Finally, Zijian applied RiboExM to hESC-iNeurons. Others have shown that most translation in distal neurites is done by monosomes. But upon treatment w/ BDNF and ACEA, which enhance local translation in distal neurites, RiboExM picked up increased abundance of polysomes. (16/18)

By tagging Rps25 and a stoichiometric 40S protein, Zijian used RiboExM to image these “specialized” Rps25-lacking ribosomes at the OMM! And because our lab had previously shown differential binding of Rps25-lacking ribosomes to certain mRNAs… (14/18)

When we knocked down Lsg1 and used ALIBi to capture ER ribosome-associated mRNAs, we were intrigued to see that the association of specific mRNAs with ER ribosomes was disproportionately affected–for example, Vamp3 and Robo4, whose protein levels also change accordingly. (10/18)

Using ALIBi to affinity purify ER ribosomes for TMT-MS, we found that 60S ribosomal proteins were relatively abundant, which corroborated Zijian’s result. A few other proteins were also enriched in ER translational machinery compared to cytosolic translational machinery… (8/18)

Oddly enough, I’d just come to a similar conclusion using a tool that I’d developed with @rna-bound.bsky.social and Kelsie Wysong: ALIBi, an optogenetically activated split “BirA” biotin ligase that enabled us to proximity biotinylate ribosomes at an organelle of interest. (7/18)

Across several benchmarking experiments, Zijian’s image processing pipeline correctly identified individual free subunits, monosomes, and polysomes based on the size, intensity, and colocalization of immunofluorescent signals coming from the tagged 40S and 60S proteins. (5/18)

In pursuit of this question, my labmate Zijian Zhang with Yunhao Bai developed RiboExM, an expansion microscopy technique using tagged ribosomal proteins to visualize single 40S and 60S ribosome subunits at ~25 nm effective resolution–on a confocal 🔬! (4/18)

Belated post! If you haven’t seen our recent work www.science.org/doi/10.1126/... , here’s a 🧵 highlighting the two techniques we developed to study subcellular populations of mammalian ribosomes and some cool findings these tools helped us uncover: (1/18)