Visualisation of ribosomes in Drosophila axons using Ribo-BiFC

Abstract

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.

The Zinc-BED Transcription Factor Bedwarfed Promotes Proportional Dendritic Growth and Branching through Transcriptional and Translational Regulation in Drosophila

“…Given the complexity of dendritic arbors, this is achieved through local protein translation at distal dendrites and axons [ 59 ]. The presence of ribosomes in dendrites and axons has long been established [ 59 , 60 , 61 ]. Studies have also demonstrated that activity-dependent local protein translation leads to the remodeling of the actin cytoskeleton which in turn modulates dendritic spine morphology [ 62 ].…”

The Zinc-BED transcription factor Bedwarfed promotes proportional dendritic growth and branching through transcriptional and translational regulation inDrosophila

“…Given the complexity of dendritic arbors, this is achieved through local protein translation at distal dendrites and axons (Holt, Martin and Schuman, 2019). The presence of ribosomes in dendrites and axons has long been established (Holt, Martin and Schuman, 2019; Singh et al ., 2019; Lottes and Cox, 2020). Studies have also demonstrated that activity dependent local protein translation leads to remodeling of the actin cytoskeleton which in turn modulates dendritic spine morphology (Nakahata and Yasuda, 2018).…”

The cap epitranscriptome: Early directions to a complex life as mRNA

“…In the absence of cOMe in Drosophila, both the CBC and EJC are reduced at synapses indicating that cOMe provides a mark for certain mRNAs to be transported in an untranslated state to synapses for local translation. [14,71] Intriguingly, CMTr2 targets in Drosophila are enriched in transcripts also targeted by FMR1, the homologue of human FMRP which has been shown to regulate protein synthesis. [14,72] In fact, the translational repressive role of FMRP in arresting ribosomes on mRNAs is consistent with a role of cOMe in directing untranslated mRNAs to synapses.…”

“…In the absence of cOMe in Drosophila , both the CBC and EJC are reduced at synapses indicating that cOMe provides a mark for certain mRNAs to be transported in an untranslated state to synapses for local translation. [ 14,71 ]…”