Super-resolution expansion microscopy reveals nanoscale protein domains and CO2-dependent remodeling of Chlamydomonas pyrenoid-traversing membranes

Within the algal carbon-assimilating organelle, the pyrenoid, specialized traversing membranes perform the essential function of delivering concentrated CO2 to Rubisco. In Chlamydomonas reinhardtii, these membranes consist of peripheral cylindrical tubules that connect to a central reticulated region. However, due to resolution limitations, the spatial distribution of their structural and functional proteins has remained unclear. Here, we achieve an [~]11-fold improvement in resolution by combining ultrastructure expansion microscopy with super-resolution instantaneous structured illumination microscopy, revealing protein localizations and condition-dependent remodeling of these membranes. At air levels of CO2, the tubule-initiating protein SAGA1 forms narrow rings at the pyrenoid edge, the tubule-extending protein MITH1 surrounds the peripheral tubules, and the putative transporter BST4 surrounds more centrally located tubule segments, suggesting that the cylindrical tubules contain multiple distinct protein domains. The CO2-delivering carbonic anhydrase CAH3 localizes to the luminal face of the central reticulated region, suggesting that this region is specialized for CO2 delivery. CAH3 remains in the reticulated region at high CO2, suggesting that the cell maintains a minimal CO2-delivery apparatus even when dispensable. Finally, at high CO2, cylindrical tubules are narrower, and MITH1 relocalizes throughout the pyrenoid-traversing membrane network. Together, our study elucidates sub-pyrenoid protein organization and CO2-dependent reorganization.