RESUMEN
The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs-Expanded (Crb-Ex) and/or Merlin-Kibra (Mer-Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6-hexanediol treatment. Overexpressing Ex or Kib induces formation of micron-scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low-complexity domains and purified Hpo-Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated "signalosomes" induced by clustering of upstream pathway components.
Asunto(s)
Proteínas de Drosophila , Vía de Señalización Hippo , Animales , Humanos , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Neurofibromina 2/metabolismo , Drosophila melanogaster/metabolismo , Mamíferos , Proteínas Serina-Treonina Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ or WW-domain-containing transcriptional activator [WWTR1]), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. Here, we show that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. We find similar results in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasa/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transactivadores/genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Animales , Fenómenos Biomecánicos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Polaridad Celular , Proliferación Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Células Epiteliales/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Mecanotransducción Celular , Ratones , Proteínas Nucleares/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transactivadores/metabolismo , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E-cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. We show that these pseudostratified epithelial cells downregulate E-cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho-kinase-mediated actomyosin contractility blocks downregulation of E-cadherin during mitosis. Mitotic activation of Rho-kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity.
Asunto(s)
Uniones Adherentes , Huso Acromático , Animales , Drosophila/genética , Células Epiteliales , MitosisRESUMEN
Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. Here, we show that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. We find no evidence for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, our results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Mecánico , Alas de Animales/embriología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/embriología , Discos Imaginales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Mecanotransducción Celular/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas/genética , Transducción de Señal/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Alas de Animales/metabolismo , Proteínas Señalizadoras YAPRESUMEN
The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mecanotransducción Celular/fisiología , Folículo Ovárico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Espectrina/metabolismo , Animales , Línea Celular , Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Folículo Ovárico/citología , Proteínas Serina-Treonina Quinasas/genética , Espectrina/genética , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Señalizadoras YAPRESUMEN
In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Microvellosidades/metabolismo , Miosina Tipo V/genética , Proteínas de Unión al GTP rab/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Cadherinas/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Polaridad Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Humanos , Microtúbulos/genética , Microvellosidades/genética , Miosina Tipo V/metabolismo , Transporte de Proteínas/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.
Asunto(s)
Proteínas del Ojo , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Nucleósido-Fosfato Quinasa , Secuencias de Aminoácidos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Guanilato-Quinasas/química , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nucleósido-Fosfato Quinasa/química , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Mask family proteins were discovered in Drosophila to promote the activity of the transcriptional coactivator Yorkie (Yki), the sole fly homolog of mammalian YAP (YAP1) and TAZ (WWTR1). The molecular function of Mask, or its mammalian homologs Mask1 (ANKHD1) and Mask2 (ANKRD17), remains unclear. Mask family proteins contain two ankyrin repeat domains that bind Yki/YAP as well as a conserved nuclear localisation sequence (NLS) and nuclear export sequence (NES), suggesting a role in nucleo-cytoplasmic transport. Here we show that Mask acts to promote nuclear import of Yki, and that addition of an ectopic NLS to Yki is sufficient to bypass the requirement for Mask in Yki-driven tissue growth. Mammalian Mask1/2 proteins also promote nuclear import of YAP, as well as stabilising YAP and driving formation of liquid droplets. Mask1/2 and YAP normally colocalise in a granular fashion in both nucleus and cytoplasm, and are co-regulated during mechanotransduction.
Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Animales , Proteínas de Unión al ADN/genética , Drosophila , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , Señales de Clasificación de Proteína , Transactivadores/genética , Proteínas Señalizadoras YAPRESUMEN
Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. Here, we show that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. Our results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia.
Asunto(s)
Membrana Celular/metabolismo , Polaridad Celular , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Células Epiteliales/citología , Células Epiteliales/enzimología , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Animales , Células CACO-2 , Proteínas de Drosophila/metabolismo , Humanos , Ratones , Fosforilación , Proteína Quinasa C/metabolismo , Interferencia de ARN , Quinasas p21 Activadas/químicaRESUMEN
The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. Here we show that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. We propose that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. Our findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions.
Asunto(s)
Aurora Quinasas/metabolismo , Proteínas de Drosophila/metabolismo , Células Epiteliales/fisiología , Mitosis , Proteína Quinasa C/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Citoplasma/metabolismo , Drosophila , Células-Madre Neurales/fisiología , Fosforilación , Huso Acromático/fisiología , Alas de Animales/crecimiento & desarrolloRESUMEN
Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Movimiento Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Capping de la Actina/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Polaridad Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Retroalimentación Fisiológica/fisiología , Femenino , Proteínas Fluorescentes Verdes/genética , Masculino , Proteínas Nucleares/metabolismo , Fosforilación/fisiología , Proteínas Quinasas/metabolismo , Transactivadores/metabolismo , Transgenes/fisiología , Proteínas Señalizadoras YAPRESUMEN
Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib). Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity. We have built a computer model of apicobasal polarity that suggests that the combination of positive feedback among apical determinants plus mutual antagonism between apical and basal determinants is essential for polarization. In agreement with this model, in vivo experiments define a positive feedback loop in which Crb self-recruits via Crb-Crb extracellular domain interactions, recruitment of Sdt-aPKC-Par6-cdc42, aPKC phosphorylation of Crb, and recruitment of Expanded (Ex) and Kibra (Kib) to prevent endocytic removal of Crb from the plasma membrane. Lgl antagonizes the operation of this feedback loop, explaining why apical determinants do not normally spread into the basolateral domain. Once Crb is removed from the plasma membrane, it undergoes recycling via Rab11 endosomes. Our results provide a dynamic model for understanding how epithelial polarity is maintained in Drosophila follicle cells.