RESUMO
The ubiquitin-proteasome system (UPS) is an important regulator of the main cellular processes. The components of the UPS are involved in the regulation of the cell cycle, signal transduction, the cell response to DNA damage, metabolism, and transcription control. E3 ubiquitin ligases (the enzymes that covalently attaches ubiquitin to target proteins) play a key role in the functioning of the UPS. The Drosophila tumor suppressor Hyd (hyperplastic discs) is one of the most interesting E3 ligases; it is required for the regulation of proliferation, growth, and cell differentiation. The study of hyd mutations in different tissues of Drosophila demonstrated that depending on the cellular context, Hyd can not only perform proteolytic functions associated with protein degradation, but can also, interacting with other proteins and/or nucleic acids, act as an important regulator of cellular processes.
Assuntos
Drosophila , Ubiquitina-Proteína Ligases , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The GAGA protein of drosophila is a factor involved in epigenetic transcription regulation of a large gene group controlling developmental processes. In this paper, the role of GAGA factor in germ cell migration is demonstrated as well as its effect on the gonad development in drosophila embryogenesis. Mutations in the Trl gene, encoding GAGA factor, prematurely induces the active migration program and relocation of the primordial cells inward the embryo before the beginning of gastrulation. The germ cells that prematurely separated from the main group migrate ectopically, lose orientation, and stay out of gonad development. Expression pattern of the Trl gene suggests its activity in epithelial cells of the embryonic blastoderm, part of which contact primordial cells. Thus, GAGA factor influences migration of these cells in an indirect manner via their somatic environment.
Assuntos
Movimento Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/embriologia , Células Germinativas/metabolismo , Gônadas/embriologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/citologia , Células Germinativas/citologia , Gônadas/citologia , Humanos , Masculino , Fatores de Transcrição/genéticaRESUMO
Drosophila Merlin, an ortholog of the merlin protein encoded by the human Neurofibromatosis 2 (NF2) gene, is important for the regulation of cell proliferation and differentiation in the eye and wing. Also, it has been shown to be involved in male fertility in flies. In the present study, we formation using the comparative light and electron microscopic research of merlin mutants (mer3 and mer4) and ectopic expression of the Mer+ construct. Our work defines specific functions for Merlin in the mitochondria association and aggregation during the nebenkern formation and unfurling mitochondrial derivates during spermatid elongation. Possible role of Merlin as an adaptor protein that can link mitochondria with cytoskeleton is discussed.
Assuntos
Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Divisão Celular , Proliferação de Células , Drosophila/citologia , Drosophila/embriologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Olho/metabolismo , Expressão Gênica , Genes da Neurofibromatose 2/fisiologia , Humanos , Masculino , Microscopia Eletrônica , Mitocôndrias/metabolismo , Morfogênese/genética , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Espermátides/metabolismo , Espermátides/ultraestrutura , Asas de Animais/fisiologiaRESUMO
The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulse-labeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.
Assuntos
Ciclo Celular/genética , Drosophila melanogaster/genética , Mutação , Neurofibromina 2 , Asas de Animais , Animais , Proliferação de Células , Larva/genética , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
Drosophila protein GAGA (GAF) is a factor of epigenetic transcription regulation of a large group of genes with a wide variety of cellular functions. GAF is encoded by the Trithorax-like (Trl) gene, which is important for the formation of various organs and tissues at all stages of ontogenesis. In our previous works, we showed that this protein is necessary for the development of the reproductive system, both in males and females of Drosophila. Decreased expression of the Trl gene led to multiple disorders of spermatogenesis and oogenesis. One of the significant disorders was associated with massive degradation and loss of cells in the germline. In this work, we carried out a more detailed cytological study to determine what type of germ cell death is characteristic of Trl mutants, and whether there are disturbances or changes in this process compared to the norm. The results obtained showed that the lack of GAF protein causes massive germ cell death in both females and males of Drosophila, but this death manifests itself in different ways, depending on the sex. In Trl females, this process does not differ phenotypically from the norm. In the dying egg chambers, signs of apoptosis and autophagy were revealed, as well as morphological features that are characteristic of the wild type. In males, Trl mutations induce mass germ cell death through autophagy, which is not typical of Drosophila spermatogenesis, and has not been previously described, neither in the norm nor in other genes' mutations. Thus, GAF lack in Trl mutants leads to increased germ cell death through apoptosis and autophagy. Ectopic cell death and germ line atrophy are probably associated with impaired expression of the GAGA factor target genes, among which there are genes that regulate both apoptosis and autophagy.
RESUMO
Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.
Assuntos
Diferenciação Celular , Proliferação de Células , Mutação , Neoplasias/metabolismo , Neurofibromina 2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Asas de Animais/metabolismo , Animais , Adesão Celular/genética , Movimento Celular/genética , Drosophila melanogaster , Neoplasias/genética , Neoplasias/patologia , Neurofibromina 2/genética , Proteínas Supressoras de Tumor/genética , Asas de Animais/patologiaRESUMO
The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer(DBB) together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endocitose/fisiologia , Neurofibromina 2/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proliferação de Células , Clatrina/genética , Clatrina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Neoplasias da Orelha/genética , Neoplasias da Orelha/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Olho/embriologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurofibromina 2/genética , Neoplasias do Sistema Nervoso Periférico/genética , Neoplasias do Sistema Nervoso Periférico/metabolismo , Receptores de Peptídeos de Invertebrados/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Asas de Animais/embriologiaRESUMO
The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr5 residue of the Drosophila Merlin protein was homologous to Ser518 of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr559 of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT559D (an analog of the phosphorylated form) and MerT559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT559A mutant form, and mycMer345-635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case ofectopic expression of the MerT559D analog of the phosphorylated form. Ectopic expression of the mycMer3, mycMerABB, and mycMer-379 truncate variants led to disturbance of meiotic cytokinesis.
Assuntos
Meiose/fisiologia , Neurofibromina 2/metabolismo , Espermatogênese/fisiologia , Substituição de Aminoácidos , Animais , Drosophila melanogaster , Humanos , Masculino , Mutação de Sentido Incorreto , Neurofibromina 2/genética , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
Cell migration is an important morphogenetic process necessary at different stages of individual development and body functioning. The initiation and maintenance of the cell movement state requires the activation of many factors involved in the regulation of transcription, signal transduction, adhesive interactions, modulation of membranes and the cytoskeleton. However, cell movement depends on the status of both migrating and surrounding cells, interacting with each other during movement. The surrounding cells or cell matrix not only form a substrate for movement, but can also participate in the spatio-temporal regulation of the migration. At present, there is no exact understanding of the genetic mechanisms of this regulation. To determine the role of the cell environment in the regulation of individual cell migration, we studied the migration of primordial germline cells (PGC) during early embryogenesis in Drosophila melanogaster. Normally, PGC are formed at the 3rd stage of embryogenesis at the posterior pole of the embryo. During gastrulation (stages 6-7), PGC as a consolidated cell group passively transfers into the midgut primordium. Further, PGC are individualized, acquire an amoeboid form, and actively move through the midgut epithelium and migrate to the 5-6 abdominal segment of the embryo, where they form paired embryonic gonads. We screened for genes expressed in the epithelium surrounding PGC during early embryogenesis and affecting their migration. We identified the myc, Hph, stat92E, Tre-1, and hop genes, whose RNA interference leads to premature active PGC migration at stages 4-7 of embryogenesis. These genes can be divided into two groups: 1) modulators of JAK/STAT pathway activity inducing PGC migration (stat92E, Tre-1, hop), and 2) myc and Hph involved in epithelial morphogenesis and polarization, i. e. modifying the permeability of the epithelial barrier. Since a depletion of each of these gene products resulted in premature PGC migration, we can conclude that, normally, the somatic environment negatively regulates PGC migration during early Drosophila embryogenesis.
RESUMO
The function of the gene gilgamesh (89B9-12) encoding a casein kinase in Drosophila spermatogenesis was studied. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with gamma-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed.
Assuntos
Caseína Quinase I/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Espermatogênese/fisiologia , Animais , Caseína Quinase I/classificação , Caseína Quinase I/genética , Proteínas de Drosophila/classificação , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/genética , Masculino , Filogenia , Proteínas Recombinantes de Fusão/genéticaRESUMO
The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.
Assuntos
Caseína Quinase I/metabolismo , Proteínas de Drosophila/metabolismo , Filogenia , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Caseína Quinase I/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermátides/patologiaRESUMO
In majority species of dicotyledonous plants cytokinesis in PMCs occurs once after completion of two caryokinesis cycles, that is a simultaneous type. This paper represents cytological picture and frequency characteristics of abnormality which resulted in cytokinesis triggering after first meiotic division in a part of transgenic tobacco PMCs. It was shown that the main process of cytoskeleton reorganization typical for simultaneous cytokinesis remained without any alterations in such cells. However, in most cases premature cell division occurred with abnormalities such as membrane "tunnel" or "gash" formation. It was ascertained that initiation of additional round ofcytokinesis did not block nuclear cycle and cytokinesis after second meiotic division. Thus, transition of cell division from simultaneous type to successive one occurs under this abnormality.
Assuntos
Citocinese , Nicotiana/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Citoesqueleto/fisiologia , Genes de Plantas , Meiose , Pólen/fisiologiaRESUMO
A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Membrana/metabolismo , Neurofibromina 2/metabolismo , Organogênese/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Asas de Animais/embriologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Membrana/genética , Neurofibromina 2/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The Drosophila Pnut protein belongs to the family of septins, which are conserved GTPases participating in cytokinesis and many more other fundamental cellular processes. Because of their filamentous appearance, membrane association, and functions, septins are considered as the fourth component of the cytoskeleton, along with actin, microtubules, and intermediate filaments. However, septins are much less studied than the other cytoskeleton elements. We had previously demonstrated that the deletion of the pnut gene leads to mitotic abnormalities in somatic cells. The goal of this work was to study the role of the pnut in Drosophila spermatogenesis. We designed a construct for pnut RNA interference allowing pnut expression to be suppressed ectopically. We analyzed the effect of pnut RNA interference on Drosophila spermatogenesis. Germline cells at the earliest stages of spermatogenesis were the most sensitive to Pnut depletion: the suppression of the pnut expression at these stages leads to male sterility as a result of immotile sperm. The testes of these sterile males did not show any significant meiotic defects; the axonemes and mitochondria were normal. We also analyzed the effect of mutations in the Pnut's conservative domains on Drosophila spermatogenesis. Mutations in the GTPase domain resulted in cyst elongation defects. Deletions of the C-terminal domain led to abnormal testis morphology. Both the GTPase domain and C-terminal domain mutant males were sterile and produced immotile sperm. To summarize, we showed that Pnut participates in spermiogenesis, that is, the late stages of spermatogenesis, when major morphological changes in spermatocytes occur.
RESUMO
Rearrangements of microtubular cytoskeleton during telophase in pollen mother cells of some dicotyledon plants with the simultaneous cytokinesis during normal and abnormal meiosis were studied. At telophase I, a potentially functional phragmoplast forms between daughter nuclei, but no cell plate is present. During interkinesis, the phragmoplast plays the role of an interphase cytoskeleton array. Dynamics of microtubule reorganization in polar regions of the telophase spindle is discussed in addition to the role played by microtubule convergence centers in cytoskeleton rearrangements during meiosis.
Assuntos
Citocinese , Citoesqueleto/metabolismo , Meiose , Fenômenos Fisiológicos Vegetais , Citoesqueleto/ultraestrutura , Microtúbulos , Pólen/fisiologia , TelófaseRESUMO
In this study we analysed the terminal step of cytoskeleton cycle in higher plant meiosis: transition from phragmoplast to radial interphase configuration. Wild type meiosis in a range of mono- and dicotyledonous species was studied. A number of cytoskeleton abnormalities on this stage was described in meiotic mutants, haploids and wide hybrids of various species. We described processes of cytoskeleton rearrangements on this stage: disjunction of phragmoplast MTs, their shortening and the role of daughter cell membranes. The independence of the interphase radial MT system formation from the previous steps of cytoskeleton cycle and from nuclear envelope cycle is proposed.
Assuntos
Citoesqueleto/ultraestrutura , Meiose , Fenômenos Fisiológicos Vegetais , Quimera , Microtúbulos/ultraestrutura , Fuso Acromático/ultraestruturaRESUMO
This review involves consideration of morphological and functional aspects of cytokinesis, such as role of microtubule and actin cytoskeleton as well as mechanisms of cell plate formation and phragmoplast expansion, spatial control of division plan and cell cycle regulation, in addition to newly discovered mutations. Recent progress in understanding mechanisms of cytokinesis in the plant cell is discussed.
Assuntos
Divisão Celular/fisiologia , Células Vegetais , Actinas/fisiologia , Citocinese/genética , Citocinese/fisiologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , MutaçãoRESUMO
Our study of cytological phenotype of meiotic mutation pam resulted in detecting a failure of cytokinesis in mutant pollen mother cells in the form of a block of fusion of membrane vesicles of the cell plate, and an impossibility of formation of daughter cell membranes. The mutation does not disturb the division spindle structure and function. Asynchrony of meiosis in pam is the result of arrest of pollen mother cells at metaphase 1 and metaphase 2.
Assuntos
Meiose/genética , Mutação , Zea mays/citologia , Divisão Celular , Zea mays/genéticaRESUMO
The morphological phenotype of meimutation acd (abnormal cytoskeleton dynamics) in Beta vulgaris was analysed using a classic method of achromatic figure visualization (Navashin fixative). The mutation demonstrates two different phenotypes under different growth conditions. The first variant is characterized by formation of chaotic network of microtubule bundles instead of two bipolar spindles at metaphase II. Such abnormality leads to the formation of polyads and monads. The second variant of phenotype demonstrates desorientation of spindles at metaphase II and partial asynapsis of homologous chromosomes.
Assuntos
Chenopodiaceae/ultraestrutura , Microtúbulos/ultraestrutura , Mutação , Chenopodiaceae/genética , Meiose/genética , Metáfase , FenótipoRESUMO
Five major mictotubule arrays characteristic of cell cycle in the higher plants were noticed: cortical coils, preprophase band, radial array, division spindle, and phragmoplast. The organization of mocrotubules into ordered arrays; their dynamics and function during plant cell division are discussed in this review.