ABSTRACT
Nuclear markers for live imaging are useful for counting and tracking cells, visualizing cell division, and examining the regulation of proteins that are controlled via entry or exit from the nucleus. Near-infrared fluorescent proteins have advantages over shorter wavelength fluorescent proteins, including reduced phototoxicity, less light scattering, and enabling multicolor live imaging. We have constructed and tested transgenic Drosophila expressing Histone H2Av iRFP fusion proteins, and confirmed that they can be used to label nuclei in both fixed and live tissue at multiple stages of development.
ABSTRACT
The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.
Subject(s)
Drosophila Proteins/physiology , Drosophila/metabolism , LIM Domain Proteins/physiology , Animals , Cytoskeleton/chemistry , Cytoskeleton/physiology , Drosophila/growth & development , Drosophila Proteins/analysis , Drosophila Proteins/genetics , LIM Domain Proteins/analysis , LIM Domain Proteins/genetics , LIM-Homeodomain Proteins/analysis , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/physiology , Signal Transduction , Transcription Factors/metabolism , Wings, Animal/growth & development , alpha Catenin/metabolismABSTRACT
Adherens junctions provide attachments between neighboring epithelial cells and a physical link to the cytoskeleton, which enables them to sense and transmit forces and to initiate biomechanical signaling. Examination of the Ajuba LIM protein Jub in Drosophila embryos revealed that it is recruited to adherens junctions in tissues experiencing high levels of myosin activity, and that the pattern of Jub recruitment varies depending upon how tension is organized. In cells with high junctional myosin, Jub is recruited to puncta near intercellular vertices, which are distinct from Ena-containing puncta, but can overlap Vinc-containing puncta. We identify roles for Jub in modulating tension and cellular organization, which are shared with the cytohesin Step, and the cytohesin adapter Sstn, and show that Jub and Sstn together recruit Step to adherens junctions under tension. Our observations establish Jub as a reporter of tension experienced at adherens junctions, and identify distinct types of tension-dependent and tension-independent junctional complexes. They also identify a role for Jub in mediating a feedback loop that modulates the distribution of tension and cellular organization in epithelia.
Subject(s)
Adherens Junctions/metabolism , Cytoskeleton/metabolism , Drosophila Proteins/metabolism , LIM Domain Proteins/metabolism , Animals , Cadherins/metabolism , Cell Adhesion/genetics , Drosophila , Drosophila Proteins/genetics , Epithelium/embryology , Female , LIM Domain Proteins/genetics , Male , Mechanotransduction, Cellular , Myosins/metabolism , Protein DomainsABSTRACT
The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for the biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions. Here, we investigate the mechanism by which the Drosophila Ajuba family protein Jub is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. We identify α-catenin as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. We also show that increased Jub recruitment to α-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. Our observations establish α-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to α-catenin as a key contributor to biomechanical regulation of Hippo signaling.
Subject(s)
Actin Cytoskeleton/metabolism , Adherens Junctions/metabolism , Drosophila Proteins/metabolism , Drosophila/physiology , LIM Domain Proteins/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Wings, Animal/physiology , alpha Catenin/metabolism , Actins/metabolism , Animals , Binding Sites/genetics , Biomechanical Phenomena , Cell Adhesion , Drosophila Proteins/genetics , Gene Expression Regulation, Developmental/genetics , Intracellular Signaling Peptides and Proteins/metabolism , LIM Domain Proteins/genetics , Mechanotransduction, Cellular , Nuclear Proteins/genetics , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Trans-Activators/genetics , YAP-Signaling ProteinsABSTRACT
Visualization of in vivo protein levels and localization is essential to analysis and elucidation of Hippo signaling mechanisms and its roles in diverse tissues. This is best done by imaging proteins using fluorescent labels. Fluorescent labeling of a protein can be achieved by direct conjugation to an intrinsically fluorescent protein, like GFP, or by use of antibodies conjugated to fluorescent dyes. Immunofluorescence imaging in Drosophila typically begins with dissection and fixation of a sample tissue, followed by a series of washes and incubations with primary antibodies, directed against proteins of interest, and dye-labeled secondary antibodies, directed against the primary antibodies. This may be followed by fluorescent dyes that label cellular components, such as DNA-labeling dyes to mark nuclei. After staining and washing is completed, samples are placed in a mounting media, transferred to a microscope slide, and imaged on a confocal microscope.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Fluorescent Antibody Technique , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Animals , Microscopy, Fluorescence , Staining and LabelingABSTRACT
Tissue growth needs to be properly controlled for organs to reach their correct size and shape, but the mechanisms that control growth during normal development are not fully understood. We report here that the activity of the Hippo signaling transcriptional activator Yorkie gradually decreases in the central region of the developing Drosophila wing disc. Spatial and temporal changes in Yorkie activity can be explained by changes in cytoskeletal tension and biomechanical regulators of Hippo signaling. These changes in cellular biomechanics correlate with changes in cell density, and experimental manipulations of cell density are sufficient to alter biomechanical Hippo signaling and Yorkie activity. We also relate the pattern of Yorkie activity in older discs to patterns of cell proliferation. Our results establish that spatial and temporal patterns of Hippo signaling occur during wing development, that these patterns depend upon cell-density modulated tissue mechanics and that they contribute to the regulation of wing cell proliferation.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Wings, Animal/embryology , Wings, Animal/metabolism , Animals , Basement Membrane/cytology , Basement Membrane/metabolism , Biomechanical Phenomena , Cell Count , Cell Proliferation , Cytoskeleton/metabolism , Drosophila melanogaster/cytology , Imaginal Discs/cytology , Imaginal Discs/embryology , Imaginal Discs/metabolism , Time Factors , Wings, Animal/cytologyABSTRACT
Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. We report that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein Jub, a negative regulator of Warts within the Hippo pathway. We further show that Jub associates with α-catenin and that its localization to adherens junctions and association with α-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. Our observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth.
Subject(s)
Cytoskeleton/metabolism , Drosophila Proteins/metabolism , Drosophila/growth & development , LIM Domain Proteins/metabolism , Signal Transduction , Wings, Animal/growth & development , Animals , Biomechanical Phenomena , Drosophila/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/metabolism , Wings, Animal/metabolism , YAP-Signaling ProteinsABSTRACT
The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1ε encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.
Subject(s)
Cadherins/metabolism , Drosophila Proteins/metabolism , Drosophila/embryology , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Tumor Suppressor Proteins/metabolism , Amino Acid Motifs/genetics , Amino Acid Motifs/physiology , Animals , Animals, Genetically Modified , Blotting, Western , Casein Kinase 1 epsilon/genetics , Drosophila/genetics , Drosophila Proteins/genetics , Histological Techniques , Humans , Immunoprecipitation , Mutation/genetics , Myosins/metabolism , Phosphorylation , Plasmids/geneticsABSTRACT
The Hippo signaling pathway has a conserved role in growth control and is of fundamental importance during both normal development and oncogenesis. Despite rapid progress in recent years, key steps in the pathway remain poorly understood, in part due to the incomplete identification of components. Through a genetic screen, we identified the Drosophila Zyxin family gene, Zyx102 (Zyx), as a component of the Hippo pathway. Zyx positively regulates the Hippo pathway transcriptional co-activator Yorkie, as its loss reduces Yorkie activity and organ growth. Through epistasis tests, we position the requirement for Zyx within the Fat branch of Hippo signaling, downstream of Fat and Dco, and upstream of the Yorkie kinase Warts, and we find that Zyx is required for the influence of Fat on Warts protein levels. Zyx localizes to the sub-apical membrane, with distinctive peaks of accumulation at intercellular vertices. This partially overlaps the membrane localization of the myosin Dachs, which has similar effects on Fat-Hippo signaling. Co-immunoprecipitation experiments show that Zyx can bind to Dachs and that Dachs stimulates binding of Zyx to Warts. We also extend characterization of the Ajuba LIM protein Jub and determine that although Jub and Zyx share C-terminal LIM domains, they regulate Hippo signaling in distinct ways. Our results identify a role for Zyx in the Hippo pathway and suggest a mechanism for the role of Dachs: because Fat regulates the localization of Dachs to the membrane, where it can overlap with Zyx, we propose that the regulated localization of Dachs influences downstream signaling by modulating Zyx-Warts binding. Mammalian Zyxin proteins have been implicated in linking effects of mechanical strain to cell behavior. Our identification of Zyx as a regulator of Hippo signaling thus also raises the possibility that mechanical strain could be linked to the regulation of gene expression and growth through Hippo signaling.
Subject(s)
Cell Adhesion Molecules/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Zyxin/metabolism , Animals , Cell Membrane/metabolism , Drosophila melanogaster/cytology , Epistasis, Genetic , Models, Biological , Protein Binding , Protein Transport , Wings, Animal/cytology , Wings, Animal/growth & development , Wings, Animal/metabolismABSTRACT
The Drosophila optic lobe develops from neuroepithelial cells, which function as symmetrically dividing neural progenitors. We describe here a role for the Fat-Hippo pathway in controlling the growth and differentiation of Drosophila optic neuroepithelia. Mutation of tumor suppressor genes within the pathway, or expression of activated Yorkie, promotes overgrowth of neuroepithelial cells and delays or blocks their differentiation; mutation of yorkie inhibits growth and accelerates differentiation. Neuroblasts and other neural cells, by contrast, appear unaffected by Yorkie activation. Neuroepithelial cells undergo a cell cycle arrest before converting to neuroblasts; this cell cycle arrest is regulated by Fat-Hippo signaling. Combinations of cell cycle regulators, including E2f1 and CyclinD, delay neuroepithelial differentiation, and Fat-Hippo signaling delays differentiation in part through E2f1. We also characterize roles for Jak-Stat and Notch signaling. Our studies establish that the progression of neuroepithelial cells to neuroblasts is regulated by Notch signaling, and suggest a model in which Fat-Hippo and Jak-Stat signaling influence differentiation by their acceleration of cell cycle progression and consequent impairment of Delta accumulation, thereby modulating Notch signaling. This characterization of Fat-Hippo signaling in neuroepithelial growth and differentiation also provides insights into the potential roles of Yes-associated protein in vertebrate neural development and medullablastoma.
Subject(s)
Cell Differentiation/genetics , Drosophila , Signal Transduction/physiology , Animals , Cell Cycle/genetics , Drosophila/genetics , Drosophila/metabolism , Drosophila/physiology , Eye/metabolism , Fats/metabolism , Neuroepithelial Cells/metabolism , Neurons/cytology , Neurons/metabolism , Optic Lobe, Nonmammalian/metabolism , Signal Transduction/geneticsABSTRACT
Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. We show that the Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.
Subject(s)
Cell Adhesion Molecules/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Signal Transduction , Wings, Animal/growth & development , Animals , Bromodeoxyuridine , Cell Adhesion Molecules/genetics , Cell Polarity , Cell Proliferation , Clone Cells , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Models, Biological , Protein Transport , Wings, Animal/cytologyABSTRACT
Recent studies in Drosophila melanogaster of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. Here, we characterize functional relationships among D. melanogaster tumor suppressors and identify the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded, hippo, salvador and mob as tumor suppressor also share multiple Fat pathway phenotypes but regulate Warts activity independently. Our results functionally link what had been four disparate groups of D. melanogaster tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals.
Subject(s)
Cell Adhesion Molecules/metabolism , Drosophila Proteins/metabolism , Genes, Tumor Suppressor , Signal Transduction , Animals , Cell Adhesion Molecules/genetics , Cell Cycle Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Myosins/genetics , Myosins/metabolism , Neurofibromin 2/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , YAP-Signaling ProteinsABSTRACT
The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.
Subject(s)
Cell Adhesion Molecules/genetics , Drosophila Proteins/genetics , Drosophila/growth & development , Drosophila/genetics , Gene Expression Regulation, Developmental , Myosins/genetics , Wings, Animal/growth & development , Amino Acid Sequence , Animals , Cell Polarity , Cloning, Molecular , Molecular Sequence Data , PhenotypeABSTRACT
Notch signaling controls formation of joints at leg segment borders and growth of the developing Drosophila leg. Here, we identify the odd-skipped gene family as a key group of genes that function downstream of the Notch receptor to promote morphological changes associated with joint formation during leg development. odd, sob, drm, and bowl are expressed in a segmental pattern in the developing leg, and their expression is regulated by Notch signaling. Ectopic expression of odd, sob, or drm can induce invaginations in the leg disc epithelium and morphological changes in the adult leg that are characteristic of endogenous invaginating joint cells. These effects are not due to an alteration in the expression of other genes of the developing joint. While odd or drm mutant clones do not affect leg segmentation, and thus appear to act redundantly, bowl mutant clones do perturb leg development. Specifically, bowl mutant clones result in a failure of joint formation from the distal tibia to tarsal segment 5, while more proximal clones cause melanotic protrusions from the leg cuticle. Together, these results indicate that the odd-skipped family of genes mediates Notch function during leg development by promoting a specific aspect of joint formation, an epithelial invagination. As the odd-skipped family genes are involved in regulating cellular morphogenesis during both embryonic segmentation and hindgut development, we suggest that they may be required in multiple developmental contexts to induce epithelial cellular changes.