BPS2026 – Direct visualization of peroxisome biogenesis using live-cell single-molecule tracking

Peroxisomes are ubiquitous organelles in eukaryotes that play a variety of roles in lipid metabolism and redox stress response. Remarkably, these structures will bud as pre-peroxisomal vesicles (ppVs) from the endoplasmic reticulum (ER) in de novo peroxisome biogenesis, which is required to maintain the cellular pool of mature peroxisomes. The mechanisms and controlling factors of the organization of peroxisome biogenesis factors on the ER membrane, however, remain unclear, as visualizing ppV budding events through conventional light or electron microscopy has presented ongoing challenges. Here, we present the first single particle tracking of the peroxisome biogenesis factor proteins in the ER. Using HaloTag, SNAPTag, and mStayGold fusions, we show realtime interactions of the peroxisome biogenesis machinery within nanodomains of the ER surface. Employing Airyscan superresolution microscopy and correlative single particle tracking-photoactivation localization microscopy (spt-PALM), we find MCTP2 enriched within substructures of the ER membrane, which could represent future sites of ppV budding events. Notably, at these MCTP2-enriched subdomains, we observe altered trajectories consistent with confined behavior for the subsequent peroxisome biogenesis factors PEX3 and PEX16. The presence of PEX3 at these subdomains is reflective of functional PEX16 within the neighborhood. These results are the first direct visualization at the single-molecule level of the early stages of peroxisome biogenesis.