File(s) stored somewhere else
Please note: Linked content is NOT stored on Janelia Research Campus and we can't guarantee its availability, quality, security or accept any liability.
Cryo-structured illumination microscopy (cryo-SIM) of A431 epithelial cells with correlative near-isotropic, reconstructed volume electron microscopy (FIB-SEM) (aic _desmosome-2)
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 cytoplasmic plaque. 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% Os0, 0.1% UA 3% HO 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), registration by John Bogovic (HHMI/Janelia).
Acquisition ID: aic desmosome-2
Voxel size (nm): 8 x 8 x 8 (X. Y. Z)
Data dimensions (um): 42.3 x 10.4 × 52.5 (X, Y, Z)
Imaging start date: 2021-01-27
Dataset URL (Redirect):https://data.janelia.org/l5NlB
EM DOI: https://doi.org/10.25378/janelia.22670176
Visualization Website: https://openorganelle.janelia.org/datasets/aic desmosome-2
Publication: "Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle" by Bharathan et al., 2022
Imaging duration (days): 4
Landing energy (eV): 1200
Imaging current (nA): 2.0
Scanning speed ( MHz): 0.500