ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0245454.].
ABSTRACT
Genome association studies in human and genetic studies in mouse implicated members of the transmembrane protein 132 (TMEM132) family in multiple conditions including panic disorder, hearing loss, limb and kidney malformation. However, the presence of five TMEM132 paralogs in mammalian genomes makes it extremely challenging to reveal the full requirement for these proteins in vivo. In contrast, there is only one TMEM132 homolog, detonator (dtn), in the genome of fruit fly Drosophila melanogaster, enabling straightforward research into its in vivo function. In the current study, we generate multiple loss-of-function dtn mutant fly strains through a polycistronic tRNA-gRNA approach, and show that most embryos lacking both maternal and paternal dtn fail to hatch into larvae, indicating an essential role of dtn in Drosophila reproduction.
Subject(s)
CRISPR-Cas Systems , Drosophila melanogaster/genetics , Gene Editing , RNA, Guide, Kinetoplastida/genetics , RNA, Transfer/genetics , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/physiology , Clustered Regularly Interspaced Short Palindromic Repeats , Drosophila Proteins/genetics , Drosophila melanogaster/physiology , Female , Fertility , Gene Editing/methods , Loss of Function Mutation , Male , ReproductionABSTRACT
While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, we identified qualitative and quantitative phenotypes in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, we found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. We further observed disruptions in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. Our findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions.
Subject(s)
DNA Copy Number Variations , Drosophila Proteins/genetics , Gene Expression Regulation, Developmental , Neurodevelopmental Disorders/genetics , Animals , Compound Eye, Arthropod/embryology , Compound Eye, Arthropod/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Organ Specificity , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction , Wings, Animal/embryology , Wings, Animal/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolismABSTRACT
In contrast to steady-state erythropoiesis, which generates new erythrocytes at a constant rate, stress erythropoiesis rapidly produces a large bolus of new erythrocytes in response to anemic stress. In this study, we illustrate that Yes-associated protein (Yap1) promotes the rapid expansion of a transit-amplifying population of stress erythroid progenitors in vivo and in vitro. Yap1-mutated erythroid progenitors failed to proliferate in the spleen after transplantation into lethally irradiated recipient mice. Additionally, loss of Yap1 impaired the growth of actively proliferating erythroid progenitors in vitro. This role in proliferation is supported by gene expression profiles showing that transiently amplifying stress erythroid progenitors express high levels of genes associated with Yap1 activity and genes induced by Yap1. Furthermore, Yap1 promotes the proliferation of stress erythroid progenitors in part by regulating the expression of key glutamine-metabolizing enzymes. Thus, Yap1 acts as an erythroid regulator that coordinates the metabolic status with the proliferation of erythroid progenitors to promote stress erythropoiesis.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Cell Cycle Proteins/physiology , Erythroid Precursor Cells/physiology , Erythropoiesis/physiology , Regeneration/physiology , Adaptor Proteins, Signal Transducing/genetics , Alleles , Animals , Cell Division , Cells, Cultured , Enzyme Induction , Erythroid Precursor Cells/cytology , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation , Mice , Mice, Inbred C57BL , RNA, Messenger/biosynthesis , Radiation Chimera , Radiation Tolerance , Recombinant Proteins/metabolism , Spleen/cytology , Stress, Physiological/genetics , Transcription Factors/genetics , YAP-Signaling ProteinsABSTRACT
Anemic stress induces the proliferation of stress erythroid progenitors in the murine spleen that subsequently differentiate to generate erythrocytes to maintain homeostasis. This process relies on the interaction between stress erythroid progenitors and the signals generated in the splenic erythroid niche. In this study, we demonstrate that although growth-differentiation factor 15 (Gdf15) is not required for steady-state erythropoiesis, it plays an essential role in stress erythropoiesis. Gdf15 acts at 2 levels. In the splenic niche, Gdf15-/- mice exhibit defects in the monocyte-derived expansion of the splenic niche, resulting in impaired proliferation of stress erythroid progenitors and production of stress burst forming unit-erythroid cells. Furthermore, Gdf15 signaling maintains the hypoxia-dependent expression of the niche signal, Bmp4, whereas in stress erythroid progenitors, Gdf15 signaling regulates the expression of metabolic enzymes, which contribute to the rapid proliferation of stress erythroid progenitors. Thus, Gdf15 functions as a comprehensive regulator that coordinates the stress erythroid microenvironment with the metabolic status of progenitors to promote stress erythropoiesis.
Subject(s)
Erythroid Precursor Cells/metabolism , Erythropoiesis/genetics , Growth Differentiation Factor 15/genetics , Stem Cell Niche , Stress, Physiological , Animals , Cell Differentiation , Cell Proliferation , Growth Differentiation Factor 15/metabolism , Mice , Mice, Knockout , Models, Biological , Signal TransductionABSTRACT
The considerable amount of experimental evidence has defined the Hippo pathway as a tumor suppressive pathway and increased expression and/or activity of its oncogenic effectors is frequently observed in cancer. However, clinical studies have failed to attribute cancer development and progression to mutations in the pathway. In explaining this conundrum, we investigated the expression and functions of a C-terminally truncated isoform of large tumor suppressor kinase 1 (LATS1) called short LATS1 (sLATS1) in human cell lines and Drosophila. Intriguingly, through overexpression of sLATS1, we demonstrated that sLATS1 either activates or suppresses the activity of Yes-associated protein (YAP), one of the effectors of the Hippo pathway, in a cell type-specific manner. The activation is mediated through inhibition of full-length LATS1, whereas suppression of YAP is accomplished through sLATS1-YAP interaction. In HEK293T cells, the former mechanism may affect the cellular response more dominantly, whereas in U2OS cells and developing tissues in Drosophila, the latter mechanism may be solely carried out. Finally, to find the clinical relevance of this molecule, we examined the expression of sLATS1 in breast cancer patients. The transcriptome analysis showed that the ratio of sLATS1 to LATS1 was increased in tumor tissues comparing to their adjacent normal tissues.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Blotting, Western , Carcinogenesis/metabolism , Cell Culture Techniques , Cell Fractionation , Cell Proliferation/genetics , Drosophila , Drosophila Proteins/metabolism , Female , Flow Cytometry , Fluorescent Antibody Technique , HEK293 Cells , Hippo Signaling Pathway , Humans , Immunoprecipitation , Nuclear Proteins/metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction , Trans-Activators/metabolism , Transcription Factors , YAP-Signaling ProteinsABSTRACT
Protein-protein interactions provide a common mechanism for regulating protein functions and also serve as the fundamental step of many biochemical reactions. To accurately determine the involvement and function of protein-protein interactions, it is crucial to detect the interactions with the minimum number of artifacts. In this chapter, we report the method of bimolecular fluorescence complementation (BiFC) in tissue culture and developing tissues of Drosophila, which allows the visualization of subcellular localization of protein-protein interactions in living cells.
Subject(s)
Drosophila/metabolism , Fluorescent Antibody Technique , Molecular Imaging , Protein Interaction Mapping , Animals , Cell Line , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression , Humans , Imaginal Discs , Microscopy, Fluorescence/methods , Molecular Imaging/methods , Protein Interaction Mapping/methods , Tissue Culture Techniques , Wings, AnimalABSTRACT
How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. In this study, we found that functional loss of a gene in Drosophila, named Drosophila defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Our study demonstrated that loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model.
Subject(s)
Drosophila Proteins/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/genetics , Genes, Insect , Morphogenesis/genetics , Animals , Apoptosis/genetics , Biocatalysis , Cell Proliferation , Clone Cells , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Gene Knockdown Techniques , Glycosylation , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Models, Biological , Mutation/genetics , Protein Subunits/metabolism , Subcellular Fractions/metabolismABSTRACT
Mammalian pancreatic ß-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in ß-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of ß-cells, the viability of ß-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect ß-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when ß-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in ß-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/physiology , Fatty Acids, Nonesterified/pharmacology , Phosphoproteins/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Connective Tissue Growth Factor/pharmacology , Cytochalasin D/pharmacology , HEK293 Cells , Humans , Immunohistochemistry , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Mice , Microscopy, Fluorescence , Palmitic Acid/pharmacology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , RNA Interference , RNA, Small Interfering/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Thiazolidines/pharmacology , Transcription Factors , YAP-Signaling ProteinsABSTRACT
In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.
Subject(s)
Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/genetics , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Cycle Proteins , Computational Biology , Genes, Neoplasm , Humans , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins/chemistry , LIM Domain Proteins/metabolism , Mice , Mutation , Neoplasms/genetics , Neoplasms/pathology , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Serine-Threonine Kinase 3 , Transferases (Other Substituted Phosphate Groups)/chemistry , Transferases (Other Substituted Phosphate Groups)/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , YAP-Signaling ProteinsABSTRACT
The loss of or decreased functional pancreatic ß-cell is a major cause of type 1 and type 2 diabetes. Previous studies have shown that adult ß-cells can maintain their ability for a low level of turnover through replication and neogenesis. Thus, a strategy to prevent and treat diabetes would be to enhance the ability of ß-cells to increase the mass of functional ß-cells. Consequently, much effort has been devoted to identify factors that can effectively induce ß-cell expansion. This review focuses on recent reports on small molecules and protein factors that have been shown to promote ß-cell expansion.
Subject(s)
Cell Proliferation , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 2/genetics , Insulin-Secreting Cells/chemistry , Cell Communication/genetics , Cell Differentiation/genetics , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 2/pathology , Humans , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathologyABSTRACT
Abnormal activation of Wnt/ß-catenin-mediated transcription is associated with a variety of human cancers. Here, we report that LATS2 inhibits oncogenic Wnt/ß-catenin-mediated transcription by disrupting the ß-catenin/BCL9 interaction. LATS2 directly interacts with ß-catenin and is present on Wnt target gene promoters. Mechanistically, LATS2 inhibits the interaction between BCL9 and ß-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 is downregulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an antimicrotubule drug, potently induces LATS2 to suppress tumor growth in vivo by targeting ß-catenin/BCL9. Our results suggest that LATS2 is not only a key tumor suppressor in human cancer but may also be an important target for anticancer therapy.
Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , HEK293 Cells , Humans , Protein Binding , Transcription Factors , Transcription, GeneticABSTRACT
Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.
Subject(s)
Actin Cytoskeleton/physiology , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Proliferation , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation , Hippo Signaling Pathway , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Coactivator with PDZ-Binding Motif Proteins , YAP-Signaling ProteinsABSTRACT
The role of Large tumor suppressor LATS/Warts in human cancer is not clearly understood. Here we show that hLATS1/2 cancer mutations affect their expression and kinase activity. hLATS1/2 mutants exhibit a decreased activity in inhibiting YAP and tissue growth. Therefore, hLATS1/2 alleles from human cancer can be loss-of-function mutations.
Subject(s)
Genes, Tumor Suppressor , Neoplasms/genetics , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/genetics , Animals , Animals, Genetically Modified , Drosophila/genetics , Drosophila/growth & development , Female , Humans , Male , Models, Genetic , Mutation , RNA Interference , Wings, Animal/growth & developmentABSTRACT
The evolutionarily conserved Hippo signaling pathway plays an important role in regulating normal development as well as tumorigenesis in animals. How this growth-inhibitory signaling is maintained at an appropriate level through feedback mechanisms is less understood. In this report, we show that bantam microRNA functions to increase the level of the Mob as tumor suppressor protein Mats, a core component of the Hippo pathway, but does not regulate mats at the transcript level. Genetic analysis also supports that bantam plays a positive role in regulating mats function for tissue growth control. Our data support a model that bantam up-regulates Mats expression through an unidentified factor that may control Mats stability.
Subject(s)
Drosophila Proteins/genetics , Drosophila/metabolism , MicroRNAs/genetics , Tumor Suppressor Proteins/genetics , Animals , Drosophila/growth & development , Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental , MicroRNAs/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Up-RegulationABSTRACT
The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo-YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions.
Subject(s)
Cell Differentiation , Cyclic AMP-Dependent Protein Kinases/metabolism , Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Acyltransferases , Adipogenesis , Animals , Cell Line , Cell Proliferation , Cyclic AMP/metabolism , Drosophila Proteins/antagonists & inhibitors , Drosophila melanogaster/enzymology , Drosophila melanogaster/metabolism , Enzyme Activation , Humans , Mice , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Phosphorylation , Second Messenger Systems/physiology , Serine-Threonine Kinase 3 , Trans-Activators/antagonists & inhibitors , Trans-Activators/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , YAP-Signaling Proteins , rho GTP-Binding Proteins/metabolismABSTRACT
BRMS1 was first discovered as a human breast carcinoma metastasis suppressor gene. However, the mechanism of BRMS1 in tumor metastasis and its developmental role remain unclear. In this paper, we first report the identification of the Drosophila ortholog of human BRMS1, dBrms1. Through a genetic approach, the role of dBrms1 during development has been investigated. We found that dBrms1 is an essential gene and loss of dBrms1 function results in lethality at early developmental stages. dBrms1mutants displayed phenotypes such as developmental delay and failure to initiate metamorphosis. Further analysis suggests that these phenotypes are contributed by defective ecdysone signaling and expression of target genes of the ecdysone pathway. Therefore, dBrms1 is required for growth control by acting as a modulator of ecdysone signaling in Drosophila and is required for metamorphosis for normal development.
Subject(s)
Ecdysone/physiology , Gene Expression Regulation, Developmental , Genes, Tumor Suppressor , Neoplasm Proteins/genetics , Animals , Drosophila , Metamorphosis, Biological , Mutation , Repressor Proteins , Signal Transduction , Time Factors , TransgenesABSTRACT
Hippo (Hpo) signaling plays a critical role in restricting tissue growth and organ size in both invertebrate and vertebrate animals. However, how the Hpo kinase is regulated during development has not been clearly understood. Using a Bimolecular Fluorescence Complementation assay, we have investigated the functional significance of Hpo homo-dimer formation and subcellular localization in living cells. We found that Hpo dimerization and membrane association are critical for its activation in growth inhibition. As dimerization facilitates Hpo to access its binding partner, Hpo kinases in the homo-dimer trans-phosphorylate each other to increase their enzymatic activity. Moreover, loss- and gain-of-function studies indicate that upstream regulators, Expanded, Merlin and Kibra, play a critical role in promoting Hpo dimerization as well as association to the cortical F-actin beneath the plasma membrane. Enforced Hpo localization to the plasma membrane increases Hpo dimerization and activity. Therefore, homo-dimerization and plasma membrane association are two important mechanisms for Hpo activation in growth control during animal development.