RESUMO
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.
Assuntos
Proteínas de Transporte/genética , Movimento Celular/fisiologia , Drosophila melanogaster/fisiologia , Proteínas dos Microfilamentos/genética , Miosinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Drosophila melanogaster/genética , Proteínas dos Microfilamentos/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Drosophila melanogaster/genética , Proteínas dos Microfilamentos/metabolismo , Oócitos/citologia , Animais , Animais Geneticamente Modificados , Adesão Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Microscopia Confocal , Oogênese , Interferência de RNA , Transdução de SinaisRESUMO
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.
Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Aspirina/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Drosophila/crescimento & desenvolvimento , Oócitos/citologia , Oócitos/metabolismo , Oogênese , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Transcrição Gênica/fisiologia , Actinas/genética , Animais , Núcleo Celular/genética , Cromatina/genética , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Transporte Proteico/fisiologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Proteínas de Drosophila/metabolismo , Oócitos/metabolismo , Oogênese/fisiologia , Actinas/análise , Animais , Núcleo Celular/química , Drosophila , Proteínas de Drosophila/análise , Feminino , Oócitos/químicaRESUMO
Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Pseudópodes/fisiologia , Células 3T3 , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Drosophila , Células HeLa , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Invasividade Neoplásica/patologia , Neoplasias/patologia , Membrana Nuclear/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de ProteínaRESUMO
Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B-12 (S10B-12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts.
Assuntos
Proteínas de Transporte/metabolismo , Nucléolo Celular/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Folículo Ovariano/fisiologia , Prostaglandinas/fisiologia , Transdução de Sinais , Animais , Proteínas de Transporte/fisiologia , Drosophila , Feminino , Humanos , Proteínas dos Microfilamentos/fisiologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Transporte ProteicoRESUMO
Prostaglandins (PGs) are lipid signals that are produced at their sites of action by cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs), and PG-type specific synthases. Active PGs serve as ligands for G protein-coupled receptors (GPCRs). The functions of PGs have largely been elucidated using pharmacologic, expression-based (synthesis and signaling components), and genetic studies. In this review, we discuss the in vivo roles of PGs in cancer, development, and reproduction that have been characterized using genetic knockout/knockdown and overexpression approaches in mice, zebrafish, and invertebrate model systems, and how pharmacologic inhibition of PG synthesis affects cardiovascular health/disease and cancer incidence and progression.
Assuntos
Prostaglandinas/genética , Prostaglandinas/metabolismo , Transdução de Sinais/genética , Animais , Doença , Modelos Animais de Doenças , Humanos , Modelos Biológicos , Prostaglandinas/biossínteseRESUMO
Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.
Assuntos
Actinas , Proteínas de Transporte , Proteínas de Drosophila , Proteínas dos Microfilamentos , Peroxidases , Prostaglandina-Endoperóxido Sintases , Prostaglandinas , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Germinativas/citologia , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , Oogênese/genética , Peroxidases/genética , Peroxidases/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Síndrome Brânquio-Otorrenal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatases/genética , Transcrição Gênica , Alelos , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Drosophila/fisiologia , Proteínas do Olho/fisiologia , Genes Dominantes , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Cinética , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Proteínas Nucleares/fisiologia , Fenótipo , Células Fotorreceptoras de Invertebrados , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/fisiologia , Homologia de Sequência de Aminoácidos , Ativação Transcricional , TransgenesRESUMO
Ets transcription factors play crucial roles in regulating diverse cellular processes including cell proliferation, differentiation and survival. Coordinated regulation of the Drosophila Ets transcription factors YAN and POINTED is required for eliciting appropriate responses to Receptor Tyrosine Kinase (RTK) signaling. YAN, a transcriptional repressor, and POINTED, a transcriptional activator, compete for regulatory regions of common target genes, with the ultimate outcome likely influenced by context-specific interactions with binding partners such as MAE. Previous work in cultured cells has led us to propose that MAE attenuates the transcriptional activity of both YAN and POINTED, although its effects on POINTED remain controversial. Here we describe a new layer of complexity to this regulatory hierarchy whereby mae expression is itself directly regulated by the opposing action of YAN and POINTED. In addition, we report that MAE can antagonize POINTED function during eye development; a finding that suggests MAE operates as a dual positive and negative regulator of RTK-mediated signaling in vivo. Together our results lead us to propose that a combination of protein-protein and transcriptional interactions between MAE, YAN and POINTED establishes a complex regulatory circuit that ensures that both down-regulation and activation of the RTK pathway occur appropriately according to specific developmental context.
Assuntos
Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Proteínas do Olho/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases/metabolismo , Receptores de Peptídeos de Invertebrados/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ligação a DNA/metabolismo , Olho/embriologia , Olho/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
ETS family transcription factors serve as downstream effectors of signal transduction pathways, mediating cellular proliferation, differentiation and, when misregulated, tumorigenesis. The transcriptional repressor YAN prevents inappropriate responses to Receptor Tyrosine Kinase signaling by outcompeting POINTED for access to target gene promoters. We demonstrate that the molecular mechanism underlying downregulation of YAN involves CRM1-mediated nuclear export and define a novel role in this context for MAE, a co-factor previously implicated in facilitating MAPK phosphorylation of YAN. In addition to promoting YAN downregulation, MAE also participates in an inhibitory feedback loop that attenuates POINTED-P2 activation. Thus, we propose that MAE plays multiple independent roles in fine-tuning the levels of POINTED and YAN activity in accordance with changing RTK signaling conditions.