The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.
The localization of oskar (osk) RNA to the posterior pole of the developing fruit fly (Drosophila) oocyte induces the assembly of pole plasm, causing development of the abdomen and germ line. Failure to localize oskar RNA results in embryos that lack abdomen and germ cells. Conversely, mis-targeting of oskar RNA to the anterior of the oocyte causes formation of ectopic abdomen and germ cells at the anterior pole. Maternal mutants that have reduced pole plasm activity produce sterile adults with normal abdominal development, suggesting that germ cells are more sensitive than abdomen to defects in pole plasm assembly. Thus mutations in genes that reduce oskar RNA localization or activity can be recovered as viable sterile adults. In a screen for mutants defective in germ cell formation, we isolated nine alleles of the tropomyosin II gene. Here we show that mutations in tropomyosin II (TmII) virtually abolish oskar RNA localization to the posterior pole, suggesting an involvement of the actin network in oskar RNA localization.
Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. We have identified a centriole-containing centrosome close to the oocyte nucleus. Remarkably, the centrosomal components, ␥-tubulin and Drosophila pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. We further report that the MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. We propose that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization.
Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz), which encodes a homeodomain protein (Ftz), is required zygotically for the formation of alternate segments in the developing embryo. Here we show that the orphan nuclear receptor alphaFtz-F1 (ref. 3), which is deposited in the egg during oogenesis, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.
We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not latestage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until ®ssion.
Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia-infected Drosophila cell line and genome-wide RNA interference (RNAi) screening to identify host factors that influence Wolbachia titer. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. Host gene knockdowns that reduced Wolbachia titer spanned a broad array of biological pathways including genes that influenced mitochondrial function and lipid metabolism. In addition, knockdown of seven genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte. The presence of Wolbachia in either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance of Wolbachia titer relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated that Wolbachia is intimately associated with the host ER and dramatically alters the morphology of this organelle. Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia's potent ability to prevent RNA virus replication.
The Drosophila oocyte is a highly polarized cell. Secretion occurs towards restricted neighboring cells and asymmetric transport controls the localization of several mRNAs to distinct cortical compartments. Here, we describe a role for the Drosophila ortholog of the Rab6 GTPase, Drab6, in establishing cell polarity during oogenesis. We found that Drab6 localizes to Golgi and Golgi-derived membranes and interacts with BicD. We also provide evidence that Drab6 and BicD function together to ensure the correct delivery of secretory pathway components, such as the TGF␣ homolog Gurken, to the plasma membrane. Moreover, in the absence of Drab6, osk mRNA localization and the organization of microtubule plus-ends at the posterior of the oocyte were both severely affected. Our results point to a possible connection between Rab protein-mediated secretion, organization of the cytoskeleton and mRNA transport.
Microtubules (MTs) are essential for many cell features, such as polarity, motility, shape, and vesicle trafficking. Therefore, in a multicellular organism, their organization differs between cell types and during development; however, the control of this process remains elusive. Here, we show that during Drosophila tracheal morphogenesis, MT reorganization is coupled to relocalization of the microtubule organizing centers (MTOC) components from the centrosome to the apical cell domain from where MTs then grow. We reveal that this process is controlled by the trachealess patterning gene in a two-step mechanism. MTOC components are first released from the centrosome by the activity of the MT-severing protein Spastin, and then anchored apically through the transmembrane protein Piopio. We further show that these changes are essential for tracheal development, thus stressing the functional relevance of MT reorganization for morphogenesis.
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