Blocked endocytotic uptake by the oocyte causes accumulation of vitellogenins in the haemolymph of the female-sterile mutants quitPX61 and stand stillPS34 of Drosophila
ABSTRACT The developmental lesions in two female-sterile mutants, quitPX61 (qui) and stand stillPS34 (stil), of Drosophila have been analysed. Previtellogenic development is normal in mutant qui ovarioles but, during vitellogenic stages, only small quantities of yolk accumulate in the oocyte. The nurse-cell cytoplasm does not stream into the oocyte. However, the follicle cells continue their developmental program and synthesize an excessive quantity of eggshell material. In the mutant stil, the oocyte remains small and contains only a fraction of the yolk proteins present in wild-type follicles. Histological and ultrastructural observations and the failure to incorporate trypan blue indicate that the yolk proteins present in the mutant follicles are neither derived from the fat body nor from the follicle cells. Since, in both mutants, the uptake mechanism of vitellogenin is affected, the 3 polypeptides accumulate in the haemolymph (in stil, the protein concentration is up to 4 times higher than in wild-type females) and the haemolymph volume increases. Reciprocal transplantations of ovarioles show that the developmental lesions in both mutants are ovary-autonomous. Furthermore, genetic chimeras of stil show that the activity of the stil gene is required in the germline cells and not in the somatic tissues.
- SourceAvailable from: Olga Pekar
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- "Interestingly, other fly mutants defective in endocytosis also display male or female sterility, and the latter is associated with abnormal oogenesis, e.g. yolkless, quitPK61, stillPS34, Merlin and clathrinheavy-chain 4 (Bazinet et al., 1993; DiMario and Mahowald, 1987; Dorogova et al., 2008; Gutzeit and Arendt, 1994), and mutants in the C. elegans EHD ortholog rme-1 failed to recycle yolk receptors (Grant et al., 2001). These results highlight the importance of Past1, and endocytosis, in development and maturation of the germlines. "
ABSTRACT: Endocytosis, which is a key process in eukaryotic cells, has a central role in maintaining cellular homeostasis, nutrient uptake, development and downregulation of signal transduction. This complex process depends on several protein-protein interactions mediated by specific modules. One such module is the EH domain. The EH-domain-containing proteins comprise a family that includes four vertebrate members (EHD1-EHD4) and one Drosophila ortholog, Past1. We used Drosophila as a model to understand the physiological role of this family of proteins. We observed that the two predicted Past1 transcripts are differentially expressed both temporally and spatially during the life cycle of the fly. Endogenous Past1 as well as Past1A and Past1B, expressed from plasmids, were localized mainly to the membrane of Drosophila-derived cells. We generated mutants in the Past1 gene by excising a P-element inserted in it. The Past1 mutants reached adulthood but died precociously. They were temperature sensitive and infertile because of lesions in the reproductive system. Garland cells that originated from Past1 mutants exhibited a marked decrease in their ability to endocytose fluorescently labeled avidin. Genetic interaction was found between Past1 and members of the Notch signaling pathway, suggesting a role for Past1 in this developmentally crucial signaling pathway.Journal of Cell Science 02/2009; 122(Pt 4):471-80. DOI:10.1242/jcs.038521 · 5.43 Impact Factor
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- "For live imaging, 0.4% Trypan Blue dye in Graces insect medium was injected into the females abdomen. The dye was endocytosed with the yolk by the oocytes and labels late endosomes containing yolk granules (Danilchik and Denegre, 1991; Gutzeit and Arndt, 1994). After 2 h incubation, the ovaries were dissected under halocarbon oil and covered with a YSI Do membrane (Yellow Springs Instrument, Ohio) for live imaging. "
ABSTRACT: The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.Mechanisms of Development 02/2008; 125(1-2):142-52. DOI:10.1016/j.mod.2007.09.008 · 2.44 Impact Factor
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- "For most endosome movies, 1 mg/ml Trypan Blue dye was injected into female fly abdomens and allowed to incubate for 2-7 hours. The dye, endocytosed with yolk by the oocyte, served as a bright fluorescence marker to make yolk granules more visible (B.-J.C., unpublished) (Danilchik and Denegre, 1991; Gutzeit and Arendt, 1994). Ovaries were dissected under halocarbon oil as described (Theurkauf, 1994b; Theurkauf and Hazelrigg, 1998). "
ABSTRACT: Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some large eukaryotic cells. In Drosophila oocytes there are two forms of microtubule-based streaming. Slow, poorly ordered streaming occurs during stages 8-10A, while pattern formation determinants such as oskar mRNA are being localized and anchored at specific sites on the cortex. Then fast well-ordered streaming begins during stage 10B, just before nurse cell cytoplasm is dumped into the oocyte. We report that the plus-end-directed microtubule motor kinesin-1 is required for all streaming and is constitutively capable of driving fast streaming. Khc mutations that reduce the velocity of kinesin-1 transport in vitro blocked streaming yet still supported posterior localization of oskar mRNA, suggesting that streaming is not essential for the oskar localization mechanism. Inhibitory antibodies indicated that the minus-end-directed motor dynein is required to prevent premature fast streaming, suggesting that slow streaming is the product of a novel dynein-kinesin competition. As F-actin and some associated proteins are also required to prevent premature fast streaming, our observations support a model in which the actin cytoskeleton triggers the shift from slow to fast streaming by inhibiting dynein. This allows a cooperative self-amplifying loop of plus-end-directed organelle motion and parallel microtubule orientation that drives vigorous streaming currents and thorough mixing of oocyte and nurse-cell cytoplasm.Development 09/2005; 132(16):3743-52. DOI:10.1242/dev.01956 · 6.46 Impact Factor