Janelia Research Campus

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.

Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of A431 epithelial cells (aic_desmosome-2)

posted on 2023-05-23, 17:22 authored by David 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 P. Kowalczyk, Woohyun Park, Alyson Petruncio, Stephan PreibischStephan Preibisch, Stephan Saalfeld, Sara N. Stahley, Eric 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 Iyer (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 by COSEM Project Team (HHMI/Janelia) and Kowalczyk Lab (Pennsylvania State College of Medicine), and automated segmentations by COSEM Project Team.

Acquisition ID: aic_desmosome-2

Voxel size (nm): 8 x 8 x 8 (X, Y, Z)

Data dimensions (µm): 42.3 x 10.4 x 52.5 (X, Y, Z)

Imaging start date: 2021-01-27

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

Visualization Website: https://openorganelle.janelia.org/datasets/aic_desmosome-2

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

DOI: https://doi.org/10.1101/2022.07.07.499185

Imaging duration (days): 4

Landing energy (eV): 1200

Imaging current (nA): 2.0

Scanning speed (MHz): 0.500