A novel role for dp115 in the organization of tER sites in Drosophila

Here, we describe that depletion of the Drosophila homologue of p115 (dp115) by RNA interference in Drosophila S2 cells led to important morphological changes in the Golgi stack morphology and the transitional ER (tER) organization. Using conventional and immunoelectron microscopy and confocal immunofluorescence microscopy, we show that Golgi stacks were converted into clusters of vesicles and tubules, and that the tERs (marked by Sec23p) lost their focused organization and were now dispersed throughout the cytoplasm. However, we found that this morphologically altered exocytic pathway was nevertheless largely competent in anterograde protein transport using two different assays. The effects were specific for dp115. Depletion of the Drosophila homologues of GM130 and syntaxin 5 (dSed5p) did not lead to an effect on the tER organization, though the Golgi stacks were greatly vesiculated in the cells depleted of dSed5p. Taken together, these studies suggest that dp115 could be implicated in the architecture of both the Golgi stacks and the tER sites

Ecdysone Triggers the Expression of Golgi Genes in Drosophila Imaginal Discs via Broad-Complex

AbstractOne of the most significant morphogenic events in the development of Drosophila melanogaster is the elongation of imaginal discs during puparium formation. We have shown that this macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. We have shown that the fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks (V. Kondylis, S. E. Goulding, J. C. Dunne, and C. Rabouille, 2001, Mol. Biol. Cell, 12, 2308). Using mRNA in situ hybridisation, we show here that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, dsed5, and drab1) and downregulates the expression of others (such as dergic53, dβ'COP, anddrab6). We show that the transcription factor Broad-complex, itself an “early” ecdysone target, mediates this regulation. And we show that the ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing our understanding of the relationship between gene expression and organelle biogenesis

The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster

The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5E13) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5E13 oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless

Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

BACKGROUND: In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. RESULTS: To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for "more and smaller Golgi") upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. CONCLUSIONS: This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

<p>Abstract</p> <p>Background</p> <p>Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood.</p> <p>Results</p> <p>We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils.</p> <p>Conclusion</p> <p>Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.</p

KRS:A cut away from release in exosomes

Cancer cells often trigger an inflammatory process, which in some cases may be driven by the presence of lysyltRNA synthetase (KRS) in the medium. Kim et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201605118) now demonstrate that cleavage of the KRS by caspase-8 inside cells triggers its interaction with syntenin and its release in inflammatory exosomes

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

Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues