In situ architecture of plasmodesmata in Physcomitrium patens resolved by cryo-electron tomography

Plasmodesmata are nanoscopic channels that traverse plant cell walls, enabling direct intercellular exchange through membrane and cytosolic continuity. Although numerous plasmodesmal components have been identified, their molecular organization remains poorly defined. Here we used cryo-electron tomography to resolve the in situ architecture of plasmodesmata in Physcomitrium patens across tissues and physiological states. We show how callose-related cell wall remodelling shapes pore architecture to modulate permeability, including a previously undescribed fully sealed state, and resolve helical protein assemblies scaffolding the central, endoplasmic-reticulum-derived desmotubule. Candidate screening via proteomics and structure prediction indicates Multiple C2 Domain and Transmembrane Proteins (MCTPs) as key constituents of these assemblies. In this model, MCTPs tether the desmotubule to the plasma membrane, while their disordered linker regions with polyampholyte charge patterning may populate the cytosolic sleeve. These findings define core architectural features of plasmodesmata and provide a structural framework for understanding how membrane, protein and cell wall components coordinate intercellular connectivity in plants.