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1.
The interstitial deletion D14 affecting the importin-alpha 2 gene of Drosophila, or imp-alpha 2(D14), causes recessive female sterility characterized by a block of nurse cell-oocyte transport during oogenesis. In wild-type egg chambers, the Imp-alpha 2 protein is uniformly distributed in the nurse cell cytoplasm with a moderate accumulation along the oocyte cortex. Cytochalasin D treatment of wild-type egg chambers disrupts the in vivo association of Imp-alpha 2 with F-actin and results in its release from the oocyte cortex and its transfer into nurse cell nuclei. Binding assay shows that the interaction of Imp-alpha 2 with F-actin, albeit not monomeric actin, requires the occurrence of NLS peptides. Phenotypic analysis of imp-alpha 2(D14) ovaries reveals that the block of nurse cell-oocyte transport results from the occlusion of the ring canals that constitute cytoplasmic bridges between the nurse cells and the oocyte. Immunohistochemistry shows that, although the Imp-alpha2 protein cannot be detected on the ring canals, the Kelch protein, a known ring canal component, fails to bind to ring canals in imp-alpha 2(D14) egg chambers. Since loss-of-function mutations of kelch results in a similar dumpless phenotype, we propose that the Imp-alpha 2 protein plays a critical role in Kelch function by regulating its deposition on ring canals during their assembly. 相似文献
2.
Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis. 总被引:1,自引:0,他引:1
M G Li M Serr K Edwards S Ludmann D Yamamoto L G Tilney C M Field T S Hays 《The Journal of cell biology》1999,146(5):1061-1074
The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development. 相似文献
3.
Cortactin modulates cell migration and ring canal morphogenesis during Drosophila oogenesis 总被引:3,自引:0,他引:3
Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. We have isolated complete loss-of-function mutants of the single Drosophila cortactin gene. Mutants are viable and fertile, showing that cortactin is not an essential gene. However, cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). We find that accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. We present evidence that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the cortactin gene not being essential for development. 相似文献
4.
An essential component of normal development is controlling the transition from cell proliferation to differentiation. One such transition occurs during Drosophila oogenesis. In early oogenesis, germ cells undergo mitotic proliferation and contain a specialized organelle called a fusome, whereas later post-mitotic cells differentiate and lose the fusome as F-actin-rich ring canals form. The hts gene encodes the only Drosophila Adducin, and is a female-sterile mutant that affects both the fusome and ring canals. We show that one Hts protein, Ovhts, is a polyprotein that is cleaved to produce two products, Ovhts-Fus and Ovhts-RC. Whereas Ovhts-Fus localizes to the fusome in mitotic cells, Ovhts-RC localizes to ring canals throughout later oogenesis. We demonstrate that an uncleavable version of Ovhts delays the transition from fusome-containing cells to those that have ring canals. Ovhts is the first polyprotein shown to produce proteins that function in separate structures. 相似文献
5.
Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis. 相似文献
6.
7.
Máthé E Bates H Huikeshoven H Deák P Glover DM Cotterill S 《Developmental biology》2000,223(2):307-322
The Drosophila importin-alpha3 gene was isolated through its interaction with the large subunit of the DNA polymerase alpha in a two-hybrid screen. The predicted protein sequence of Importin-alpha3 is 65-66% identical to those of the human and mouse importin-alpha3 and alpha4 and 42.7% identical to that of Importin-alpha2 (Oho31/Pendulin), the previously reported Drosophila homologue. Both Importin-alpha3 and Importin-alpha2 interact with similar subsets of proteins in vitro, one of which is Ketel, the importin-beta homologue of Drosophila. importin-alpha3 is an essential gene, whose encoded protein is expressed throughout development. During early embryogenesis, Importin-alpha3 accumulates at the nuclear membrane of cleavage nuclei, whereas after blastoderm formation it is characteristically found within the interphase nuclei. Nuclear localisation is seen in several tissues throughout subsequent development. During oogenesis its concentration within the nurse cell nuclei increases during stages 7-10, concomitant with a decline in levels in the oocyte nucleus. Mutation of importin-alpha3 results in lethality throughout pupal development. Surviving females are sterile and show arrest of oogenesis at stages 7-10. Thus, Importin-alpha3-mediated nuclear transport is essential for completion of oogenesis and becomes limiting during pupal development. Since they have different expression patterns and subcellular localisation profiles, we suggest that the two importin-alpha homologues are not redundant in the context of normal Drosophila development. 相似文献
8.
Jagunal is required for reorganizing the endoplasmic reticulum during Drosophila oogenesis
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Vesicular traffic in the Drosophila melanogaster oocyte occurs actively during vitellogenesis. Although endocytosis in the oocyte has been well characterized, exocytic vesicular traffic is less well understood. We show that the oocyte endoplasmic reticulum (ER) becomes concentrated into subcortical clusters during vitellogenesis. This ER reorganization requires Jagunal, which is an evolutionarily conserved ER membrane protein. Loss of Jagunal reduces vesicular traffic to the oocyte lateral membrane, but does not affect posterior polarized vesicular traffic, suggesting a role for Jagunal in facilitating vesicular traffic in the subcortex. Reduced membrane traffic caused by loss of Jagunal affects oocyte and bristle growth. We propose that ER reorganization is an important mechanism used by cells to prepare for an increased demand for membrane traffic, and Jagunal facilitates this process through ER clustering. 相似文献
9.
In Drosophila, oogenesis is initiated when a germline stem cell produces a differentiating daughter cell called the cystoblast. The cystoblast undergoes four rounds of synchronous divisions with incomplete cytokinesis to generate a syncytial cyst of 16 interconnected cystocytes, in which one cystocyte differentiates into an oocyte. Strong mutations of the arrest (aret) gene disrupt cyst formation and cause the production of clusters of ill-differentiated germline cells that retain cellular and molecular characteristics of cystoblasts. These mutant germ cells express high levels of BAM-C and SXL proteins in the cytoplasm but do not accumulate markers for advanced cystocytes or differentiating oocytes, such as the nuclear localization of SXL or the accumulation of osk mRNA, orb mRNA, and cytoplasmic dynein. However, the mutant germ cells do not contain spectrosomes, the cytoplasmic structure that objectifies the divisional asymmetry of the cystoblast. The aret mutant germ cells undergo active mitosis with complete cytokinesis. Their mitosis is accompanied by massive necrosis, so that the number of germ cells in a stem cell-derived cluster ranges from one to greater than 70. These defects of aret mutants reveal a novel function of aret as the first gene with a defined function in the cystoblast to cyst transition during early oogenesis. 相似文献
10.
SET domain proteins are histone lysine methyltransferases (HMTs) that play essential roles in development. Here we show for the first time that histone methylation occurs in both the germ cells and somatic cells of the Drosophila ovary, and demonstrate in vivo that an HMT, the product of the eggless (egg) gene, is required for oogenesis. Egg is a SET domain protein that is similar to the human protein SETDB1 and its mouse ortholog ESET. These proteins are members of a small family of HMTs that contain bifurcated SET domains. Because depletion of SETDB1 in tissue culture cells is cell-lethal, and an ESET mutation causes very early periimplantation embryonic arrest, the role of SETDB1/ESET in development has proven difficult to address. We show that egg is required in the Drosophila ovary for trimethylation of histone H3 at its K9 residue. In females bearing an egg allele that deletes the SET domain, oogenesis arrests at early stages. This arrest is accompanied by reduced proliferation of somatic cells required for egg chamber formation, and by apoptosis in both germ and somatic cell populations. We propose that other closely related SET domain proteins may function similarly in gametogenesis in other species. 相似文献
11.
Entry of human adenovirus into cells is a two-step process, mediated in the first step by a specific interaction between the trimeric fiber protein and a specific receptor on the surface of susceptible cells. Because of the interest in human adenovirus as a vector for gene therapy, we have mapped domains in the fiber protein that are important for proper assembly of this trimeric structure and for proper addition of O-linked N-acetylglucosamine (0-GlcNAc). Mutants of adenovirus type 2 fiber in this study were expressed in human cells by use of a recombinant vaccinia virus expression system that yielded protein indistinguishable from the fiber produced during adenovirus infection. The N-terminal half of the protein did not appear to influence fiber trimer formation, since deletions up to 260 amino acids (aa) from the N-terminal end as well as in-frame deletions within the shaft of the molecule still allowed trimerization; internal deletions in the shaft between aa 61 and 260 appeared to alter addition of 0-GlcNAc, as judged by loss of reactivity to a monoclonal antibody specific for this carbohydrate addition. Deletions from the C terminus of the molecule (as small as 2 aa) appeared to prevent trimer formation. Additions of amino acids to the C-terminal end of the fiber showed variable results: a 6-aa addition allowed trimer formation, while a 27-aa addition did not. These trimer-defective mutants were also relatively less stable, as judged kV pulse-chase experiments. Taken together, our results indicate that trimerization of the fiber requires at least two domains, the entire head (aa 400 to 582), and at least the C-terminal-most 15 aa of the shaft. 相似文献
12.
In Drosophila melanogaster oogenesis, there are 16 germline cells that form a cyst and stay connected to each other by ring canals. Ring canals allow the cytoplasmic transport of proteins, messenger ribonucleic acids, and yolk components from the nurse cells into the oocyte. In this paper, we describe the protein Rings lost (Rngo) and show that it is required for ring canal growth in germline cysts. rngo is an essential gene, and germline clones of a rngo-null allele show defects in ovary development, including mislocalization of ring canal proteins and fusion of germline cells. Rngo appears to be a ubiquitin receptor that possesses a ubiquitin-like domain, a ubiquitin-associated domain, and a retroviral-like aspartate protease (RVP) domain. Rngo binds to ubiquitin and to the 26S proteasome and colocalizes with both in germline cells, and its RVP domain is required for dimerization of Rngo and for its function in vivo. Our results thus show, for the first time, a function for a ubiquitin receptor in Drosophila development. 相似文献
13.
chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis. 总被引:12,自引:0,他引:12
The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis. chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport. We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin. The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly. In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm. We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells. 相似文献
14.
Clathrin interactor 1 [CLINT1] (also called enthoprotin/EpsinR) is an Epsin N-terminal homology (ENTH) domain-containing adaptor protein that functions in anterograde and retrograde clathrin-mediated trafficking between the trans-Golgi network and the endosome. Removal of both Saccharomyces cerevisiae homologs, Ent3p and Ent5p, result in yeast that are viable, but that display a cold-sensitive growth phenotype and mistrafficking of various vacuolar proteins. Similarly, either knock-down or overexpression of vertebrate CLINT1 in cell culture causes mistrafficking of proteins. Here, we have characterized Drosophila CLINT1, liquid-facets Related (lqfR). LqfR is ubiquitously expressed throughout development and is localized to the Golgi and endosome. Strong hypomorphic mutants generated by imprecise P-element excision exhibit extra macrochaetae, rough eyes and are female sterile. Although essentially no eggs are laid, the ovaries do contain late-stage egg chambers that exhibit abnormal morphology. Germline clones reveal that LqfR expression in the somatic follicle cells is sufficient to rescue the oogenesis defects. Clones of mutant lqfR follicle cells have a decreased cell size consistent with a downregulation of Akt1. We find that while total Akt1 levels are increased there is also a significant decrease in activated phosphorylated Akt1. Taken together, these results show that LqfR function is required to regulate follicle cell size and signaling during Drosophila oogenesis. 相似文献
15.
The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Here we show that Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments, stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development. 相似文献
16.
Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis. 总被引:3,自引:0,他引:3
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The targeting of positional information to specific regions of the oocyte or early embryo is one of the key processes in establishing anterior-posterior and dorsal-ventral polarity. In many developmental systems, this is accomplished by localization of mRNAs. The germ line-specific Drosophila orb gene plays a critical role in defining both axes of the developing oocyte, and its mRNA is localized in a complex pattern during oogenesis. We have identified a 280-bp sequence from the orb 3' untranslated region capable of reproducing this complex localization pattern. Furthermore, we have found that multiple cis-acting elements appear to be required for proper targeting of orb mRNA. 相似文献
17.
Wispy, the Drosophila homolog of GLD-2, is required during oogenesis and egg activation 总被引:1,自引:0,他引:1
Egg activation is the process that modifies mature, arrested oocytes so that embryo development can proceed. One key aspect of egg activation is the cytoplasmic polyadenylation of certain maternal mRNAs to permit or enhance their translation. wispy (wisp) maternal-effect mutations in Drosophila block development during the egg-to-embryo transition. We show here that the wisp gene encodes a member of the GLD-2 family of cytoplasmic poly(A) polymerases (PAPs). The WISP protein is required for poly(A) tail elongation of bicoid, Toll, and torso mRNAs upon egg activation. In Drosophila, WISP and Smaug (SMG) have previously been reported to be required to trigger the destabilization of maternal mRNAs during egg activation. SMG is the major regulator of this activity. We report here that SMG is still translated in activated eggs from wisp mutant mothers, indicating that WISP does not regulate mRNA stability by controlling the translation of smg mRNA. We have also analyzed in detail the very early developmental arrest associated with wisp mutations. Pronuclear migration does not occur in activated eggs laid by wisp mutant females. Finally, we find that WISP function is also needed during oogenesis to regulate the poly(A) tail length of dmos during oocyte maturation and to maintain a high level of active (phospho-) mitogen-activated protein kinases (MAPKs). 相似文献
18.
Chromosomes segregate at mitosis along microtubules attached to the kinetochore, an organelle that assembles at the centromere. Despite major advances in defining molecular components of the yeast segregation apparatus, including discrete centromere sequences and proteins of the kinetochore, relatively little is known of corresponding elements in more complex eukaryotes. We show here that human CENP-C, a human autoantigen previously localized to the kinetochore, assembles at centromeres of divergent species, and that the specificity of this targeting is maintained by an inherent destruction mechanism that prevents the accumulation of CENP-C and toxicity of mistargeted CENP-C. The N-terminus of CENP-C is not only required for CENP-C destruction but renders unstable proteins that otherwise possess long half-lives. The conserved targeting of CENP-C is underscored by the discovery of significant homology between regions of CENP-C and Mif2, a protein of Saccharomyces cerevisiae required for the correct segregation of chromosomes. Mutations in the Mif2 homology domain of CENP-C impair the ability of CENP-C to assemble at the kinetochore. Together, these data indicate that essential elements of the chromosome segregation apparatus are conserved in eukaryotes. 相似文献
19.