Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos

Claire M. Lye 0 Huw W. Naylor 0 Be ne dicte Sanson 0 0 The Department of Physiology, Development and Neuroscience, University of Cambridge , Downing Street, Cambridge CB2 3DY, UK A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at midembryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis. - INTRODUCTION Fluorescent proteins have revolutionised our ability to observe proteins in live tissues. Single gene studies now routinely ectopically express fluorescently tagged versions of a given protein to identify its localisation. Beyond a single gene approach, efforts have been made in the past decade to fluorescently tag many proteins in parallel. In yeast, it was possible to tag 75% of endogenous proteins by inserting a GFP exon at the 3 end of open reading frames (Huh et al., 2003). Partly because of the lack of efficient homologous recombination, this feat is difficult to replicate in multicellular organisms, so large-scale production of fluorescently tagged proteins relies instead on transposon-mediated tagging. Transposons are modified to integrate an exon encoding a fluorescent protein at nearrandom locations into the genome. When the transposon inserts within an intron, this can result in a tagged protein expressed from its natural promoters. Screens for protein traps using the mobilisation of fluorescent exons were first carried out in Drosophila (Morin et al., 2001; Clyne et al., 2003; Buszczak et al., 2007; Quinones-Coello et al., 2007). These screens recovered both enhancer trap and protein trap lines, because the main transposable element used, the P-element, is biased towards insertion in sequences 5 to coding sequences. From these studies, over 449 true protein trap lines were generated, corresponding to the in-frame tagging of 226 unique genes with GFP (Aleksic et al., 2009). Outside Drosophila, large-scale tagging of full-length proteins with fluorescent exons to analyse subcellular localisation is developed in vertebrates such as zebrafish (Trinh and Fraser, 2013) and in the model plant Arabidopsis (Tanz et al., 2013). The accompanying paper reports the generation in Drosophila, of a collection of proteins tagged with YFP using new vectors based on piggyBac transposition to principally produce protein traps (Lowe et al., 2014). This new collection is composed of over 600 Cambridge Protein Trap Insertion (CPTI) lines, corresponding to just under 400 identified genes. The subcellular localisations of the CPTI lines have been characterised in many tissues by a consortium of UK groups and the information is centralized in the Flyprot website, www.flyprot.org (Lowe et al., 2014). In this paper, we aim to provide a further resource to the community by characterising the subcellular localisation of the complete CPTI collection of YFP-trap proteins in live Drosophila embryos. We had two main goals: to give clues to the function of uncharacterised proteins and to identify markers for organelles and subcellular regions. Such markers are still scarce in Drosophila but are crucial to conducting cell biology studies in live tissues, embryonic or other. To characterise the subcellular localisations, we imaged cellularising embryos (stage 5) because the cells are regularly arranged and larger than at other stages of development (Mazumdar and Mazumdar, 2002; Lecuit, 2004). For the protein traps localising at the plasma membrane or cortex, we expanded our characterisation to stages 6 to 10, to include epithelial morphogenesis during axis extension and early segmentation (Lye and Sanson, 2011). Because the tagged proteins are expressed at endogenous levels, we used spinning disk confocal microscopy coupled with an EM-CCD camera to increase the sensitivity of detection. This paper systematically identifies the subcellular localisation of hundreds of Drosophila proteins and provides a comprehensive resource for cell biology studies. and 521 (93%) at stage 15 and later (late embryogenesis) (supplementary material Table S1). Most of the lines are expressed in all tissues without obvious patterns at stage 5 and 11. The main exception are lines showing metameric patterns: at stage 5, two insertions in the Teneurin homologue Ten-m are expressed in stripes (supplementary material Fig. S1A); at stage 11, 31 lines show a metameric pattern, including genes known to be segmentally expressed such as: frizzled 2, the glypicans dally and dally-like, semaphorins 1b and 2a and netrin A and B, and genes for which this was previously unknown such as arginine kinase (supplementary material Fig. S1B). At stage 15 or later, when the larval organs have formed, we found more patterns (supplementary material Fig. S1D-H), the most frequent being expression in the central nervous system (137 lines, 26%, supplementary material Table S1), but here again the tagged proteins are in majority expressed in most tissues. All expression pattern information is summarised in supplementary material Table S1 and some notable patterns are shown in supplementary material Fig. S1 and the accompanying paper (Lowe et al., 2014). We focused on the 415 lines showing expression at stage 5 to determine their subcellular localisation and found that 258 YFP-trap proteins localised in the cytoplasm, 130 in the nucleus, 75 at the membrane and/or cortex and 13 in the extracellular space (Fig. 1 and supplementary material Table S1). CPTI lines localising in the nucleus The 130 nuclear YFP-trap protei (...truncated)