The R-SNARE Ykt6 is required for multiple events during oogenesis in Drosophila.

Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, we characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. We find that Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, we show that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface.

Distinct roles for hu li tai shao and swallow in cytoskeletal organization during Drosophila oogenesis.

BACKGROUND: Cytoskeletal organization is essential for localization of developmentally significant molecules during Drosophila oogenesis. Swallow (Swa) and an isoform of Hu li tai shao (Ovhts-RC) have been implicated in the organization of actin filaments in developing oocytes but their precise roles have been obscured by the dependence of hts RNA localization on swa function. The functional significance of hts RNA localization in the oocyte has not been established. RESULTS: In this study we examine Ovhts-RC distribution and cytoskeletal organization under conditions in which Swa protein and/or hts RNA localization are perturbed. We find Swa is required for overall actin organization and for the maintenance of a distinct subset of microtubules in the oocyte. hts RNA localization modulates the distribution of Ovhts-RC in the oocyte and, in turn, local actin filament proliferation. CONCLUSIONS: Our results support separate contributions of Swa and hts RNA localization to actin organization during oogenesis. Swa is crucial for the organization of actin networks that lead to the formation of a specialized microtubule population, while Ovhts-RC acts to modulate spatially restricted actin filament growth at the oocyte cortex. This suggests RNA localization can lead to modifications of both the actin and microtubule cytoskeletons at specific subcellular locales.

Live imaging of GFP-labeled proteins in Drosophila oocytes.

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes. The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent and provides a useful system for investigating cytoskeleton function at these stages. Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Microtubules, the ER and Exu: new associations revealed by analysis of mini spindles mutations.

During Drosophila oogenesis, organized microtubule networks coordinate the localization of specific RNAs, the positioning of the oocyte nucleus, and ooplasmic streaming events. We used mutations in mini spindles (msps), a microtubule-associated protein, to disrupt microtubule function during mid- and late-oogenesis, and show that msps is required for these microtubule-based events. Since endoplasmic reticulum (ER) organization is influenced by microtubules in other systems, we hypothesized that using msps to alter microtubule dynamics might affect the structure and organization of the ER in nurse cells and the oocyte. ER organization was monitored using GFP-tagged versions of Reticulon-like1 and protein disulfide isomerase. Analyses of living cells indicate microtubule associations mediate the movement of ER components within the oocyte. Surprisingly, the distribution and behavior of tubular ER in the oocyte differs from general ER, suggesting these two compartments of the ER interact differently with microtubules. We find that the morphology of Exu particles is msps-dependent, and that Exu is specifically associated with tubular ER in msps mutants. Our results extend previous descriptions of sponge bodies and the fusome, suggesting both are manifestations of a dynamic structure that interacts with microtubules and persists throughout oogenesis.

Identification of hypomorphic and null alleles of swallow via molecular and phenotypic analyses.

The swallow gene of Drosophila is required for the localization of two messenger RNAs, bicoid and hu-li tai shao, to the anterior pole of oocytes during the later stages of oogenesis. In addition, swallow appears to play a role in early embryogenesis, as swallow mutant embryos have defects in early nuclear cleavage and migration. In an effort to identify regions of the Swallow protein that are essential for function, we have initiated a molecular characterization of seven existing alleles of swallow. All seven alleles have been sequenced, and comparison to wild-type swallow indicates that the seven alleles include single amino acid substitutions that identify critical residues, as well as lesions that result in truncated proteins. Western blots using affinity-purified antibodies agree well with the DNA sequence data, and identify a probable null protein. In order to determine the extent to which each allele affects swallow function, females homozygous or hemizygous for each allele were tested for the range and abundance of (1) RNA localization defects, and (2) embryonic cuticular defects. Swallow alleles can be grouped into two categories: those that retain partial function, and those indistinguishable from the putative null allele. Some swallow mutant alleles partially rescue the dominant female sterility of mutations in the atypical 67C alpha-tubulin gene, supporting other studies that suggest a link between RNA localization and the microtubule cytoskeleton.