Super-resolution expansion microscopy reveals nanoscale protein domains and CO 2 -dependent remodeling of Chlamydomonas pyrenoid-traversing membranes
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Within the algal carbon-assimilating organelle, the pyrenoid, specialized traversing membranes perform the essential function of delivering concentrated CO 2 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 CO 2 , 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 CO 2 -delivering carbonic anhydrase CAH3 localizes to the luminal face of the central reticulated region, suggesting that this region is specialized for CO 2 delivery. CAH3 remains in the reticulated region at high CO 2 , suggesting that the cell maintains a minimal CO 2 -delivery apparatus even when dispensable. Finally, at high CO 2 , cylindrical tubules are narrower, and MITH1 relocalizes throughout the pyrenoid-traversing membrane network. Together, our study elucidates sub-pyrenoid protein organization and CO 2 -dependent reorganization.
