Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway.

The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.

Phosphorylation-linked complex profiling identifies assemblies required for Hippo signal integration.

While several computational methods have been developed to predict the functional relevance of phosphorylation sites, experimental analysis of the interdependency between protein phosphorylation and Protein-Protein Interactions (PPIs) remains challenging. Here, we describe an experimental strategy to establish interdependencies between protein phosphorylation and complex formation. This strategy is based on three main steps: (i) systematically charting the phosphorylation landscape of a target protein; (ii) assigning distinct proteoforms of the target protein to different protein complexes by native complex separation (AP-BNPAGE) and protein correlation profiling; and (iii) analyzing proteoforms and complexes in cells lacking regulators of the target protein. We applied this strategy to YAP1, a transcriptional co-activator for the control of organ size and tissue homeostasis that is highly phosphorylated and among the most connected proteins in human cells. We identified multiple YAP1 phosphosites associated with distinct complexes and inferred how both are controlled by Hippo pathway members. We detected a PTPN14/LATS1/YAP1 complex and suggest a model how PTPN14 inhibits YAP1 via augmenting WW domain-dependent complex formation and phosphorylation by LATS1/2.

Co-option of epidermal cells enables touch sensing.

The epidermis is equipped with specialized mechanosensory organs that enable the detection of tactile stimuli. Here, by examining the differentiation of the tactile bristles, mechanosensory organs decorating the Drosophila adult epidermis, we show that neighbouring epidermal cells are essential for touch perception. Each mechanosensory bristle signals to the surrounding epidermis to co-opt a single epidermal cell, which we named the F-Cell. Once specified, the F-Cell adopts a specialized morphology to ensheath each bristle. Functional assays reveal that adult mechanosensory bristles require association with the epidermal F-Cell for touch sensing. Our findings underscore the importance of resident epidermal cells in the assembly of functional touch-sensitive organs.

ECM degradation in the Drosophila abdominal epidermis initiates tissue growth that ceases with rapid cell-cycle exit.

During development, multicellular organisms undergo stereotypical patterns of tissue growth in space and time. How developmental growth is orchestrated remains unclear, largely due to the difficulty of observing and quantitating this process in a living organism. Drosophila histoblast nests are small clusters of progenitor epithelial cells that undergo extensive growth to give rise to the adult abdominal epidermis and are amenable to live imaging. Our quantitative analysis of histoblast proliferation and tissue mechanics reveals that tissue growth is driven by cell divisions initiated through basal extracellular matrix degradation by matrix metalloproteases secreted by the neighboring larval epidermal cells. Laser ablations and computational simulations show that tissue mechanical tension does not decrease as the histoblasts fill the abdominal epidermal surface. During tissue growth, the histoblasts display oscillatory cell division rates until growth termination occurs through the rapid emergence of G0/G1 arrested cells, rather than a gradual increase in cell-cycle time as observed in other systems such as the Drosophila wing and mouse postnatal epidermis. Different developing tissues can therefore achieve their final size using distinct growth termination strategies. Thus, adult abdominal epidermal development is characterized by changes in the tissue microenvironment and a rapid exit from the cell cycle.

Photoablation at single cell resolution and its application in the Drosophila epidermis and peripheral nervous system.

Tissues contain diverse cell populations that, together, make up physiologically functional units. A remarkable example is the animal epidermis, where neuronal and non-neuronal cells intermingle to allow somatosensory perception. In the peripheral nervous system (PNS), the tight association between heterogenous cell types poses challenges when the structural and physiological contributions of neuronal and surrounding cells need to be dissected with suitable precision. When genetic tools for cell-specific, spatiotemporally controlled gene expression are not available, targeted cell ablation represents a considerable obstacle. Here, we describe an efficient method to overcome this limitation and demonstrate its application to the study of the differentiating Drosophila epidermis and PNS. This methodology relies on the use of near infrared (NIR) femtosecond (fs) laser pulses for ablation of the desired cells at the desired time. We show how to confine the photodamage to the targeted cell to induce its death, without harming neighbouring tissues or structures. We validated our approach in the Drosophila PNS by studying the responses of photo-ablated neurons, non-neuronal cells, and the surrounding epidermis. Diverse cellular behaviours including cell extrusion, cell rearrangements and cell shape changes can be monitored in vivo immediately after damage, as well as for several hours post-ablation with high optical resolution using confocal microscopy. This methodology provides a flexible tool to ablate individual cells with high precision and study morphological responses to cell loss in targeted areas or neighbouring structures. We anticipate that this protocol can be easily adapted to other model systems and tissues.

RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering.

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

Casein kinase 1 family proteins promote Slimb-dependent Expanded degradation.

Hippo signalling integrates diverse stimuli related to epithelial architecture to regulate tissue growth and cell fate decisions. The Hippo kinase cascade represses the growth-promoting transcription co-activator Yorkie. The FERM protein Expanded is one of the main upstream Hippo signalling regulators in Drosophila as it promotes Hippo kinase signalling and directly inhibits Yorkie. To fulfil its function, Expanded is recruited to the plasma membrane by the polarity protein Crumbs. However, Crumbs-mediated recruitment also promotes Expanded turnover via a phosphodegron-mediated interaction with a Slimb/ß-TrCP SCF E3 ligase complex. Here, we show that the Casein Kinase 1 (CKI) family is required for Expanded phosphorylation. CKI expression promotes Expanded phosphorylation and interaction with Slimb/ß-TrCP. Conversely, CKI depletion in S2 cells impairs Expanded degradation downstream of Crumbs. In wing imaginal discs, CKI loss leads to elevated Expanded and Crumbs levels. Thus, phospho-dependent Expanded turnover ensures a tight coupling of Hippo pathway activity to epithelial architecture.

Hippo signalling during development.

The Hippo signalling pathway and its transcriptional co-activator targets Yorkie/YAP/TAZ first came to attention because of their role in tissue growth control. Over the past 15 years, it has become clear that, like other developmental pathways (e.g. the Wnt, Hedgehog and TGFß pathways), Hippo signalling is a 'jack of all trades' that is reiteratively used to mediate a range of cellular decision-making processes from proliferation, death and morphogenesis to cell fate determination. Here, and in the accompanying poster, we briefly outline the core pathway and its regulation, and describe the breadth of its roles in animal development.

ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail.

Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.

Upstairs, downstairs: spatial regulation of Hippo signalling.

Cellular signalling lies at the heart of every decision involved in the development and homeostasis of multicellular organisms. The Hippo pathway was discovered nearly two decades ago through seminal work in Drosophila and rapidly emerged as a crucial signalling network implicated in developmental and oncogenic growth, tissue regeneration and stem cell biology. Here, we review recent advances in the field relating to the upstream regulation of Hippo signalling and the intracellular tug-of-war that tightly controls its main target, the transcriptional co-activator Yorkie/YAP.

Stable MOB1 interaction with Hippo/MST is not essential for development and tissue growth control.

The Hippo tumor suppressor pathway is essential for development and tissue growth control, encompassing a core cassette consisting of the Hippo (MST1/2), Warts (LATS1/2), and Tricornered (NDR1/2) kinases together with MOB1 as an important signaling adaptor. However, it remains unclear which regulatory interactions between MOB1 and the different Hippo core kinases coordinate development, tissue growth, and tumor suppression. Here, we report the crystal structure of the MOB1/NDR2 complex and define key MOB1 residues mediating MOB1's differential binding to Hippo core kinases, thereby establishing MOB1 variants with selective loss-of-interaction. By studying these variants in human cancer cells and Drosophila, we uncovered that MOB1/Warts binding is essential for tumor suppression, tissue growth control, and development, while stable MOB1/Hippo binding is dispensable and MOB1/Trc binding alone is insufficient. Collectively, we decrypt molecularly, cell biologically, and genetically the importance of the diverse interactions of Hippo core kinases with the pivotal MOB1 signal transducer.The Hippo tumor suppressor pathway is essential for development and tissue growth control. Here the authors employ a multi-disciplinary approach to characterize the interactions of the three Hippo kinases with the signaling adaptor MOB1 and show how they differently affect development, tissue growth and tumor suppression.

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division.

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. We show that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery.

EpiTools: An Open-Source Image Analysis Toolkit for Quantifying Epithelial Growth Dynamics.

Epithelia grow and undergo extensive rearrangements to achieve their final size and shape. Imaging the dynamics of tissue growth and morphogenesis is now possible with advances in time-lapse microscopy, but a true understanding of their complexities is limited by automated image analysis tools to extract quantitative data. To overcome such limitations, we have designed a new open-source image analysis toolkit called EpiTools. It provides user-friendly graphical user interfaces for accurately segmenting and tracking the contours of cell membrane signals obtained from 4D confocal imaging. It is designed for a broad audience, especially biologists with no computer-science background. Quantitative data extraction is integrated into a larger bioimaging platform, Icy, to increase the visibility and usability of our tools. We demonstrate the usefulness of EpiTools by analyzing Drosophila wing imaginal disc growth, revealing previously overlooked properties of this dynamic tissue, such as the patterns of cellular rearrangements.

The hippo pathway core cassette regulates asymmetric cell division.

Asymmetric cell division (ACD) is a crucial process during development, homeostasis, and cancer. Stem and progenitor cells divide asymmetrically, giving rise to two daughter cells, one of which retains the parent cell self-renewal capacity, while the other is committed to differentiation. Any imbalance in this process can induce overgrowth or even a cancer-like state. Here, we show that core components of the Hippo signaling pathway, an evolutionarily conserved organ growth regulator, modulate ACD in Drosophila. Hippo pathway inactivation disrupts the asymmetric localization of ACD regulators, leading to aberrant mitotic spindle orientation and defects in the generation of unequal-sized daughter cells. The Hippo pathway downstream kinase Warts, LATS1-2 in mammals, associates with the ACD modulators Inscuteable and Bazooka in vivo and phosphorylates Canoe, the ortholog of Afadin/AF-6, in vitro. Moreover, phosphosite mutant Canoe protein fails to form apical crescents in dividing neuroblasts in vivo, and the lack of Canoe phosphorylation by Warts leads to failures of Discs Large apical localization in metaphase neuroblasts. Given the relevance of ACD in stem cells during tissue homeostasis, and the well-documented role of the Hippo pathway as a tumor suppressor, these results represent a potential route for perturbations in the Hippo signaling to induce tumorigenesis via aberrant stem cell divisions.

Differential control of Yorkie activity by LKB1/AMPK and the Hippo/Warts cascade in the central nervous system.

The Hippo (Hpo) pathway is a highly conserved tumor suppressor network that restricts developmental tissue growth and regulates stem cell proliferation and differentiation. At the heart of the Hpo pathway is the progrowth transcriptional coactivator Yorkie [Yki-Yes-activated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) in mammals]. Yki activity is restricted through phosphorylation by the Hpo/Warts core kinase cascade, but increasing evidence indicates that core kinase-independent modes of regulation also play an important role. Here, we examine Yki regulation in the Drosophila larval central nervous system and uncover a Hpo/Warts-independent function for the tumor suppressor kinase liver kinase B1 (LKB1) and its downstream effector, the energy sensor AMP-activated protein kinase (AMPK), in repressing Yki activity in the central brain/ventral nerve cord. Although the Hpo/Warts core cascade restrains Yki in the optic lobe, it is dispensable for Yki target gene repression in the late larval central brain/ventral nerve cord. Thus, we demonstrate a dramatically different wiring of Hpo signaling in neighboring cell populations of distinct developmental origins in the central nervous system.

Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4.

Signalling through the Hippo (Hpo) pathway involves a kinase cascade, which leads to the phosphorylation and inactivation of the pro-growth transcriptional co-activator Yorkie (Yki). Despite the identification of a large number of pathway members and modulators, our understanding of the molecular events that lead to activation of Hpo and the downstream kinase Warts (Wts) remain incomplete. Recently, targeted degradation of several Hpo pathway components has been demonstrated as a means of regulating pathway activity. In particular, the stability of scaffold protein Salvador (Sav), which is believed to promote Hpo/Wts association, is crucially dependent on its binding partner Hpo. In a cell-based RNAi screen for ubiquitin regulators involved in Sav stability, we identify the HECT domain protein Herc4 (HECT and RLD domain containing E3 ligase) as a Sav E3 ligase. Herc4 expression promotes Sav ubiquitylation and degradation, while Herc4 depletion stabilises Sav. Interestingly, Hpo reduces Sav/Herc4 interaction in a kinase-dependent manner. This suggests the existence of a positive feedback loop, where Hpo stabilises its own positive regulator by antagonising Herc4-mediated degradation of Sav.

Zyxin antagonizes the FERM protein expanded to couple F-actin and Yorkie-dependent organ growth.

BACKGROUND: Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. RESULTS: We generated a zyx mutant in Drosophila using TALEN endonucleases and used this to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, we show that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). CONCLUSIONS: Our results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity.

Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye.

Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. We show here that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. We uncover a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and show that apical Magi recruits the RASSF8-ASPP complex during AJ remodeling in IOCs. We further show that this Magi complex is required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and ß-catenin. We propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad complexes at junction sites.