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Manual segmentations of subcellular structures in near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of A431 epithelial cells (aic_desmosome-3)
Sample: Cultured A431 (ATCC CRL-1555) cells stably expressing Desmoplakin-eGFP (Addgene #32227) and mApple-VAPB, stained with MitoView650
Sample Description: The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signaling, and lipid transfer. Using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometer proximity to keratin filaments and the desmosome ctoplasmic plague. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization and mobility. These findings indicate that desmosomes and the keratin cytoskeleton pattern the distribution of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.
Protocol: High pressure freezing, freeze-substitution resin embedding with 2% OsO, 0.1% UA 3% H,O in acetone; resin embedding in Eponate 12.
Contributions: Sample prepared by Jesse Aaron and Satya Khuon (AIC Janelia), staining and resin embedding by Nirmala lyer (HHMI/Janelia), trimming and imaging by Jesse Aaron and Satya Khoun (AIC Janelia), post-processing by Eric Trautman and Stephan Preibisch (HHMI/Janelia), manual segmentations Kowalczyk Lab (Pennsylvania State College of Medicine).
Acquisition ID: aic desmosome-3
Voxel size (nm): 4 x 4 x 4 (X, Y, Z)
Data dimensions (um): 14.9 x 12.9 x 48.4 (X, Y, Z)
Imaging start date: 2021-03-18
Dataset URL (Redirect): https://data.janelia.org/VoDT2
Visualization Website: https://openorganelle.janelia.org/datasets/aic_desmosome-3
Imaging duration (days): 8
Landing energy (eV): 700
Imaging current (nA): .25
Scanning speed (MHz): 0.200