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.
Epithelial tissues are composed of polarized cells with distinct apical and basolateral membrane domains 1. In the Drosophila ovarian follicle cell epithelium, apical membranes are specified by ...Crumbs (Crb), Stardust (Sdt), and the aPKC-Par6-cdc42 complex 1–7. Basolateral membranes are specified by Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib) 8, 9. Apical and basolateral determinants are known to act in a mutually antagonistic fashion, but it remains unclear how this interaction generates polarity 1. 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.
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► Positive feedback plus mutual antagonism generates polarity in a computer model ► Apical determinants self-recruit to the plasma membrane in a positive feedback loop ► Self-recruitment involves Crb-Crb interactions and aPKC phosphorylation ► Ex and Kib stabilize Crb at the apical membrane
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.
•Aurora kinases directly phosphorylate Lgl during mitosis•Aurora phosphorylation of Lgl drives relocalization to the cytoplasm at mitosis•Relocalization of Lgl promotes mitotic spindle orientation in epithelia•Removal of Lgl from the plasma membrane may promote Pins-Dlg interactions
Bell et al. describe a new role for the Lgl tumor suppressor in mitotic spindle orientation. They show that Aurora kinases phosphorylate Lgl to drive a dramatic relocalization of the protein during mitosis that is necessary for normal orientation of mitotic spindles in epithelial cells.
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.
Synopsis
In Drosophila, both the apical and the basolateral Spectrin cytoskeleton are able to activate the Hippo signaling pathway in a tissue‐dependent manner. In cultured human cells this depends on cell density, suggesting a role for Spectrins in mechanosensing.
The Spectrin cytoskeleton is required for Hippo signalling in Drosophila.
Loss of Spectrins causes mild tissue overgrowth.
Apical Spectrins bind to Expanded, Merlin and Kibra.
Spectrins are potential mechanosensors.
In Drosophila, both the apical and the basolateral Spectrin cytoskeleton are able to activate the Hippo signaling pathway in a tissue‐dependent manner. In cultured human cells this depends on cell density, suggesting a role for Spectrins in mechanosensing.
Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole
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
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
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.
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.
Sterile inflammation can be initiated by innate immune recognition of markers of tissue injury termed damage-associated molecular patterns (DAMPs). DAMP recognition by dendritic cells (DCs) has also ...been postulated to lead to T cell responses to foreign antigens in tumors or allografts. Many DAMPs represent intracellular contents that are released upon cell damage, notably after necrosis. In this regard, we have previously described DNGR-1 (CLEC9A) as a DC-restricted receptor specific for an unidentified DAMP that is exposed by necrotic cells and is necessary for efficient priming of cytotoxic T cells against dead cell-associated antigens. Here, we have shown that the DNGR-1 ligand is preserved from yeast to man and corresponds to the F-actin component of the cellular cytoskeleton. The identification of F-actin as a DNGR-1 ligand suggests that cytoskeletal exposure is a universal sign of cell damage that can be targeted by the innate immune system to initiate immunity.
► DNGR-1 recognizes a cell-associated ligand conserved from yeast to man ► F-actin is the ligand for DNGR-1 ► F-actin exposure permits DNGR-1 recognition of necrotic cells
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.
Mask family proteins were discovered in
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.
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.
Synopsis
The conserved Hippo signalling pathway regulates the activity of Yorkie (YAP/TAZ in mammals) transcriptional co‐activator via Hippo (MST1/2) and Warts (LATS1/2) kinases. This study shows that Hippo pathway components form large cytoplasmic punctae in epithelial cells through a process that involves the formation of biomolecular condensates.
Formation of Hippo kinase condensates is promoted by apically localised upstream signalling components such as Kibra or Expanded.
Mechanical strain at the apical domain of epithelial cells inhibits Hippo kinase condensate formation.
Hippo kinase condensate formation is inhibited by growth factor signalling via the PI3K‐Akt pathway.
The organisation of Hippo kinase complexes into condensates and their regulation is conserved between Drosophila and mammalian epithelia.
Mechanical strain and growth factor signalling regulate the condensation of Hippo pathway components in Drosophila and mammalian epithelia.
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