RESUMEN
The Notch communication pathway, discovered in Drosophila over 100 years ago, regulates a wide range of intra-lineage decisions in metazoans. The division of the Drosophila mechanosensory organ precursor is the archetype of asymmetric cell division in which differential Notch activation takes place at cytokinesis. Here, we review the molecular mechanisms by which epithelial cell polarity, cell cycle and intracellular trafficking participate in controlling the directionality, subcellular localization and temporality of mechanosensitive Notch receptor activation in cytokinesis.
Asunto(s)
Drosophila melanogaster , Receptores Notch , Animales , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Epitelio/metabolismo , Polaridad Celular , Proteínas de Drosophila/metabolismo , Órganos de los Sentidos/metabolismo , Órganos de los Sentidos/citología , Transducción de Señal , Células Epiteliales/metabolismo , Células Epiteliales/citologíaRESUMEN
Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterised. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in Drosophila pupal notum, alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-actin, and non-muscle myosin II in the plane of adherens junctions. On the other hand, ß-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector ß-Heavy Spectrin Karst, and ß-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the SJ, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Epiteliales/metabolismo , Uniones Intercelulares/metabolismo , Integrinas/metabolismoRESUMEN
Although the molecular mechanisms governing abscission of isolated cells have largely been elucidated, those underlying the abscission of epithelial progenitors surrounded by epidermal cells (ECs), connected via cellular junctions, remain largely unexplored. Here, we investigated the remodeling of the paracellular diffusion barrier ensured by septate junctions (SJs) during cytokinesis of Drosophila sensory organ precursors (SOPs). We found that SOP cytokinesis involves the coordinated, polarized assembly and remodeling of SJs in the dividing cell and its neighbors, which remain connected to the former via membrane protrusions pointing towards the SOP midbody. SJ assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to quicker disentanglement of neighboring cell membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for the transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions and is required for SJ integrity, and alteration of SJ integrity leads to premature abscission. Our study uncovers cell-intrinsic and -extrinsic functions of Shrub in coordinating remodeling of the SJs and SOP abscission.
Asunto(s)
Citocinesis , Proteínas de Drosophila , Drosophila , Proteínas del Tejido Nervioso , Animales , Movimiento Celular , Difusión , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas del Tejido Nervioso/genética , Proteínas de Drosophila/genéticaRESUMEN
In multiple cell lineages, Delta-Notch signalling regulates cell fate decisions owing to unidirectional signalling between daughter cells. In Drosophila pupal sensory organ lineage, Notch regulates the intra-lineage pIIa/pIIb fate decision at cytokinesis. Notch and Delta that localise apically and basally at the pIIa-pIIb interface are expressed at low levels and their residence time at the plasma membrane is in the order of minutes. How Delta can effectively interact with Notch to trigger signalling from a large plasma membrane area remains poorly understood. Here, we report that the signalling interface possesses a unique apico-basal polarity with Par3/Bazooka localising in the form of nano-clusters at the apical and basal level. Notch is preferentially targeted to the pIIa-pIIb interface, where it co-clusters with Bazooka and its cofactor Sanpodo. Clusters whose assembly relies on Bazooka and Sanpodo activities are also positive for Neuralized, the E3 ligase required for Delta activity. We propose that the nano-clusters act as snap buttons at the new pIIa-pIIb interface to allow efficient intra-lineage signalling.
Asunto(s)
División Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Órganos de los Sentidos/metabolismo , Células Madre/metabolismo , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Polaridad Celular , Citocinesis , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Receptores Notch/genética , Órganos de los Sentidos/citología , Transducción de Señal , Factores de Tiempo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.
Asunto(s)
Imagenología Tridimensional , Organogénesis , Organoides , Animales , Diferenciación Celular/fisiología , Cilios , Ratones , Organoides/diagnóstico por imagenRESUMEN
Epithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. AURKA triggers the degradation of Inner Mitochondrial Membrane/matrix proteins by interacting with core components of the autophagy pathway. On the inner mitochondrial membrane, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2, which triggers mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex and restores normal ATP production levels. Altogether, these data provide evidence for a role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.
Asunto(s)
Aurora Quinasa A/metabolismo , Mitocondrias/metabolismo , Mitofagia/genética , Animales , Aurora Quinasa A/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HEK293 , Humanos , Células MCF-7 , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/fisiología , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/metabolismo , Mitofagia/fisiología , Prohibitinas , Proteínas Represoras/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Current super-resolution microscopy (SRM) methods suffer from an intrinsic complexity that might curtail their routine use in cell biology. We describe here random illumination microscopy (RIM) for live-cell imaging at super-resolutions matching that of 3D structured illumination microscopy, in a robust fashion. Based on speckled illumination and statistical image reconstruction, easy to implement and user-friendly, RIM is unaffected by optical aberrations on the excitation side, linear to brightness, and compatible with multicolor live-cell imaging over extended periods of time. We illustrate the potential of RIM on diverse biological applications, from the mobility of proliferating cell nuclear antigen (PCNA) in U2OS cells and kinetochore dynamics in mitotic S. pombe cells to the 3D motion of myosin minifilaments deep inside Drosophila tissues. RIM's inherent simplicity and extended biological applicability, particularly for imaging at increased depths, could help make SRM accessible to biology laboratories.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Iluminación , Animales , Microscopía Fluorescente/métodos , DrosophilaRESUMEN
Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We have previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signalling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores Notch/metabolismo , Órganos de los Sentidos/metabolismo , Células Madre/metabolismo , Animales , Linaje de la Célula , Núcleo Celular/metabolismo , Polaridad Celular , Mutación con Ganancia de Función , Penetrancia , FenotipoRESUMEN
In epithelia, tricellular junctions (TCJs) serve as pivotal sites for barrier function and integration of both biochemical and mechanical signals [1-3]. In Drosophila, TCJs are composed of the transmembrane protein Sidekick at the adherens junction (AJ) level, which plays a role in cell-cell contact rearrangement [4-6]. At the septate junction (SJ) level, TCJs are formed by Gliotactin (Gli) [7], Anakonda (Aka) [8, 9], and the Myelin proteolipid protein (PLP) M6 [10, 11]. Despite previous data on TCJ organization [12-14], TCJ assembly, composition, and links to adjacent bicellular junctions (BCJs) remain poorly understood. Here, we have characterized the making of TCJs within the plane of adherens junctions (tricellular adherens junction [tAJ]) and the plane of septate junctions (tricellular septate junction [tSJ]) and report that their assembly is independent of each other. Aka and M6, whose localizations are interdependent, act upstream to localize Gli. In turn, Gli stabilizes Aka at tSJ. Moreover, tSJ components are not only essential at vertex, as we found that loss of tSJ integrity induces micron-length bicellular SJ (bSJ) deformations. This phenotype is associated with the disappearance of SJ components at tricellular contacts, indicating that bSJs are no longer connected to tSJs. Reciprocally, SJ components are required to restrict the localization of Aka and Gli at vertex. We propose that tSJs function as pillars to anchor bSJs to ensure the maintenance of tissue integrity in Drosophila proliferative epithelia.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Proteolipídica de la Mielina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Depuradores/metabolismo , Animales , Animales Modificados Genéticamente , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Uniones Intercelulares/genética , Microscopía Intravital , Proteínas de la Membrana/genética , Proteína Proteolipídica de la Mielina/genética , Proteínas del Tejido Nervioso/genética , Estabilidad Proteica , Receptores Depuradores/genéticaRESUMEN
Many epithelial cancers show cell cycle dysfunction tightly correlated with the overexpression of the serine/threonine kinase Aurora A (AURKA). Its role in mitotic progression has been extensively characterised, and evidence for new AURKA functions emerges. Here, we reveal that AURKA is located and imported in mitochondria in several human cancer cell lines. Mitochondrial AURKA impacts on two organelle functions: mitochondrial dynamics and energy production. When AURKA is expressed at endogenous levels during interphase, it induces mitochondrial fragmentation independently from RALA. Conversely, AURKA enhances mitochondrial fusion and ATP production when it is over-expressed. We demonstrate that AURKA directly regulates mitochondrial functions and that AURKA over-expression promotes metabolic reprogramming by increasing mitochondrial interconnectivity. Our work paves the way to anti-cancer therapeutics based on the simultaneous targeting of mitochondrial functions and AURKA inhibition.
Asunto(s)
Aurora Quinasa A/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Aurora Quinasa A/química , Biocatálisis , Línea Celular Tumoral , Respiración de la Célula , Citosol/metabolismo , Drosophila melanogaster/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Células HEK293 , Humanos , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Modelos Biológicos , Péptidos/metabolismo , Transporte de Proteínas , Proteolisis , Regulación hacia ArribaRESUMEN
In Drosophila, the sensory organ precursor (SOP or pI cell) divides asymmetrically to give birth to daughter cells, the fates of which are governed by the differential activation of the Notch pathway. Proteolytic activation of Notch induced by ligand is based on the correct polarized sorting and localization of the Notch ligand Delta, the Notch receptor and its trafficking partner Sanpodo (Spdo). Here, we have identified Stratum (Strat), a presumptive guanine nucleotide exchange factor for Rab GTPases, as a regulator of Notch activation. Loss of Strat causes cell fate transformations associated with an accumulation of Notch, Delta and Spdo in the trans-Golgi network (TGN), and an apical accumulation of Spdo. The strat mutant phenotype is rescued by the catalytically active as well as the wild-type form of Rab8, suggesting a chaperone function for Strat rather than that of exchange factor. Strat is required to localize Rab8 at the TGN, and rab8 phenocopies strat We propose that Strat and Rab8 act at the exit of the Golgi apparatus to regulate the sorting and the polarized distribution of Notch, Delta and Spdo.
Asunto(s)
Proteínas de Drosophila/metabolismo , GTP Fosfohidrolasas/metabolismo , Receptores Notch/metabolismo , Red trans-Golgi/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , GTP Fosfohidrolasas/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Transporte de Proteínas/fisiología , Receptores Notch/genética , Red trans-Golgi/genéticaRESUMEN
The orientation of the mitotic spindle (MS) is tightly regulated, but the molecular mechanisms are incompletely understood. Here we report a novel role for the multifunctional adaptor protein ALG-2-interacting protein X (ALIX) in regulating MS orientation in addition to its well-established role in cytokinesis. We show that ALIX is recruited to the pericentriolar material (PCM) of the centrosomes and promotes correct orientation of the MS in asymmetrically dividing Drosophila stem cells and epithelial cells, and symmetrically dividing Drosophila and human epithelial cells. ALIX-deprived cells display defective formation of astral microtubules (MTs), which results in abnormal MS orientation. Specifically, ALIX is recruited to the PCM via Drosophila Spindle defective 2 (DSpd-2)/Cep192, where ALIX promotes accumulation of γ-tubulin and thus facilitates efficient nucleation of astral MTs. In addition, ALIX promotes MT stability by recruiting microtubule-associated protein 1S (MAP1S), which stabilizes newly formed MTs. Altogether, our results demonstrate a novel evolutionarily conserved role of ALIX in providing robustness to the orientation of the MS by promoting astral MT formation during asymmetric and symmetric cell division.
Asunto(s)
Centrosoma/fisiología , Proteínas de Drosophila/fisiología , Proteínas de Microfilamentos/fisiología , Huso Acromático/fisiología , Animales , Encéfalo/citología , Drosophila/fisiología , Células Epiteliales/fisiología , Femenino , Células HeLa , Humanos , Masculino , Microtúbulos/fisiología , Mitosis/fisiología , Ovario/citología , Células Madre/fisiologíaRESUMEN
How permeability barrier function is maintained when epithelial cells divide is largely unknown. Here, we have investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. We report that, following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. We propose that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia.
Asunto(s)
Comunicación Celular , Proliferación Celular , Citocinesis , Drosophila melanogaster/fisiología , Embrión no Mamífero/fisiología , Epitelio/fisiología , Uniones Intercelulares/fisiología , Animales , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Epitelio/crecimiento & desarrollo , Discos Imaginales/citología , Discos Imaginales/fisiología , Alas de Animales/citología , Alas de Animales/fisiologíaRESUMEN
Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosin cables formed in apoptotic and neighboring cells drives cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. We propose a model in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion.
Asunto(s)
Apoptosis/fisiología , Adhesión Celular/fisiología , Drosophila/embriología , Epitelio/embriología , Estrés Mecánico , Resistencia a la Tracción , Uniones Adherentes/fisiología , Animales , Animales Modificados Genéticamente , Fenómenos Biomecánicos , Polaridad Celular , Forma de la Célula , Embrión no Mamífero , Epitelio/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
Intercellular bridges called "ring canals" (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs' anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin-containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs' anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin-dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth.
Asunto(s)
Cadherinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Oogénesis/fisiología , Complejo 1 de Proteína Adaptadora/genética , Complejo 1 de Proteína Adaptadora/metabolismo , Animales , Cadherinas/genética , Membrana Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , FemeninoRESUMEN
Epithelia are compact tissues comprising juxtaposed cells that function as mechanical and chemical barriers between the body and the environment. This barrier relies, in part, on adhesive contacts within adherens junctions, which are formed and stabilized by E-cadherin and catenin proteins linked to the actomyosin cytoskeleton. During development and throughout adult life, epithelia are continuously growing or regenerating, largely as a result of cell division. Although persistence of adherens junctions is needed for epithelial integrity, these junctions are continually remodelled during cell division. In this Commentary, we will focus on cytokinesis, the final step of mitosis, a multiparty phenomenon in which the adherens junction belt plays an essential role and during which a new cell-cell interface is generated between daughter cells. This new interface is the site of intense remodelling, where new adhesive contacts are assembled and cell polarity is transmitted from mother to daughter cells, ultimately becoming the site of cell signalling.
Asunto(s)
División Celular , Células Epiteliales/citología , Uniones Adherentes/metabolismo , Animales , Polaridad Celular , Citocinesis , Células Epiteliales/metabolismo , MitosisRESUMEN
In metazoans, unequal partitioning of the cell-fate determinant Numb underlies the generation of distinct cell fates following asymmetric cell division [1-5]. In Drosophila, during asymmetric division of the sensory organ precursor (SOP) cell, Numb is unequally inherited by the pIIb daughter cell, where it antagonizes Notch [1, 6-8]. Numb inhibits Notch partly through inhibiting the plasma membrane localization of Sanpodo (Spdo), a transmembrane protein required for Notch signaling during asymmetric cell division [9, 10]. Numb, by binding to Spdo and α-Adaptin, was proposed to mediate Spdo endocytosis alone or bound to Notch in the pIIb cell, thereby preventing Notch activation [11-16]. However, in addition to endocytosis, Numb also controls the postendocytic trafficking and degradation of Notch in mammals [17, 18] and negatively regulates basolateral recycling in C. elegans [19, 20]. Thus, whether Numb promotes the endocytosis of Spdo is a question that requires experimental demonstration and is therefore investigated in this article. Based on internalization assays, we show that Spdo endocytosis is restricted to cells in interphase and requires AP-2 activity. Surprisingly, the bulk endocytosis of Spdo occurs properly in numb mutant SOP, indicating that Numb does not regulate the steady-state localization of Spdo via Spdo internalization. We report that Numb genetically and physically interacts with AP-1, a complex regulating the basolateral recycling of Spdo [21]. In numb mutant organs, Spdo is efficiently internalized and recycled back to the plasma membrane. We propose that Numb acts in concert with AP-1 to control the endocytic recycling of Spdo to regulate binary-fate decisions.
Asunto(s)
División Celular Asimétrica/fisiología , Diferenciación Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Endocitosis/fisiología , Hormonas Juveniles/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora/metabolismo , Animales , Membrana Celular/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Inmunoprecipitación , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Células-Madre Neurales/fisiología , Receptores Notch/antagonistas & inhibidores , Células Receptoras Sensoriales/fisiología , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-2/metabolismoRESUMEN
How adhesive contacts with neighbors may affect epithelial cell cytokinesis is unknown. We report that in Drosophila, septins are specifically required for planar (but not orthogonal) cytokinesis. During planar division, cytokinetic furrowing initiates basally, resulting in a contractile ring displaced toward the adherens junction (AJ). The formation of new AJ between daughter cells requires the disengagement of E-Cadherin complexes between mitotic and neighboring cells at the cleavage furrow, followed by the assembly of E-Cadherin complexes on the daughter-daughter interface. The strength of adhesion with neighbors directly impacts both the kinetics of AJ disengagement and the length of the new AJ. Loss of septins causes a reduction in the contractility of the actomyosin ring and prevents local disengagement of AJ in the cleavage furrow. By modulating the strength of tension induced by neighbors, we uncover a mechanical function for septins to overcome the extrinsic tension induced by neighboring interphasic cells.
Asunto(s)
Actomiosina , Uniones Adherentes , Citocinesis/genética , Septinas , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Uniones Adherentes/genética , Uniones Adherentes/fisiología , Animales , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/genética , Polaridad Celular , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Desarrollo Embrionario , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Contracción Muscular/genética , Septinas/genética , Septinas/metabolismo , Tórax/citología , Tórax/crecimiento & desarrolloRESUMEN
Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.
Asunto(s)
Proteínas de Ciclo Celular/aislamiento & purificación , Citoesqueleto/química , Factores de Intercambio de Guanina Nucleótido/aislamiento & purificación , Nanopartículas de Magnetita/química , Proteínas Nucleares/aislamiento & purificación , Proteína de Unión al GTP ran/aislamiento & purificación , Animales , Proteínas de Ciclo Celular/química , Diferenciación Celular , Núcleo Celular/química , Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Proteínas Nucleares/química , Transducción de Señal , Xenopus laevis/metabolismo , Proteína de Unión al GTP ran/químicaRESUMEN
Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells.