ABSTRACT
Lipid droplets (LDs), crucial regulators of lipid metabolism, accumulate during oocyte development. However, their roles in fertility remain largely unknown. During Drosophila oogenesis, LD accumulation coincides with the actin remodeling necessary for follicle development. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when the prostaglandin (PG) synthase Pxt is missing. Dominant genetic interactions and PG treatment of follicles indicate that ATGL acts upstream of Pxt to regulate actin remodeling. Our data suggest that ATGL releases arachidonic acid (AA) from LDs to serve as the substrate for PG synthesis. Lipidomic analysis detects AA-containing triglycerides in ovaries, and these are increased when ATGL is lost. High levels of exogenous AA block follicle development; this is enhanced by impairing LD formation and suppressed by reducing ATGL. Together, these data support the model that AA stored in LD triglycerides is released by ATGL to drive the production of PGs, which promote the actin remodeling necessary for follicle development. We speculate that this pathway is conserved across organisms to regulate oocyte development and promote fertility.
Subject(s)
Drosophila Proteins , Prostaglandins , Animals , Lipid Droplets , Actins , Adipogenesis , Drosophila , Lipase , Peroxidases , Drosophila Proteins/geneticsABSTRACT
Germ cells undergo distinct nuclear processes as they differentiate into gametes. Although these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Our efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly conserved Importin ß/karyopherin family member Importin-9 (Ipo9, herein referring to Ranbp9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization revealed that Ipo9KO mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9KO mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins, and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis. This article has an associated First Person interview with the first author of the paper.
Subject(s)
Chromosome Segregation , Drosophila , Animals , Chromosome Segregation/genetics , Drosophila/genetics , Female , Germ Cells , In Situ Hybridization, Fluorescence , Karyopherins , MaleABSTRACT
BACKGROUND: The actin bundling protein Fascin is essential for developmental cell migrations and promotes cancer metastasis. In addition to bundling actin, Fascin has several actin-independent roles; how these other functions contribute to cell migration remains unclear. Border cell migration during Drosophila oogenesis provides an excellent model to study Fascin's various roles during invasive, collective cell migration. RESULTS: On-time border cell migration during Stage 9 requires Fascin (Drosophila Singed). Fascin functions not only within the migrating border cells, but also within the nurse cells, the substrate for this migration. Fascin genetically interacts with the actin elongation factor Enabled to promote on-time Stage 9 migration and overexpression of Enabled suppresses the defects seen with loss of Fascin. Loss of Fascin results in increased, shorter and mislocalized protrusions during migration. Additionally, loss of Fascin inhibits border cell delamination and increases E-Cadherin (Drosophila Shotgun) adhesions on both the border cells and nurse cells. CONCLUSIONS: Overall, Fascin promotes on-time border cell migration during Stage 9 and contributes to multiple aspects of this invasive, collective cell migration, including both protrusion dynamics and delamination. These findings have implications beyond Drosophila, as border cell migration has emerged as a model to study mechanisms mediating cancer metastasis.
Subject(s)
Actins/metabolism , Cadherins/metabolism , Carrier Proteins/metabolism , Cell Movement , Drosophila melanogaster/genetics , Microfilament Proteins/metabolism , Oocytes/cytology , Animals , Animals, Genetically Modified , Cell Adhesion , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Female , Green Fluorescent Proteins/metabolism , Male , Microscopy, Confocal , Oogenesis , RNA Interference , Signal TransductionABSTRACT
Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Drosophila/embryology , Oogenesis/physiology , Ovarian Follicle/growth & development , Animals , Drosophila Proteins/biosynthesis , Drosophila Proteins/genetics , Female , Microscopy, Confocal , Staining and Labeling , Transcription Factors/biosynthesis , Transcription Factors/genetics , Utrophin/biosynthesis , Utrophin/geneticsABSTRACT
In this chapter, we highlight examples of the diverse array of developmental, cellular, and biochemical insights that can be gained by using Drosophila melanogaster oogenesis as a model tissue. We begin with an overview of ovary development and adult oogenesis. Then we summarize how the adult Drosophila ovary continues to advance our understanding of stem cells, cell cycle, cell migration, cytoplasmic streaming, nurse cell dumping, and cell death. We also review emerging areas of study, including the roles of lipid droplets, ribosomes, and nuclear actin in egg development. Finally, we conclude by discussing the growing conservation of processes and signaling pathways that regulate oogenesis and female reproduction from flies to humans.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Female , Actins/metabolism , Drosophila/genetics , Drosophila/metabolism , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Oogenesis/genetics , OvaryABSTRACT
A key regulator of collective cell migration is prostaglandin (PG) signaling. However, it remains largely unclear whether PGs act within the migratory cells or their microenvironment to promote migration. Here we use Drosophila border cell migration as a model to uncover the cell-specific roles of two PGs in collective migration. Prior work shows PG signaling is required for on-time migration and cluster cohesion. We find that the PGE2 synthase cPGES is required in the substrate, while the PGF2α synthase Akr1B is required in the border cells for on-time migration. Akr1B acts in both the border cells and their substrate to regulate cluster cohesion. One means by which Akr1B regulates border cell migration is by promoting integrin-based adhesions. Additionally, Akr1B limits myosin activity, and thereby cellular stiffness, in the border cells, whereas cPGES limits myosin activity in both the border cells and their substrate. Together these data reveal that two PGs, PGE2 and PGF2α, produced in different locations, play key roles in promoting border cell migration. These PGs likely have similar migratory versus microenvironment roles in other collective cell migrations.
ABSTRACT
Introduction: A key regulator of collective cell migration is prostaglandin (PG) signaling. However, it remains largely unclear whether PGs act within the migratory cells or their microenvironment to promote migration. Here we use Drosophila border cell migration as a model to uncover the cell-specific roles of two PGs in collective migration. The border cells undergo a collective and invasive migration between the nurse cells; thus, the nurse cells are the substrate and microenvironment for the border cells. Prior work found PG signaling is required for on-time border cell migration and cluster cohesion. Methods: Confocal microscopy and quantitative image analyses of available mutant alleles and RNAi lines were used to define the roles of the PGE2 and PGF2α synthases in border cell migration. Results: We find that the PGE2 synthase cPGES is required in the substrate, while the PGF2α synthase Akr1B is required in the border cells for on-time migration. Akr1B acts in both the border cells and their substrate to regulate cluster cohesion. One means by which Akr1B may regulate border cell migration and/or cluster cohesion is by promoting integrin-based adhesions. Additionally, Akr1B limits myosin activity, and thereby cellular stiffness, in the border cells, whereas cPGES limits myosin activity in both the border cells and their substrate. Decreasing myosin activity overcomes the migration delays in both akr1B and cPGES mutants, indicating the changes in cellular stiffness contribute to the migration defects. Discussion: Together these data reveal that two PGs, PGE2 and PGF2α, produced in different locations, play key roles in promoting border cell migration. These PGs likely have similar migratory versus microenvironment roles in other collective cell migrations.
ABSTRACT
Prostaglandins (PGs), locally acting lipid signals, regulate female reproduction, including oocyte development. However, the cellular mechanisms of PG action remain largely unknown. One cellular target of PG signaling is the nucleolus. Indeed, across organisms, loss of PGs results in misshapen nucleoli, and changes in nucleolar morphology are indicative of altered nucleolar function. A key role of the nucleolus is to transcribe ribosomal RNA (rRNA) to drive ribosomal biogenesis. Here we take advantage of the robust, in vivo system of Drosophila oogenesis to define the roles and downstream mechanisms whereby PGs regulate the nucleolus. We find that the altered nucleolar morphology due to PG loss is not due to reduced rRNA transcription. Instead, loss of PGs results in increased rRNA transcription and overall protein translation. PGs modulate these nucleolar functions by tightly regulating nuclear actin, which is enriched in the nucleolus. Specifically, we find that loss of PGs results in both increased nucleolar actin and changes in its form. Increasing nuclear actin, by either genetic loss of PG signaling or overexpression of nuclear targeted actin (NLS-actin), results in a round nucleolar morphology. Further, loss of PGs, overexpression of NLS-actin or loss of Exportin 6, all manipulations that increase nuclear actin levels, results in increased RNAPI-dependent transcription. Together these data reveal PGs carefully balance the level and forms of nuclear actin to control the level of nucleolar activity required for producing fertilization competent oocytes.
ABSTRACT
A key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, we identify, for the first time, that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in: increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascin's actin bundling activity is required to limit Myosin activation. Surprisingly, we find that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to uncover that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This understudied means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration.
Subject(s)
Carrier Proteins/genetics , Cell Movement/physiology , Drosophila melanogaster/physiology , Microfilament Proteins/genetics , Myosins/metabolism , Animals , Carrier Proteins/metabolism , Drosophila melanogaster/genetics , Microfilament Proteins/metabolismABSTRACT
Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Eye Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Antibodies, Phospho-Specific/immunology , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Embryonic Induction , Eye/embryology , Eye/enzymology , Eye/metabolism , Eye Proteins/chemistry , Eye Proteins/genetics , Gene Expression Regulation , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Phosphorylation , Protein Conformation , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Substrate Specificity , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Fascin, an actin-binding protein, regulates many developmental migrations and contributes to cancer metastasis. Specifically, Fascin promotes cell motility, invasion, and adhesion by forming filopodia and invadopodia through its canonical actin bundling function. In addition to bundling actin, Fascin has non-canonical roles in the cell that are thought to promote cell migration. These non-canonical functions include regulating the activity of other actin-binding proteins, binding to and regulating microtubules, mediating mechanotransduction to the nucleus via interaction with the Linker of the Nucleoskeleton and Cytoskeleton (LINC) Complex, and localizing to the nucleus to regulate nuclear actin, the nucleolus, and chromatin modifications. The many functions of Fascin must be coordinately regulated to control cell migration. While much remains to be learned about such mechanisms, Fascin is regulated by post-translational modifications, prostaglandin signaling, protein-protein interactions, and transcriptional means. Here, we review the structure of Fascin, the various functions of Fascin and how they contribute to cell migration, the mechanisms regulating Fascin, and how Fascin contributes to diseases, specifically cancer metastasis.
ABSTRACT
Homeostasis of Drosophila germline stem cells (GSC) depends upon the integration of intrinsic and extrinsic signals. This review highlights emerging data that support nuclear architecture as an intrinsic regulator of GSC maintenance and germ cell differentiation. Here, we focus on the nuclear lamina (NL) and the nucleolus, two compartments that undergo alterations in composition upon germ cell differentiation. Loss of NL or nucleolar components leads to GSC loss, resulting from activation of GSC quality control checkpoint pathways. We suggest that the NL and nucleolus integrate signals needed for the switch between GSC maintenance and germ cell differentiation, and propose regulation of nuclear actin pools as one mechanism that connects these compartments.
Subject(s)
Cell Differentiation , Drosophila , Oogonial Stem Cells/cytology , Animals , Cell Cycle Checkpoints , Cell Nucleolus , Female , Nuclear LaminaABSTRACT
While prostaglandins (PGs), short-range lipid signals, regulate single cell migration, their roles in collective migration remain unclear. To address this, we use Drosophila border cell migration, an invasive, collective migration that occurs during Stage 9 of oogenesis. Pxt is the Drosophila cyclooxygenase-like enzyme responsible for PG synthesis. Loss of Pxt results in both delayed border cell migration and elongated clusters, whereas somatic Pxt knockdown causes delayed migration and compacted clusters. These findings suggest PGs act in both the border cells and nurse cells, the substrate on which the border cells migrate. As PGs regulate the actin bundler Fascin, and Fascin is required for on-time migration, we assessed whether PGs regulate Fascin to promote border cell migration. Coreduction of Pxt and Fascin results in delayed migration and elongated clusters. The latter may be due to altered cell adhesion, as loss of Pxt or Fascin, or coreduction of both, decreases integrin levels on the border cell membranes. Conversely, integrin localization is unaffected by somatic knockdown of Pxt. Together these data lead to the model that PG signaling controls Fascin in the border cells to promote migration and in the nurse cells to maintain cluster cohesion.
Subject(s)
Cell Movement , Prostaglandins/metabolism , Animals , Carrier Proteins/metabolism , Cell Adhesion , Cell Aggregation , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Integrins/metabolism , Microfilament Proteins/metabolism , Peroxidases/metabolism , Signal TransductionABSTRACT
The inability to effectively stimulate cardiomyocyte proliferation remains a principle barrier to regeneration in the adult human heart. A tightly regulated, acute inflammatory response mediated by a range of cell types is required to initiate regenerative processes. Prostaglandin E2 (PGE2), a potent lipid signaling molecule induced by inflammation, has been shown to promote regeneration and cell proliferation; however, the dynamics of PGE2 signaling in the context of heart regeneration remain underexplored. Here, we employ the regeneration-competent zebrafish to characterize components of the PGE2 signaling circuit following cardiac injury. In the regenerating adult heart, we documented an increase in PGE2 levels, concurrent with upregulation of cox2a and ptges, two genes critical for PGE2 synthesis. Furthermore, we identified the epicardium as the most prominent site for cox2a expression, thereby suggesting a role for this tissue as an inflammatory mediator. Injury also drove the opposing expression of PGE2 receptors, upregulating pro-restorative ptger2a and downregulating the opposing receptor ptger3. Importantly, treatment with pharmacological inhibitors of Cox2 activity suppressed both production of PGE2, and the proliferation of cardiomyocytes. These results suggest that injury-induced PGE2 signaling is key to stimulating cardiomyocyte proliferation during regeneration.
Subject(s)
Dinoprostone/metabolism , Heart Injuries/metabolism , Heart/physiology , Regeneration , Animals , Animals, Genetically Modified , Cell Proliferation , Down-Regulation , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , In Situ Hybridization , Inflammation , Lipids/chemistry , Myocytes, Cardiac/metabolism , Signal Transduction , ZebrafishABSTRACT
Prostaglandins (PGs) are lipid signaling molecules with numerous physiologic functions, including pain/inflammation, fertility, and cancer. PGs are produced downstream of cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs). In numerous systems, PGs regulate actin cytoskeletal remodeling, however, their mechanisms of action remain largely unknown. To address this deficiency, we undertook a pharmaco-genetic interaction screen during late-stage Drosophila oogenesis. Drosophila oogenesis is as an established model for studying both actin dynamics and PGs. Indeed, during Stage 10B, cage-like arrays of actin bundles surround each nurse cell nucleus, and during Stage 11, the cortical actin contracts, squeezing the cytoplasmic contents into the oocyte. Both of these cytoskeletal properties are required for follicle development and fertility, and are regulated by PGs. Here we describe a pharmaco-genetic interaction screen that takes advantage of the fact that Stage 10B follicles will mature in culture and COX inhibitors, such as aspirin, block this in vitro follicle maturation. In the screen, aspirin was used at a concentration that blocks 50% of the wild-type follicles from maturing in culture. By combining this aspirin treatment with heterozygosity for mutations in actin regulators, we quantitatively identified enhancers and suppressors of COX inhibition. Here we present the screen results and initial follow-up studies on three strong enhancers - Enabled, Capping protein, and non-muscle Myosin II Regulatory Light Chain. Overall, these studies provide new insight into how PGs regulate both actin bundle formation and cellular contraction, properties that are not only essential for development, but are misregulated in disease.
Subject(s)
Actins/metabolism , Drosophila Proteins/metabolism , Drosophila/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Animals , Aspirin/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Drosophila/growth & development , Oocytes/cytology , Oocytes/metabolism , Oogenesis , Prostaglandin-Endoperoxide Synthases/chemistry , Prostaglandin-Endoperoxide Synthases/metabolism , Prostaglandins/metabolism , Signal Transduction/drug effectsABSTRACT
Post-translational modifications such as phosphorylation provide versatile context-specific strategies for modulating transcription factor activity. In the prevailing view of this process, modifying enzymes indirectly influence gene expression by shuttling to and from the nucleus where they alter the activity of their target transcription factors. However, a new paradigm has recently been suggested from studies of Eyes absent (EYA), a member of a conserved network of transcriptional regulators implicated in the development of numerous tissues and organs including the eye, ear, muscle and kidney. These findings indicate that EYA operates both as a transcriptional coactivator and as the prototype of a novel class of protein tyrosine phosphatases. The regulatory potential of such a bifunctional transcription factor is enormous and suggests a new layer of dynamic regulation in which transcription factors themselves might provide intrinsic enzymatic activities to fine-tune nuclear output.
Subject(s)
Drosophila Proteins/physiology , Eye Proteins/physiology , Protein Tyrosine Phosphatases/physiology , Transcription Factors/physiology , Animals , Drosophila melanogaster , Gene Expression Regulation/physiologyABSTRACT
While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses induce the nuclear localization and alter the structure of actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. Here, we explore the early discovery of actin in the nucleus and discuss the forms and functions of nuclear actin in both normal and disease contexts. Anat Rec, 301:1999-2013, 2018. © 2018 Wiley Periodicals, Inc.
Subject(s)
Actins/metabolism , Cell Nucleus/metabolism , Chromatin/metabolism , Transcription, Genetic/physiology , Actins/genetics , Animals , Cell Nucleus/genetics , Chromatin/genetics , Cytoplasm/genetics , Cytoplasm/metabolism , Humans , Protein Transport/physiologyABSTRACT
While nuclear actin was reported ~50 years ago, it's in vivo prevalence and structure remain largely unknown. Here, we use Drosophila oogenesis, that is, follicle development, to characterize nuclear actin. We find that three different reagents-DNase I, anti-actin C4, and anti-actin AC15-recognize distinct pools of nuclear actin. DNase I labels monomeric or G-actin, and, during follicle development, G-actin is present in the nucleus of every cell. Some G-actin is recognized by the C4 antibody. In particular, C4 nuclear actin colocalizes with DNase I to the nucleolus in anterior escort cells, follicle stem cells, some mitotic follicle cells, and a subset of nurse cells during early oogenesis. C4 also labels polymeric nuclear actin in the nucleoplasm of the germline stem cells, early cystoblasts, and oocytes. The AC15 antibody labels a completely distinct pool of nuclear actin from that of DNase I and C4. Specifically, AC15 nuclear actin localizes to the chromatin in the nurse and follicle cells during mid-to-late oogenesis. Within the oocyte, AC15 nuclear actin progresses from localizing to puncta surrounding the DNA, to forming a filamentous cage around the chromosomes. Together these findings reveal that nuclear actin is highly prevalent in vivo, and multiple pools of nuclear actin exist and can be recognized using different reagents. Additionally, our localization studies suggest that nuclear actin may regulate stemness, nucleolar structure and function, transcription, and nuclear structure. Such findings call for further studies to explore the prevalence, diversity, and functions of nuclear actin across tissues and organisms. Anat Rec, 301:2014-2036, 2018. © 2018 Wiley Periodicals, Inc.
Subject(s)
Actins/metabolism , Cell Nucleus/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/physiology , Drosophila Proteins/metabolism , Oocytes/metabolism , Oogenesis/physiology , Actins/analysis , Animals , Cell Nucleus/chemistry , Drosophila , Drosophila Proteins/analysis , Female , Oocytes/chemistryABSTRACT
Eyes absent (EYA) proteins are defined by a conserved C-terminal EYA domain (ED) that both contributes to its function as a transcriptional coactivator by mediating protein-protein interactions and possesses intrinsic protein tyrosine phosphatase activity. Mutations in human EYA1 result in an autosomal dominant disorder called branchio-oto-renal (BOR) syndrome as well as congenital cataracts and ocular defects (OD). Both BOR- and OD-associated missense mutations alter residues in the conserved ED as do three missense mutations identified from Drosophila eya alleles. To investigate the molecular mechanisms whereby these mutations disrupt EYA function, we tested their activity in a series of assays that measured in vivo function, phosphatase activity, transcriptional capability, and protein-protein interactions. We find that the OD-associated mutations retain significant in vivo activity whereas those derived from BOR patients show a striking decrease or loss of in vivo functionality. Protein-protein interactions, either with its partner transcription factor Sine oculis or with EYA itself, were not significantly compromised. Finally, the results of the biochemical assays suggest that both loss of protein tyrosine phosphatase activity and reduced transcriptional capability contribute to the impaired EYA function associated with BOR/OD syndrome, thus shedding new light into the molecular mechanisms underlying this disease.
Subject(s)
Branchio-Oto-Renal Syndrome/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Eye Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Protein Tyrosine Phosphatases/genetics , Transcription, Genetic , Alleles , Amino Acid Sequence , Animals , Blotting, Western , Drosophila Proteins/physiology , Eye Proteins/physiology , Genes, Dominant , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/physiology , Kinetics , Molecular Sequence Data , Mutation , Mutation, Missense , Nuclear Proteins/physiology , Phenotype , Photoreceptor Cells, Invertebrate , Protein Binding , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/physiology , Sequence Homology, Amino Acid , Transcriptional Activation , TransgenesABSTRACT
Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.