mRNA localization in the Drosophila germline.

Localization and the associated translational control of mRNA is a well established mechanism for segregating cellular protein expression. Drosophila has been instrumental in deciphering the prevailing mechanisms of mRNA localization and regulation. This review will discuss the diverse roles of mRNA localization in the Drosophila germline, the cis-elements and cellular components regulating localization and the superimposition of translational regulatory mechanisms. Despite a history of discovery, there are still many fundamental questions regarding mRNA localization that remain unanswered. Take home messages, outstanding questions and future approaches that will likely lead to resolving these unknowns in the future are summarized at the end.This work was supported by the University of Cambridge, ISSF to T.T.W. [grant number 097814].This is the final version of the article. It first appeared from Taylor & Francis via http://dx.doi.org/10.4161/rna.3609

Subcellular mRNA localisation at a glance.

mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms.This work was supported by a Wellcome Trust Senior Research Fellowship to I.D. supporting R.M.P. [grant number: 096144], a studentship from the Wellcome Trust to A.D. [grant number: 097304], the University of Cambridge, ISSF to T.T.W. [grant number 097814].This is the final version of the article. It first appeared from the Company of Biologists via http://dx.doi.org/10.1242/jcs.11427

Localized Translation of gurken/TGF-α mRNA during Axis Specification Is Controlled by Access to Orb/CPEB on Processing Bodies.

In Drosophila oocytes, gurken/TGF-α mRNA is essential for establishing the future embryonic axes. gurken remains translationally silent during transport from its point of synthesis in nurse cells to its final destination in the oocyte, where it associates with the edge of processing bodies. Here we show that, in nurse cells, gurken is kept translationally silent by the lack of sufficient Orb/CPEB, its translational activator. Processing bodies in nurse cells have a similar protein complement and ultrastructure to those in the oocyte, but they markedly less Orb and do not associate with gurken mRNA. Ectopic expression of Orb in nurse cells at levels similar to the wild-type oocyte dorso-anterior corner at mid-oogenesis is sufficient to cause gurken mRNA to associate with processing bodies and translate prematurely. We propose that controlling the spatial distribution of translational activators is a fundamental mechanism for regulating localized translation.This work was supported by a studentship from the Wellcome Trust (grant 097304 to A.D.), a Wellcome Trust Senior Research Fellowship (grant 096144 to I.D and supporting R.M.P), the University of Cambridge, ISSF (grant 097814 to T.T.W), and Wellcome Trust Strategic Awards 091911 and 107457 supporting advanced microscopy at Micron Oxford (http://micronoxford.com).This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.celrep.2016.02.03

Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP.

Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.PTC was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (105653/Z/14/Z) and by an Issac Newton Trust Research Grant from the University of Cambridge awarded to TTW (RG78799). AW, ZN and JWR were supported by a Senior Investigator Award awarded to JWR and funded by the Wellcome Trust (104575/Z/14/Z). The OMX microscope used in this study is part of the Oxford Micron Advanced Bioimaging Unit supported by a Wellcome Trust Strategic Award (091911).This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.0848

A single and rapid calcium wave at egg activation in Drosophila.

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca(2+) concentration in mammals, ascidians and polychaete worms and a single Ca(2+) peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca(2+) levels occur. Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca(2+) wave requires an intact actin cytoskeleton and an increase in intracellular Ca(2+) can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca(2+) at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca(2+) channels; a single Ca(2+) wave then propagates in an actin dependent manner; this Ca(2+) wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.This work was supported by the University of Cambridge, ISSF to T.T.W. [grant number 097814]; and Wellcome Trust Senior Research Fellowship to I.D. [grant number 096144].This is the final version of the article. It first appeared from the Company of Biologists via http://dx.doi.org/10.1242/bio.20141129

Osmolarity-regulated swelling initiates egg activation in Drosophila.

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na+ channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants

Private Sector Union Density and the Wage Premium: Past, Present, and Future

The rise and decline of private sector unionization were among the more important features of the U.S. labor market during the twentieth century. Following a dramatic spurt in unionization after passage of the depression-era National Labor Relations Act (NLRA) of 1935, union density peaked in the mid-1950s, and then began a continuous decline. At the end of the century, the percentage of private wage and salary workers who were union members was less than 10 percent, not greatly different from union density prior to the NLRA