A subcellular map of translational machinery composition and regulation at the single-molecule level.

Millions of ribosomes are packed within mammalian cells, yet we lack tools to visualize them in toto and characterize their subcellular composition. In this study, we present ribosome expansion microscopy (RiboExM) to visualize individual ribosomes and an optogenetic proximity-labeling technique (ALIBi) to probe their composition. We generated a super-resolution ribosomal map, revealing subcellular translational hotspots and enrichment of 60S subunits near polysomes at the endoplasmic reticulum (ER). We found that Lsg1 tethers 60S to the ER and regulates translation of select proteins. Additionally, we discovered ribosome heterogeneity at mitochondria guiding translation of metabolism-related transcripts. Lastly, we visualized ribosomes in neurons, revealing a dynamic switch between monosomes and polysomes in neuronal translation. Together, these approaches enable exploration of ribosomal localization and composition at unprecedented resolution. Editor's summary: Mammalian cells are packed with millions of ribosomes, tiny machines that decode genetic information into proteins. Although they are essential for this decoding process, they are often viewed as identical particles with little regulatory influence. Zhang et al. developed techniques to observe single ribosomes and examine their composition in different cellular compartments. They found that a protein called Lsg1 is involved in recruiting ribosomes near the endoplasmic reticulum to produce membrane-embedded proteins. They also found specialized ribosomes near mitochondria for making metabolism-related proteins and remodeling of ribosomal populations in neuronal processes, revealing a new layer of gene control based on ribosome type and location. —Di Jiang INTRODUCTION: Ribosomes, the universal molecular machinery for protein synthesis, have been viewed as structurally and functionally uniform entities since their initial characterization over six decades ago. However, accumulating evidence suggests that ribosomes can contain distinct combinations of ribosomal proteins (RPs) and are regulated by ribosome-associated proteins (RAPs). These heterogeneous ribosomal subpopulations are hypothesized to play specialized roles in translational control, contributing to cellular protein homeostasis. However, the field has lacked tools to directly visualize individual ribosomes, characterize their molecular composition, and map their spatial distribution in cells. Understanding how ribosomal diversity impacts translation could unveil new layers of gene expression regulation. RATIONALE: To bridge this technical gap, we developed two synergistic approaches: ribosome expansion microscopy (RiboExM) and ALIBi (an optogenetic proximity labeling method). RiboExM couples physical expansion of cells with standard confocal microscopy to achieve subribosomal resolution, allowing direct visualization of distinct ribosomal populations and their spatial organization within mammalian cells. ALIBi enables biochemical isolation of ribosomes and associated RAPs and mRNAs from specific subcellular compartments, facilitating quantitative proteomics and transcriptomics analyses. Together, these methods offer complementary insights into ribosomal composition and its functional implications, enabling a comprehensive investigation of subcellular translational machinery at the single-molecule level. RESULTS: Using RiboExM, we constructed a single-molecule panorama of the translational machinery in mammalian cells. This map revealed distinctive spatial patterns: Whereas 40S subunits were distributed more evenly in the cytoplasm, 60S subunits clustered near polysomes and were enriched at the endoplasmic reticulum (ER). ALIBi confirmed the selective enrichment of 60S subunits at the ER and also revealed RAPs that were enriched at the ER, including the presumed ribosome biogenesis factor Lsg1. Knocking down Lsg1 disrupted 60S enrichment at the ER and altered translation of specific proteins, revealing a mechanism of translational control independent of ER stress. We also applied ALIBi to examine ribosome composition in other subcellular compartments, identifying both well-described and new RAPs as a resource for studying ribosomes associated with specific organelles. ALIBi and RiboExM enabled discovery of "specialized ribosomes" with unconventional RP compositions. Focusing on the outer mitochondrial membrane (OMM), we visualized the enrichment of RPS25- and RPL29-depleted ribosomes. In addition, RiboExM enabled simultaneous visualization of ribosomes and their associated mRNAs, revealing that specialized ribosomes at the OMM preferentially bind to mRNAs involved in metabolism, specifically those in the vitamin B12 utilization pathway. This demonstrates ribosome heterogeneity–mediated translational control in subcellular contexts. In neurons, RiboExM revealed a functional dichotomy between monosomes and polysomes. Monosomes were enriched in distal neurites at baseline, whereas polysomes formed dynamically in response to external stimuli, suggesting regulation of protein synthesis in neuronal processes. CONCLUSION: Together, RiboExM and ALIBi constitute a key toolkit for studying ribosomal composition, localization, and function at single-molecule resolution. In this study, we have used these tools to unveil ribosomal heterogeneity and its implications for translational control across cellular compartments and cell types. In general, RiboExM and ALIBi offer opportunities for exploring macromolecular interactions and regulatory mechanisms in cellular biology. The versatility of these methods holds potential for advancing understanding of translational regulation in both physiological and pathological contexts. Two complementary methods reveal subcellular heterogeneity of ribosomes and mRNA translation.: Ribosome expansion microscopy (RiboExM) enables isotropic single-molecule imaging of ribosomal populations, specialized ribosomes, and associated mRNAs. AviTag-specific Location-restricted Illumination-Enhanced Biotinylation (ALIBi) is an optogenetically controlled technology that allows affinity purification of subcellular ribosome populations for multimodal downstream analysis. Here, we describe the use of these technologies to characterize ribosomes in subcellular space and in neurons. [ABSTRACT FROM AUTHOR]