Writing in Science, Tran, Carter et al. (https://doi.org/10.1126/science.abh2474) now report an approach based on cryogenic correlated light and electron microscopy combined with electron cryo-tomography (cryo-CLEM-ET) to reveal the molecular organization of ER-stress-induced IRE1α oligomers in human cells. They found that IRE1α foci localize to distinct regions of the ER they term “IRE1α subdomains,” in which they observe a network of about 28-nm-wide anastomosing membrane tubes (left in image) continuous with the ER. In electron micrographs and cryotomograms, similar tube networks are observed and confirmed to contain IRE1α labeled with immunogold. Notably, both transverse and longitudinal cross-sections of those tubes appear to show a circular density about 9 nm in diameter (middle in image, arrows: black, membrane tubes; light blue, connection to general ER; red, three-way junction). Subtomogram averaging reveals that this ‘wire in the tube’ is a left-handed double helix (right in image) that somewhat resembles a crystal structure of the Saccharomyces cerevisiae core IRE1 lumenal domain reported in 2005 (https://doi.org/10.1073/pnas.0509487102). To assess the compatibility of their data with the human IRE1α forming the observed helices, the authors built a molecular model of the hIRE1α lumenal domain and fit it into the double-helical map (right in image). The resulting model is indeed compatible with the yeast structure, with a slight modification of the helical pitch. Notably, the helical interfaces in the structural model contain several residues that have been shown to disrupt human IRE1α foci formation.
Both tubes and enclosed IRE1α double helices appear to be structurally flexible, as both curved and bent segments were frequently observed. Similarly, the spacing between the tube membrane and fibrils varies considerably, which may be explained by conformational flexibility of the 52-amino-acid linker connecting the fibril-forming IRE1α lumenal domains to their respective transmembrane helices.
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