Drosophila Cyclin J is a mitotically stable Cdk1 partner without essential functions

Cyclin J is a cyclin family member that appears to have evolved before the metazoan radiation. Its evolutionary conservation argues for an important role but functional characterizations of Cyclin J have remained very limited. In Drosophila, Cyclin J is expressed only in females. Using transgenic Drosophila lines expressing Cyclin J versions with N- or C-terminal GFP extensions, we demonstrate that it is expressed exclusively in the germline. After low level expression in all nuclei within the germarium, it gets highly enriched in the germinal vesicle within the oocyte until stage 12 of oogenesis, followed by disappearance after germinal vesicle breakdown before the first meiotic division. Surprisingly, Cyclin J is not required for female fertility. Chromosome segregation during female meiosis, as well as the rapid early embryonic cell cycles after fertilization, occurs normally in the complete absence of Cyclin J. Cyclin J with EGFP fused at either N- or C-terminus binds to Cdk1 and not to Cdk2. However, in contrast to the other known Cdk1 partners, the A- and B-type cyclins, Cyclin J is not degraded during mitosis

Microtubule Polarity Predicts Direction of Egg Chamber Rotation in Drosophila.

Whole-tissue rotations have recently been recognized as a widespread morphogenetic process important for tissue elongation [1-4]. In Drosophila ovaries, elongation of the egg chamber involves a global rotation of the follicle epithelium along the anterior-posterior axis [5]. Individual egg chambers rotate either in a clockwise or counterclockwise direction; however, how the symmetry of egg chambers is broken to allow rotation remains unknown. Here we show that at the basal side of follicle cells, microtubules are preferentially aligned perpendicular to the anterior-posterior axis of the egg chamber. Microtubule depolymerization stalls egg chamber rotation and egg chamber elongation. The preferential alignment of microtubules and egg chamber rotation depend on the atypical cadherin Fat2 and the planar polarized Fat2 localization depends on intact microtubules. Moreover, by tracking microtubule plus-end growth in vivo using EB1::GFP, we find that microtubules are highly polarized in the plane of the follicle epithelium. Polarization of microtubules precedes the onset of egg chamber rotation and predicts the direction of rotation. Our data suggest a feedback amplification mechanism between Fat2 localization and microtubule polarity involved in breaking symmetry and directing egg chamber rotation

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers.

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development. Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis. Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers' circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (>50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications

Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila.

Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, we studied a Drosophila strain with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. We also found changes in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, we analyzed posttranslational modification of H3S28 in an experimental heart failure model and observed increased H3S28 phosphorylation levels in HF hearts. Our data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure