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Integrative cryoET approaches to study cell-cell communication

Cell-cell connectivity is a fundamental feature of multicellular organization across all kingdoms of life. Specialized structures facilitate intercellular exchange of small molecules, peptides, and genetic information. In plants, connectivity is mediated by plasmodesmata (PD), nanoscopic channels with complex architecture: PD traverse the cell wall and establish continuity between the cytoplasm, plasma membranes, and ER of neighboring cells. Despite their essential role, the mechanisms by which PD regulate the selective translocation of specific molecules while restricting movement of others remain unclear.

We developed a cryo-electron tomography (cryo-ET) workflow to study PD in tissues preserved by high-pressure freezing. Using tomography in combination with transport assays, proteomics, and fluorescence microscopy, we uncover structural mechanisms underlying PD gating. Additionally, subtomogram averaging revealed a large protein complex at orifices of PD. While we continue to investigate its composition, our findings suggest the repurposing of components from other cellular machinery to form a dynamic permeability barrier at PD.