RESUMO
Abscission is the final step of cytokinesis that allows the physical separation of sister cells through the scission of the cellular membrane. This deformation is driven by ESCRT-III proteins, which can bind membranes and form dynamic helices. A crucial step in abscission is the recruitment of ESCRT-III proteins at the right time and place. Alix is one of the best characterized proteins that recruits ESCRT-III proteins from yeast to mammals. However, recent studies in vivo have revealed that pathways acting independently or redundantly with Alix are also required at abscission sites in different cellular contexts. Here, we show that Lgd acts redundantly with Alix to properly localize ESCRT-III to the abscission site in germline stem cells (GSCs) during Drosophila oogenesis. We further demonstrate that Lgd is phosphorylated at multiple sites by the CycB/Cdk1 kinase. We found that these phosphorylation events potentiate the activity of Shrub, a Drosophila ESCRT-III, during abscission of GSCs. Our study reveals that redundancy between Lgd and Alix, and coordination with the cell cycle kinase Cdk1, confers robust and timely abscission of Drosophila germline stem cells.
Assuntos
Proteínas de Drosophila , Complexos Endossomais de Distribuição Requeridos para Transporte , Células Germinativas , Células-Tronco , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclina B , Citocinese/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Germinativas/metabolismo , Mamíferos/metabolismo , Células-Tronco/metabolismoRESUMO
Although the molecular mechanisms governing abscission of isolated cells have largely been elucidated, those underlying the abscission of epithelial progenitors surrounded by epidermal cells (ECs), connected via cellular junctions, remain largely unexplored. Here, we investigated the remodeling of the paracellular diffusion barrier ensured by septate junctions (SJs) during cytokinesis of Drosophila sensory organ precursors (SOPs). We found that SOP cytokinesis involves the coordinated, polarized assembly and remodeling of SJs in the dividing cell and its neighbors, which remain connected to the former via membrane protrusions pointing towards the SOP midbody. SJ assembly and midbody basal displacement occur faster in SOPs than in ECs, leading to quicker disentanglement of neighboring cell membrane protrusions prior to midbody release. As reported in isolated cells, the endosomal sorting complex required for the transport-III component Shrub/CHMP4B is recruited at the midbody and cell-autonomously regulates abscission. In addition, Shrub is recruited to membrane protrusions and is required for SJ integrity, and alteration of SJ integrity leads to premature abscission. Our study uncovers cell-intrinsic and -extrinsic functions of Shrub in coordinating remodeling of the SJs and SOP abscission.
Assuntos
Citocinese , Proteínas de Drosophila , Drosophila , Proteínas do Tecido Nervoso , Animais , Movimento Celular , Difusão , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas do Tecido Nervoso/genética , Proteínas de Drosophila/genéticaRESUMO
Chromatin packaging and modifications are important to define the identity of stem cells. How chromatin properties are retained over multiple cycles of stem cell replication, while generating differentiating progeny at the same time, remains a challenging question. The chromatin assembly factor CAF1 is a conserved histone chaperone, which assembles histones H3 and H4 onto newly synthesized DNA during replication and repair. Here, we have investigated the role of CAF1 in the maintenance of germline stem cells (GSCs) in Drosophila ovaries. We depleted P180, the large subunit of CAF1, in germ cells and found that it was required in GSCs to maintain their identity. In the absence of P180, GSCs still harbor stem cell properties but concomitantly express markers of differentiation. In addition, P180-depleted germ cells exhibit elevated levels of DNA damage and de-repression of the transposable I element. These DNA damages activate p53- and Chk2-dependent checkpoints pathways, leading to cell death and female sterility. Altogether, our work demonstrates that chromatin dynamics mediated by CAF1 play an important role in both the regulation of stem cell identity and genome integrity.
Assuntos
Células-Tronco Adultas/citologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Instabilidade Genômica/genética , Ovário/citologia , Proteína 4 de Ligação ao Retinoblastoma/genética , Animais , Animais Geneticamente Modificados , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/fisiologia , Dano ao DNA/genética , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Feminino , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Abscission is the final event of cytokinesis that leads to the physical separation of the two daughter cells. Recent technical advances have allowed a better understanding of the cellular and molecular events leading to abscission in isolated yeast or mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood. Here, we characterized the function of the ESCRT-III protein Shrub during cytokinesis in germ cells undergoing a series of complete and incomplete divisions. We found that Shrub is required for complete abscission, and that levels of Shrub are critical for proper timing of abscission. Loss or gain of Shrub delays abscission in germline stem cells (GSCs), and leads to the formation of stem-cysts, where daughter cells share the same cytoplasm as the mother stem cell and cannot differentiate. In addition, our results indicate a negative regulation of Shrub by the Aurora B kinase during GSC abscission. Finally, we found that Lethal giant discs (lgd), known to be required for Shrub function in the endosomal pathway, also regulates the duration of abscission in GSCs.
Assuntos
Citocinese/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitose/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco/citologia , Animais , Aurora Quinase B/genética , Citoplasma/genética , Drosophila melanogaster/crescimento & desenvolvimento , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Feminino , Células Germinativas/citologia , Humanos , Ovário/citologia , Proteínas Supressoras de Tumor/genéticaRESUMO
Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.
Assuntos
Ciclo Celular/genética , Citocinese/genética , Proteínas de Drosophila/genética , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Mapas de Interação de Proteínas/genética , Células-Tronco/citologia , Células-Tronco/metabolismoAssuntos
Ciclina A , Células-Tronco , Animais , Diferenciação Celular , Drosophila , Proteínas de DrosophilaRESUMO
Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. Here we use Drosophila border cells to examine how collective guidance is performed. We report that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cgamma are used redundantly, and we find that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group.
Assuntos
Movimento Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas do Citoesqueleto , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes Essenciais/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Fosfolipase C gama/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptores de Peptídeos de Invertebrados/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteínas rac de Ligação ao GTP/metabolismo , Quinases raf/genética , Quinases raf/metabolismoRESUMO
In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. We found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. Our results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division.
Assuntos
Divisão Celular , Proteínas de Drosophila , Drosophila melanogaster , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteases Específicas de Ubiquitina , Animais , Citocinese/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Feminino , Células Germinativas/citologia , Células Germinativas/fisiologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Cell migration within a natural context is tightly controlled, often by specific transcription factors. However, the switch from stationary to migratory behavior is poorly understood. Border cells perform a spatially and temporally controlled invasive migration during Drosophila oogenesis. Slbo, a C/EBP family transcriptional activator, is required for them to become migratory. We purified wild-type and slbo mutant border cells as well as nonmigratory follicle cells and performed comparative whole-genome expression profiling, followed by functional tests of the contributions of identified targets to migration. About 300 genes were significantly upregulated in border cells, many dependent on Slbo. Among these, the microtubule regulator Stathmin was strongly upregulated and was required for normal migration. Actin cytoskeleton regulators were also induced, including, surprisingly, a large cluster of "muscle-specific" genes. We conclude that Slbo induces multiple cytoskeletal effectors, and that each contributes to the behavioral changes in border cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/fisiologia , Perfilação da Expressão Gênica , Oogênese/fisiologia , Ovário/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas do Citoesqueleto/fisiologia , Drosophila , Proteínas de Drosophila/genética , Feminino , Oogênese/genética , Ovário/citologia , Ovário/metabolismo , Estatmina/fisiologia , Fatores de Transcrição/genética , Regulação para CimaRESUMO
We have found that the Drosophila gene vps25 possesses several properties of a tumor suppressor. First, vps25 mutant cells activate Notch and Dpp receptor signaling, inducing ectopic organizers in developing eyes and limbs and consequent overproliferation of both mutant and nearby wild-type cells. Second, as the mutant cells proliferate, they lose their epithelial organization and undergo apoptosis. Strikingly, when apoptosis of mutant cells is blocked, tumor-like overgrowths are formed that are capable of metastasis. vps25 encodes a component of the ESCRT-II complex, which sorts membrane proteins into multivesicular bodies during endocytic trafficking to the lysosome. Activation of Notch and Dpp receptor signaling in mutant cells results from an endocytic blockage that causes accumulation of these receptors and other signaling components in endosomes. These results highlight the importance of endocytic trafficking in regulating signaling and epithelial organization and suggest a possible role for ESCRT components in human cancer.
Assuntos
Drosophila/genética , Endossomos/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/fisiologia , Proliferação de Células , Clonagem Molecular , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Endossomos/genética , Células Epiteliais/metabolismo , Extremidades/crescimento & desenvolvimento , Olho/citologia , Olho/crescimento & desenvolvimento , Mutação , Metástase Neoplásica , Transporte Proteico/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Migration of border cells during Drosophila melanogaster oogenesis is a good model system for investigating the genetic requirements for cell migration in vivo. We present a sensitized loss-of-function screen used to identify new genes required in border cells for their migration. Chromosomes bearing FRTs on all four major autosomal arms were mutagenized by insertions of the transposable element PiggyBac, allowing multiple parallel clonal screens and easy identification of the mutated gene. For border cells, we analyzed homozygous mutant clones positively marked with lacZ and sensitized by expression of dominant-negative PVR, the guidance receptor. We identified new alleles of genes already known to be required for border cell migration, including aop/yan, DIAP1, and taiman as well as a conserved Slbo-regulated enhancer downstream of shg/DE-cadherin. Mutations in genes not previously described to be required in border cells were also uncovered: hrp48, vir, rme-8, kismet, and puckered. puckered was unique in that the migration defects were observed only when PVR signaling was reduced. We present evidence that an excess of JNK signaling is deleterious for migration in the absence of PVR activity at least in part through Fos transcriptional activity and possibly through antagonistic effects on DIAP1.
Assuntos
Movimento Celular , Elementos de DNA Transponíveis , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mutação , Oogênese , Transdução de Sinais , Transcrição GênicaRESUMO
Abscission is the last step of cytokinesis that physically separates the cytoplasm of sister cells. As the final stage of cell division, abscission is poorly characterized during animal development. Here, we show that Aurora B and Survivin regulate the number of germ cells in each Drosophila egg chamber by inhibiting abscission during differentiation. This inhibition is mediated by an Aurora B-dependent phosphorylation of Cyclin B, as a phosphomimic form of Cyclin B rescues premature abscission caused by a loss of function of Aurora B. We show that Cyclin B localizes at the cytokinesis bridge, where it promotes abscission. We propose that mutual inhibitions between Aurora B and Cyclin B regulate the duration of abscission and thereby the number of sister cells in each cyst. Finally, we show that inhibitions of Aurora B and Cyclin-dependent kinase 1 activity in vertebrate cells also have opposite effects on the timing of abscission, suggesting a possible conservation of these mechanisms.
Assuntos
Ciclina B1/metabolismo , Ciclina B/metabolismo , Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Células Germinativas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Aurora Quinases , Diferenciação Celular/fisiologia , Ciclina B/genética , Ciclina B1/genética , Ciclina B2/genética , Ciclina B2/metabolismo , Drosophila , Proteínas de Drosophila/genética , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Células Germinativas/citologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Survivina , Transfecção , VertebradosRESUMO
Despite its evolutionary conservation and functional importance, little is known of the signaling pathways that underlie development of the hypothalamus. Although mutations affecting Nodal and Hedgehog signaling disrupt hypothalamic development, the time and site of action and the exact roles of these pathways remain very poorly understood. Unexpectedly, we show here that cell-autonomous reception of Nodal signals is neither required for the migration of hypothalamic precursors within the neural plate, nor for further development of the anterior-dorsal hypothalamus. Nodal signaling is, however, cell-autonomously required for establishment of the posterior-ventral hypothalamus. Conversely, Hedgehog signaling antagonizes the development of posterior-ventral hypothalamus, while promoting anterior-dorsal hypothalamic fates. Besides their distinct roles in the regionalization of the diencephalon, we reveal cooperation between Nodal and Hedgehog pathways in the maintenance of the anterior-dorsal hypothalamus. Finally we show that it is the prechordal plate and not the head endoderm that provides the early signals essential for establishment of the hypothalamus.
Assuntos
Hipotálamo/embriologia , Transativadores/genética , Fator de Crescimento Transformador beta/genética , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero , Indução Embrionária , Endoderma , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hipotálamo/metabolismo , Mutação , Proteína Nodal , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/genéticaRESUMO
Nodal signalling is essential for many developmental events during vertebrate development, including the establishment of left-right asymmetry, of dorsoventral axis of the central nervous system, and endoderm and mesoderm formation. The zebrafish TGFbeta-related type I receptor, TARAM-A (Tar), is expressed in the prospective mesendodermal territory and, when activated, can transfate early blastomeres into endoderm, suggesting that Nodal and Tar may represent similar signalling pathways. We have analysed the functional relationships between those two pathways in zebrafish. We first demonstrate that tar and the zebrafish nodal genes cyc and sqt functionally interact. We also show that a dominant-negative isoform of Tar, TarMR, interferes specifically with the function of Cyc and Sqt in vitro, but does not interfere with the function of BMP2, another TGFbeta-related molecule. TarMR interferes also with Nodal signalling in vivo since it enhances the phenotype of embryos with weakened Nodal signalling. Overexpression of tarMR in wild-type embryos interfered with the formation of endoderm-derived structures. Conversely, overexpression of tar enlarged the presumptive mesendodermal region at the onset of gastrulation. Together, our results point to Tar as an essential factor for endoderm formation and an important modulator of Nodal signalling, potentially representing one of the Nodal receptors. (c)2001 Elsevier Science.
Assuntos
Padronização Corporal/fisiologia , Endoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/fisiologia , Proteínas Serina-Treonina Quinases , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas de Peixe-Zebra , Ativinas/metabolismo , Animais , Biomarcadores , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Embrião não Mamífero/fisiologia , Genes Dominantes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mutagênese , Proteína Nodal , Ligantes da Sinalização Nodal , Fenótipo , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. Here, we address the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. First, we find that Fgf signalling is required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. Second, combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with our model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations.