In vivo proximity biotin ligation identifies the interactome of Egalitarian, a Dynein cargo adaptor

ABSTRACT Numerous motors of the Kinesin family contribute to plus-end-directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In selected cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. We used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be linked to Dynein in an Egalitarian-dependent manner. We therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. We also devised and validated a nanobody-based proximity biotinylation strategy that can be used to define the interactome of any GFP-tagged protein.

Acetylated microtubules are required for maintenance of the barrier between two adjacent tissues

Microtubule acetylation is found in populations of stable, long-lived microtubules, occurring on the conserved lysine 40 (K40) residue of α-tubulin, catalyzed by alpha-tubulin acetyltransferases (αTATs). K40 acetylation has been shown to stabilize microtubules via enhancing microtubule resilience against mechanical stress. Here we show that Drosophila CG17003/leaky (Lky), an αTAT, is required for proper oogenesis. We found that loss of lky disrupted the cell junction between germline cyst and follicle epithelial cells, adjacent cells that form an egg chamber. This resulted in leakage of germline contents into somatic follicle cells. The follicle cells that received germline-derived nanos gene product failed to maintain their cell fate, leading to an egg chamber fusion. The same phenotype was observed upon replacement of major α-tubulin84B K40 with α-tubulin84B K40A (non-acetylable tubulin), suggesting α-tubulin K40 acetylation is required for the boundary integrity of these two adjacent tissues. Taken together, this study provides the first in vivo function of tubulin acetylation in maintaining the integrity of a tissue barrier.

Cellular and Supracellular Planar Polarity: A Multiscale Cue to Elongate the Drosophila Egg Chamber

Tissue elongation is known to be controlled by oriented cell division, elongation, migration and rearrangement. While these cellular processes have been extensively studied, new emerging supracellular mechanisms driving tissue extension have recently been unveiled. Tissue rotation and actomyosin contractions have been shown to be key processes driving Drosophila egg chamber elongation. First, egg chamber rotation facilitates the dorsal-ventral alignment of the extracellular matrix and of the cell basal actin fibers. Both fiber-like structures form supracellular networks constraining the egg growth in a polarized fashion thus working as ‘molecular corsets’. Second, the supracellular actin fiber network, powered by myosin periodic oscillation, contracts anisotropically driving tissue extension along the egg anterior-posterior axis. During both processes, cellular and supracellular planar polarity provide a critical cue to control Drosophila egg chamber elongation. Here we review how different planar polarized networks are built, maintained and function at both cellular and supracellular levels in the Drosophila ovarian epithelium.

Orphan nuclear receptor ftz-f1 (NR5A3) promotes egg chamber survival in the Drosophila ovary

Gamete production in mammals and insects is controlled by cell signaling pathways that facilitate communication between germ cells and somatic cells. Nuclear receptor signaling is a key mediator of many aspects of reproduction, including gametogenesis. For example, the NR5A sub-family of nuclear receptors are essential for gonad development and sex steroid production in mammals. Despite the original identification of the NR5A sub-family in the model insect Drosophila melanogaster, it has been unclear whether Drosophila NR5A receptors directly control oocyte production. Ftz-f1 is expressed throughout the ovary, including in germline stem cells, germline cysts, and several populations of somatic cells. We demonstrate that ftz-f1 is required in follicle cells prior to stage 10 to promote egg chamber survival at the mid-oogenesis checkpoint. Our data suggest that egg chamber death in the absence of ftz-f1 is due, at least in part, to failure of follicle cells to exit the mitotic cell cycle or failure to accumulate oocyte-specific factors in the germline. Taken together, these results demonstrate that, as in mammals, the NR5A sub-family promotes maximal reproductive output in Drosophila. Our data underscore the importance of nuclear receptors in the control of reproduction and highlight the utility of Drosophila oogenesis as a key model for unraveling the complexity of nuclear receptor signaling in gametogenesis.

Buried treasure—marine turtles do not ‘disguise’ or ‘camouflage’ their nests but avoid them and create a decoy trail

After laying their eggs and refilling the egg chamber, sea turtles scatter sand extensively around the nest site. This is presumed to camouflage the nest, or optimize local conditions for egg development, but a consensus on its function is lacking. We quantified activity and mapped the movements of hawksbill ( Eretmochelys imbricata ) and leatherback ( Dermochelys coriacea ) turtles during sand-scattering. For leatherbacks, we also recorded activity at each sand-scattering position. For hawksbills, we recorded breathing rates during nesting as an indicator of metabolic investment and compared with published values for leatherbacks. Temporal and inferred metabolic investment in sand-scattering was substantial for both species. Neither species remained near the nest while sand-scattering, instead moving to several other positions to scatter sand, changing direction each time, progressively displacing themselves from the nest site. Movement patterns were highly diverse between individuals, but activity at each sand-scattering position changed little between completion of egg chamber refilling and return to the sea. Our findings are inconsistent with sand-scattering being to directly camouflage the nest, or primarily for modifying the nest-proximal environment. Instead, they are consistent with the construction of a series of dispersed decoy nests that may reduce the discovery of nests by predators.

Fusion of Drosophila oocytes with specified germline sister cells

ABSTRACTIn many animals, oocytes develop together with sister germline cells that pass products to the developing oocyte. In Drosophila, fifteen sister germline (nurse) cells in each egg chamber are known to apoptose by stage 12-13, but we discovered that two specific nurse cells that are juxtaposed to the oocyte are eliminated precociously at stage 10B. These nurse cells fuse with the oocyte and their nuclei extrude through an opening that forms in the oocyte. These nuclei extinguish in the ooplasm, and at stage 11, egg chambers have thirteen nucleated nurse cells and the plasma membrane of the oocyte is mostly restored. In infrequent egg chambers in which nurse cells are not eliminated, oocytes do not develop normally and are not fertilized. Precocious elimination is common to other Drosophila species. We conclude that nurse cells are distinguished by position and identity, and that nurse cell dissolution proceeds in two stages.