RESUMEN
Intracellular trafficking of secretory proteins plays key roles in animal development and physiology, but so far, tools for investigating the dynamics of membrane trafficking have been limited to cultured cells. Here, we present a system that enables acute manipulation and real-time visualization of membrane trafficking through the reversible retention of proteins in the endoplasmic reticulum (ER) in living multicellular organisms. By adapting the "retention using selective hooks" (RUSH) approach to Drosophila, we show that trafficking of GPI-linked, secreted, and transmembrane proteins can be controlled with high temporal precision in intact animals and cultured organs. We demonstrate the potential of this approach by analyzing the kinetics of ER exit and apical secretion and the spatiotemporal dynamics of tricellular junction assembly in epithelia of living embryos. Furthermore, we show that controllable ER retention enables tissue-specific depletion of secretory protein function. The system is broadly applicable to visualizing and manipulating membrane trafficking in diverse cell types in vivo.
Asunto(s)
Drosophila , Aparato de Golgi , Animales , Transporte de Proteínas/fisiología , Aparato de Golgi/metabolismo , Transporte Biológico , ExocitosisRESUMEN
E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Polarised E-cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E-cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E-cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E-cadherin secretion. Our data reveal that Myosin V and F-actin are required for the formation of a continuous apicolateral E-cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E-cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal-most region of the lateral membrane. E-cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E-cadherin by showing how Rab7 and Snx16 cooperate in moving E-cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E-cadherin secretion maintains epithelial architecture and prevents metastasis.
Asunto(s)
Cadherinas/metabolismo , Miosina Tipo V/metabolismo , Actinas/metabolismo , Uniones Adherentes/metabolismo , Animales , Adhesión Celular , Endosomas/metabolismo , Exocitosis , Humanos , Metástasis de la Neoplasia/prevención & controlRESUMEN
Epithelial barriers can open their junctions to enhance paracellular flux. A new article by Isasti-Sanchez et al. in this issue of Developmental Cell shows how changes in cell adhesion and relaxation of acto-myosin tension cooperate in opening the cell vertices of the Drosophila follicular epithelium.
Asunto(s)
Drosophila , Células Epiteliales , Animales , Adhesión Celular , Epitelio , MiosinasRESUMEN
Degradation of endocytosed proteins involves the formation of transient connections between late endosomes and lysosomes in a process called "kiss and run." Genes and proteins controlling this mechanism are unknown. Here, we identify the small guanosine triphosphatase (GTPase) RabX1 as an organizer of a late endosomal compartment that forms dynamic tubular connections to lysosomes. By analyzing trafficking of the adhesion protein Fasciclin2 in the Drosophila follicular epithelium, we show that a reduction of RabX1 function leads to defects in Fasciclin2 degradation. RabX1 mutants fail to form normal lysosomes and accumulate Fasciclin2 in a swelling late-endosomal compartment. RabX1 protein localizes to late endosomes, where it induces the formation of tubular connections to lysosomes. We propose that these tubules facilitate influx of lysosomal content into late endosomes and that this influx leads to the formation of endolysosomes, in which Fasciclin2 is degraded. We show that the formation of RabX1 tubules is dependent on the V-ATPase proton pump. Moreover, we provide evidence that V-ATPase activity is upregulated during epithelial differentiation. This upregulation intensifies RabX1 tubulation and thereby boosts the capacity of the endolysosomal pathway. Enhanced endolysosomal capacity is required for the removal of Fasciclin2 from the epithelium, which is part of a developmental program promoting epithelial morphogenesis.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de Unión al GTP rab/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endocitosis/fisiología , Femenino , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cytoplasmic dynein-1 is a large minus-end-directed microtubule motor complex involved in membrane trafficking, organelle positioning, and microtubule organization. The roles of dynein light intermediate chains (DLICs; DLIC1 and DLIC2) within the complex are, however, still largely undefined. In this study, we investigated the possible roles of DLICs in epithelial homeostasis and colon cancer development. Mutant clonal analysis of Drosophila Dlic in the follicular epithelium of Drosophila ovary showed defects in nuclear positioning, epithelial integrity, and apical cell polarity. Consistently, knockdown of human DLIC1 and DLIC2 in colon carcinoma cells resulted in damaged epithelial organization, disturbed lumen formation, and impaired apical polarity establishment in three-dimensional cell culture. Depletion of DLIC1 and DLIC2 led to reduced proliferation, enhanced apoptosis rates, disrupted mitotic spindle assembly, and induction of G2/M arrest in cell cycle progression. Moreover, reduced levels of DLIC1 in contrast to DLIC2 impaired the migratory ability. On the other hand, immunohistochemical examination of human colorectal tissue samples and further colorectal cancer dataset analysis showed a significant upregulation for DLIC1 in tumors, whereas DLIC2 expression was unchanged. In addition, the overexpression of DLIC1 caused increased proliferation, decreased apoptosis and enhanced migration, whereas DLIC2 overexpression did not result in any significant changes. Together, these results indicate that DLIC1 and DLIC2 contribute to the establishment and maintenance of epithelial homeostasis. Furthermore, these findings present the first evidence that DLIC1 and DLIC2 have distinct roles in colon cancer development and that DLIC1 may contribute to proliferative overgrowth and migratory characteristics.
Asunto(s)
Movimiento Celular , Proliferación Celular , Colon/patología , Neoplasias del Colon/patología , Dineínas Citoplasmáticas/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Animales , Apoptosis , Estudios de Casos y Controles , Células Cultivadas , Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Dineínas Citoplasmáticas/genética , Drosophila , Regulación Neoplásica de la Expresión Génica , Humanos , Regulación hacia ArribaRESUMEN
In vivo RNAi in Drosophila facilitates simple and rapid analysis of gene functions in a cell- or tissue-specific manner. The versatility of the UAS-GAL4 system allows to control exactly where and when during development the function of a gene is depleted. The epithelium of the ovary is a particularly good model to study in a living animal how stem cells are maintained and how their descendants proliferate and differentiate. Here I provide basic information about the publicly available reagents for in vivo RNAi, and I describe how the oogenesis system can be applied to analyze stem cells and epithelial development at a histological level. Moreover, I give helpful hints to optimize the use of the UAS-GAL4 system for RNAi induction in the follicular epithelium. Finally, I provide detailed step-by-step protocols for ovary dissection, antibody stainings, and ovary mounting for microscopic analysis.
Asunto(s)
Drosophila/genética , Epitelio/metabolismo , Folículo Ovárico/citología , Interferencia de ARN , Células Madre/citología , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Proliferación Celular , Femenino , Ovario/citologíaRESUMEN
E-cadherin localization to the zonula adherens is fundamental for epithelial differentiation but the mechanisms controlling localization are unclear. Using the Drosophila follicular epithelium we genetically dissect E-cadherin transport in an in vivo model. We distinguish three mechanisms mediating E-cadherin accumulation at the zonula adherens. Two membrane trafficking pathways deliver newly synthesized E-cadherin to the plasma membrane. One is Rab11 dependent and targets E-cadherin directly to the zonula adherens, while the other transports E-cadherin to the lateral membrane. Lateral E-cadherin reaches the zonula adherens by endocytosis and targeted recycling. We show that this pathway is dependent on RabX1, which provides a functional link between early and recycling endosomes. Moreover, we show that lateral E-cadherin is transported to the zonula adherens by an apically directed flow within the plasma membrane. Differential activation of these pathways could facilitate cell shape changes during morphogenesis, while their misregulation compromises cell adhesion and tissue architecture in differentiated epithelia.
Asunto(s)
Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Células Epiteliales/metabolismo , Transporte de Proteínas/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/genética , Animales , Drosophila , Endocitosis/genética , Endosomas/metabolismo , Epitelio/metabolismo , Femenino , Folículo Ovárico/citologíaRESUMEN
The Drosophila oogenesis system provides an excellent model to study the development of epithelial tissues. Here, we report the first genome-scale in vivo RNA interference (RNAi) screen for genes controlling epithelial development. By directly analysing cell and tissue architecture we identified 1125 genes, which we assigned to seven different functions in epithelial formation and homeostasis. We validated the significance of our screen by generating mutants for Vps60, a component of the endosomal sorting complexes required for transport (ESCRT) machinery. This analysis provided new insights into spatiotemporal control of cell proliferation in the follicular epithelium. Previous studies have identified signals controlling divisions in the follicle stem cell niche. However, 99% of cell divisions occur outside of the niche and it is unclear how these divisions are controlled. Our data distinguish two new domains outside of the stem cell niche where there are differing controls on proliferation. One domain abuts the niche and is characterised by ESCRT, Notch and JAK/STAT-mediated control of proliferation. Adjacent to this domain, another domain is defined by loss of the impact of ESCRT on cell division. Thus, during development epithelial cells pass through a variety of microenvironments that exert different modes of proliferation control. The switch between these modes might reflect a decrease in the 'stemness' of epithelial cells over time.
Asunto(s)
Proliferación Celular , Drosophila melanogaster/genética , Células Epiteliales/fisiología , Nicho de Células Madre , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Epitelio/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Genoma de los Insectos , Oogénesis , Folículo Ovárico/citología , Folículo Ovárico/fisiología , Fenotipo , Interferencia de ARN , Receptores Notch/genética , Receptores Notch/metabolismo , Ubiquitina/metabolismoRESUMEN
Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. In this paper, we report a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. We show that the inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, we identify here a mechanism controlling the morphogenesis of a squamous epithelium.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Forma de la Célula , Proteínas de Drosophila/fisiología , Drosophila melanogaster/enzimología , Endocitosis , Células Epiteliales/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Membrana Celular/metabolismo , Polaridad Celular , Codón sin Sentido , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Mutagénesis , Folículo Ovárico/citología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de ProteínasRESUMEN
The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.
Asunto(s)
Drosophila/embriología , Coloración y Etiquetado/métodos , Animales , Drosophila/anatomía & histología , Larva/anatomía & histología , Coloración y Etiquetado/economía , Pez Cebra/embriologíaRESUMEN
Many RNAs show polarized or otherwise non-random subcellular distributions. To create a method for genome-wide genetic screens for RNAs with asymmetric subcellular distributions, we have combined methods for gene tagging and live imaging of messenger RNA (mRNA). A pilot screen in a highly polarized, differentiated cell in the Drosophila larva, the branched terminal cell of the tracheal system, demonstrates the feasibility of the method for identifying new asymmetrically localized mRNAs in vivo.
Asunto(s)
Drosophila melanogaster/genética , Pruebas Genéticas , Transporte de ARN/genética , ARN/metabolismo , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/metabolismo , Levivirus/metabolismo , Especificidad de Órganos/genética , Proyectos Piloto , Biosíntesis de Proteínas/genética , Reproducibilidad de los ResultadosRESUMEN
The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, beta-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.
Asunto(s)
Polaridad Celular , Drosophila/citología , Células Epiteliales/citología , Uniones Intercelulares/química , Uniones Adherentes/química , Animales , Proteínas de Drosophila/fisiología , Células Epiteliales/ultraestructura , Epitelio , Mesodermo/citologíaRESUMEN
The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.
Asunto(s)
Actinas/fisiología , Citoplasma/fisiología , Microtúbulos/fisiología , Oocitos/fisiología , Animales , Drosophila , Inmunohistoquímica , Microscopía ConfocalRESUMEN
The Hippo pathway is a potent regulator of tissue growth. Two recent studies report a new function of the pathway in the differentiation of the follicular epithelium during Drosophila oogenesis.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Oogénesis/fisiología , Folículo Ovárico/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Tipificación del Cuerpo/efectos de los fármacos , Diferenciación Celular , Proteínas de Drosophila/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Folículo Ovárico/fisiología , Óvulo/citología , Óvulo/fisiologíaRESUMEN
The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation [1, 2]. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. Here we identify a role for the actomyosin cytoskeleton in this process. We show that myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. We demonstrate that the epithelium collapses in the absence of myosin activity and show that the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, our data indicate that cyst growth induces cell division in the epithelium. In addition, we show how apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1beta9C. In addition, our data indicate that active myosin is apically anchored by the Baz/Par-6/aPKC complex.
Asunto(s)
Actomiosina/fisiología , Citoesqueleto/fisiología , Proteínas de Drosophila/fisiología , Drosophila/fisiología , Animales , Drosophila/embriología , Femenino , Oogénesis/fisiologíaRESUMEN
The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals. During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid. To elucidate the molecular pathways triggered by Par-1, we have performed a genome-wide, high-throughput screen for Par-1 targets. Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation. We show that Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo. We identify two motifs in Exu that are phosphorylated by Par-1, and show that their mutation abolishes bicoid mRNA localisation during mid-oogenesis. Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis. These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation. Furthermore, our analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Proteínas del Huevo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Quinasas/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Secuencias de Aminoácidos , Animales , Western Blotting , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Electroforesis en Gel de Poliacrilamida , Embrión no Mamífero , Etiquetas de Secuencia Expresada , Femenino , Glucógeno Sintasa Quinasa 3 , Hibridación in Situ , Microscopía Fluorescente , Modelos Genéticos , Mutagénesis , Mutación , Ovario/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , TransgenesRESUMEN
Par-1 kinase is critical for polarization of the Drosophila melanogaster oocyte and the one-cell Caenorhabditis elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Here we show that Drosophila Par-1 phosphorylates the posterior determinant Oskar (Osk) and demonstrate genetically that Par-1 is required for accumulation of Osk protein. We show in cell-free extracts that Osk protein is intrinsically unstable and that it is stabilized after phosphorylation by Par-1. Our data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk.