The distribution of assembled, and potentially translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-subunit junctions in the assembled 80S ribosome. Here we describe transgenes expressing two novel RP pairs tagged with Venus-based BiFC fragments that considerably increase the sensitivity of this technique we termed Ribo-BiFC. This improved method should provide a convenient way of monitoring the local distribution of ribosomes in most Drosophila cells and we suggest that it could be implemented in other organisms. We visualised 80S ribosomes in different neurons, particularly photoreceptors in the larva, pupa and adult brain. Assembled ribosomes are most abundant in the various neuronal cell bodies, but they are also present along the full length of axons. They are concentrated in growth cones of developing photoreceptors and are apparent at the terminals of mature larval photoreceptors targeting the larval optical neuropil. Surprisingly, there is relatively less puromycin incorporation in the distal portion of axons in the larval optic stalk, suggesting that some of the ribosomes that have initiated translation may not be engaged in elongation in growing axons. This article has an associated First Person interview with the first author of the paper . INTRODUCTION Ribosomes are ubiquitous molecular machines that translate gene sequences into the thousands of different proteins that make and operate every organism, so ribosomal components are some of the most abundant and evolutionarily conserved macromolecular constituents of cells. Each ribosome is made up of two complex ribonucleoprotein subunits – 40S and 60S in eukaryotes – and the joining of these into 80S functional ribosomes is tightly regulated. Even when cells are replete with ribosome subunits there are physiological situations (e.g. during nutrient deprivation or other cell stresses) when relatively few are assembled into protein-translating ribosomes ( Hinnebusch, 2014 , 2017 ). The joining of ribosomal subunits is a multi-step process, requiring the coordinated activity of several initiation factors, occurring each time that translation of an mRNA is initiated ( Hinnebusch, 2017 ; Jackson et al., 2010 ). In eukaryotes, the first step is activation of the 40S subunit, which starts with its loading with methionine initiator tRNA (tRNAi met ). The resulting pre-initiation complex, typically guided by an interaction with the eukaryotic translation initiation factor eIF4G which is bound to the 5′ end cap-associated eIF4E, then attaches to the mRNA and scans its 5′UTR until the initiation codon is recognised by base pairing between the anticodon of tRNAi met and an AUG start codon ( Kozak, 1989 ). Once tRNAi met is base-paired with the AUG and is precisely placed in the peptidyl site on the 40S subunit, the 60S subunit is recruited. The assembled 80S ribosome translocates along the mRNA, catalysing protein synthesis until it reaches a stop codon. It then dissociates and the free subunits become available for new rounds of translation ( Dever and Green, 2012 ). We have used the Bimolecular Fluorescence Complementation (BiFC) technique to visualise assembled ribosomes in Drosophila cells. This is a technique that allows direct detection of diverse types of protein–protein interactions in living cells ( Hu et al., 2002 ; Kerppola, 2008 ). To apply this for ribosomes, one selects a pair of RPs on the surface of the individual subunits that only come into close and stable contact when the 80S ribosome assembles. These RPs are then tagged with functionally complementary halves of a fluorescent protein. The two non-functional halves of the fluorescent protein only make a stable contact when the 80S ribosome is assembled at initiation, so emission of fluorescence reports that translation initiation has occurred ( Al-Jubran et al., 2013 ). Initially, when we were developing the BiFC-based ribosome visualisation technique, several pairs of RPs were tagged with either the N-terminal half (YN) or the C-terminal half (YC) of Yellow Fluorescent Protein (YFP). These were co-expressed in Drosophila S2 cells and only those pairs that come together when the 80S ribosome assembles gave rise to ribosomal fluorescence ( Al-Jubran et al., 2013 ). Moreover, the fluorescence was enhanced by translation elongation inhibitors that stabilise the 80S, and was reduced by initiation inhibitors ( Al-Jubran et al., 2013 ). We then designed transgenic flies encoding one such adjacent pair of RPs under UAS regulation (RpS18[uS13]-YN and RpL11[uL5]-YC) – the names in brackets follow a newer system of naming ribosomal proteins, the prefix ‘u’ (for universal) indicates the protein is conserved in all domains of life ( Ban et al., 2014 ). Here we used the Drosophila nomenclature of our previous study to avoid confusion; however, both names are given when a protein is first mentioned in the text or in Fig. 1 . When these were expressed in salivary glands, a translationally very active tissue that secretes copious amounts of glue proteins ( Andrew et al., 2000 ; Beckendorf and Kafatos, 1976 ), the tissue showed an intense 80S ribosomal fluorescence signal ( Al-Jubran et al., 2013 ). Download powerpoint Fig. 1. Ribo-BiFC visualisation of 80S ribosomes in photoreceptors. (A) Model of the Drosophila 80S ribosome with the two BiFC tagged RP pairs on the small and large subunits highlighted: RpS18/RpL11 [uS13/uL5] and RpS6/RpL24 [eS6/eL24]; the image was generated with PyMOL using the published high-resolution Drosophila 80S structure, PDB file 4V6W ( Anger et al., 2013 ). RpS18 and RpS6 on the 40S are indicated in pale green, RpL11 and RpL24 on the 60S in pale red. (B) Diagram of the Bimolecular Fluorescence Complementation (BiFC) constructs with spacer sequences indicated, the VN and VC BiFC-compatible fragments of Venus fluorescent protein are shown as yellow boxes. (C) Schematic of the eye disc connected by the optic stalk to the brain optic lobe of Drosophila larva, showing the photoreceptor cell bodies in the retina (yellow) and their axonal projections into the brain (blue). The photoreceptors R1-R6 project their axons to the lamina region of the brain, while R7 and R8 project their axons further inside to the medulla underneath. The star shapes (red) at the end of axons indicate growth cones. Bolwig's nerve (BN, orange) passes through the lamina/medulla and innervates the larval optic neuropil in each lobe. (D) Confocal microscopy images showing the BiFC signal produced by different transgene combinations expressed in the developing photoreceptors using GMR-GAL4>RpS18VN/RpL11VC (panel I), > RpS6VN/RpL24VC (panel II) and as comparison the YFP-based > RpS18YN/RpL11YC (panel III). (E) Visualisation of the RpS18VN-RpL11VC (yellow, panel I) in tissues where the developing photoreceptors are immunostained by mAb24B10 (magenta, panel II), their colocalisation is shown in the merged image (panel III); the RpS18VN-RpL11VC BiFC signal is shown in green instead of yellow in the merged image for better contrast. Insets show magnified views of growth cone region. Labels refer to: ED, eye disc; OS, optic stalk; L, lamina; LP, lamina plexus; M, medulla; GC, growth cone; BN, Bolwig's nerve. We investigated whether a similar approach could track ribosomes in axons and synapses, and hence serve as a tool for studies of localised translation in the Drosophila nervous system ( Glock et al., 2017 ; Holt et al., 2019 ; Kim and Jung, 2015 ). Using the available transgenic flies expressing RpS18-YN and RpL11-YC, however, we were only able to detect weak 80S ribosomal fluorescence in the cell bodies of some large neurons. So we sought to improve the sensitivity of this technique we termed Ribo-BiFC. Here we describe an improved version that employs transgenic flies expressing either of two novel RP pairs (RpS18/RpL11 and RpS6[eS6]/RpL24[eL24]) – the prefix ‘e’ is for eukaryotic ribosomal proteins without bacterial homologs – that are tagged with BiFC fragments of Venus fluorescent protein ( Hudry et al., 2011 ). These Venus-based reporters greatly improved the sensitivity of the method and revealed clear ribosome signals along the full length of axons and at the axon terminals of both developing and mature neurons. In eye photoreceptor axons, which we examined in most detail, intense ribosome signals are particularly apparent in their growth cones during larval and pupal development. We suggest that these Venus-tagged RP pairs can provide a useful research tool with which to monitor the subcellular localisation and trafficking of assembled ribosomes in most Drosophila cells and tissues. RESULTS BiFC-Venus-tagged 80S ribosomes can be detected in axons and growth cones of photoreceptor neurons The ribosomal protein pairs RpS18/RpL11 (uS13/uL5) and RpS6/RpL24 (eS6/eL24) span inter-subunit potential contact points, on the surface of the ‘head’ and the ‘foot’, respectively, of the 80S ribosome ( Fig. 1 A). We generated UAS-driven Drosophila transgenes encoding these proteins that were tagged with complementing fragments of Venus fluorescent protein corresponding to the N-terminal domain (VN, 1-173 aa) and C-terminal domain (VC, 155-238 aa) ( Fig. 1 B). These yield a brighter and more specific BiFC interaction than YFP constructs ( Hudry et al., 2011 ). Moreover, our characterisation in S2 cells indicated that fluorescence from the inter-subunit Venus BiFC complex might be more stable during translation elongation than the one from the corresponding YFP complex ( Al-Jubran et al., 2013 ). We tested the new transgenes in the Drosophila larval visual system, which is an excellent model for microscopic visualisation of the axonal projections of neurons. The eye is made up of about 750 ommatidia, each having eight photoreceptor neurons (the R-cells: R1-R8). R1–R6 axons project to a synaptic layer of the brain optic lobe termed the lamina plexus, and R7 and R8 axons pass through the lamina and end in a deeper brain region termed the medulla ( Fig. 1 C) ( Mencarelli and Pichaud, 2015 ). Expression of either of our BiFC-Venus RP pairs in developing eye by using the GMR-GAL4 driver ( Freeman, 1996 ) results in a strong signal. Within the growing photoreceptors, this is brightest in the cell bodies located in the developing eye, but it is apparent along the entire length of the photoreceptor axons, both in R1-R6 (ending in the lamina) and in R7 and R8 (ending in the medulla) ( Fig. 1 D; panel I, RpS18/RpL11; Panel II, RpS6/RpL24). The RpS18/RpL11 pair was used in the experiments described below. The signal from the Venus-based reporters is much stronger than from the previous YFP-based RpS18/RpL11 transgene pair, which was only apparent in the cell bodies and proximal regions of the axons ( Fig. 1 D, panel III). This was despite the fact that substantial amounts of conventional GFP- or RFP-tagged versions of RpS18 and RpL11, which report the distributions of free ribosomal subunits as well as assembled ribosomes, are abundantly present throughout the axons when expressed with GMR-GAL4 ( Fig. S1 A). Although the expression levels of the tagged proteins could not be directly assessed in photoreceptors, as these make up only a small fraction of the cells in the tissue, our previous western blotting analysis of salivary glands indicates that these tagged proteins are at a substantially lower level than the endogenous counterparts, even when expressed in salivary glands with a strong GAL4 driver that results in a BiFC signal much brighter than that detected in the photoreceptors ( Al-Jubran et al., 2013 ). Moreover, there is no evidence of proteins being considerably toxic when expressed with GMR-GAL4 since the eye develops as expected, except for a very mild glossy eye phenotype ( Fig. S2 B). The neuronal distribution of the signal is confirmed by immunostaining with mAb24B10, which specifically recognises chaoptin, a GPI-linked cell surface glycoprotein that is present only on photoreceptor neurons and their axons ( Fig. 1 E) ( Reinke et al., 1988 ; Zipursky et al., 1985 ). There is also intense 80S ribosome signal in enlarged foci at the tips of the R7 and R8 axons in the medulla region ( Fig. 1 E), which is probably in growth cones ( Prokop and Meinertzhagen, 2006 ). Strong signals in photoreceptor growth cones are also apparent during pupal development ( Fig. S2 A). By comparing the pattern of the 80S signal with that of chaoptin, which mostly stains the periphery of the growth cones (compare insets in Fig. 1 E), it is clear that the most intense ribosome signal is inside the growth cones. Comparison of the 80S signal with that of mCD8-GFP, another plasma membrane marker ( Lee and Luo, 1999 ), which is evenly distributed along the axon ( Fig. S1 B, panel I versus panel II), also supports the conclusion that the whole interior of the growth cones must be replete with 80S ribosomes. We also found signals in the axons of functional adult fly photoreceptors ( Fig. S2 A). Although the Ribo-BiFC signal is weaker than in developing photoreceptor axons, the reduction is probably a consequence of reduced expression of the GMR-GAL4 expression in adult flies, as this is also apparent when expressing mCD8-GFP alone (unpublished data). To test further whether ribosomes are present in the axons of mature neurons, we examined the Bolwig's organ. This is the organ of sight/light-sensation of the larva. It consists of a bilateral bundle of 12 photoreceptors near the mouth-hook at the anterior of the animal, which projects their axons in a nerve that joins with the optical stalk of the eye-disc before entering the brain optic lobe and terminates in a distinctive small region of the medulla representing the larval optical neuropil in each brain hemisphere ( Fig. 1 C) ( Hofbauer and Campos-Ortega, 1990 ; Larderet et al., 2017 ). Within the neuropil, synapses are formed with the lateral neurons required for the circadian behaviour of the larva as well as the other neurons comprising the larval optical system ( Helfrich-Förster et al., 2002 ; Keene et al., 2011 ; Larderet et al., 2017 ). We detected clear Ribo-BiFC signals along the Bolwig's nerve and at its terminals in larval optical neuropil ( Fig. 2 A).
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