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Cryo-structured illumination microscopy (cryo-SIM) of A431 epithelial cells with correlative near-isotropic, reconstructed volume electron microscopy (FIB-SEM) (aic _desmosome-3)

Version 2 2023-05-23, 17:25
Version 1 2023-05-16, 21:26
posted on 2023-05-23, 17:25 authored by David AckermanDavid Ackerman, Jesse Aaron, Davis Bennett, Navaneetha Krishnan Bharathan, John Bogovic, CellMap Project TeamCellMap Project Team, Teng-Leong Chew, COSEM Project TeamCOSEM Project Team, William Giang, Larissa Heinrich, Satya Khuon, Andrew Kowalczyk, Woohyun Park, Alyson Petruncio, Stephan PreibischStephan Preibisch, Sara Stahley, Eric T. Trautman, A. Wayne Vogl, Aubrey WeigelAubrey Weigel

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% OsO₄ 0.1% UA 3% H₂O 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-3

Voxel size (nm): 4 x 4 x 4 (X, Y, Z)

Data dimensions (µm): 14.9 x 12.9 x 48.4 (X, Y, Z)

Imaging start date: 2021-03-18

Dataset URL (Redirect): https://data.janelia.org/0cEX8

EM DOI: https://doi.org/10.25378/janelia.22675078

Visualization Website: https://openorganelle.janelia.org/datasets/aic_desmosome-3

Publications: "Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle" by Bharathan et al., 2022

Imaging duration (days): 8

Landing energy (eV): 700

Imaging current (nA): .25

Scanning speed (MHz): 0.200


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