCR-direct sequencing were further analysed using Scorpion/ARMS technology. Up to 30% of the PCR-direct sequencing negative patients turned out to be positive in the Scorpion/ARMS EGFR mutation tests. For immunohistochemistry analysis of EGFR mutations, the p.E746_A750del specific antibody showed a sensitivity of 57% and a specificity of 100% for exon 19 deletions while the p.L858R point mutation specific antibody showed a sensitivity of 68% and a specificity of 95%. CONCLUSIONS: Based on this study, we proposed an algorithm for comprehensive and efficient testing of EGFR mutations on lung adenocarcinoma patients in Asia.