Manual segmentations of subcellular structures in near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of A431 epithelial cells (aic_desmosome-2)

<b>Sample</b>: Cultured A431 (ATCC CRL-1555) cells stably expressing Desmoplakin-eGFP (Addgene #32227) and mApple-VAPB, stained with MitoView650<b>Sample Description</b>: 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.<b>Protocol</b>: High pressure freezing, freeze-substitution resin embedding with 2% Os0, 0.1% UA 3% HO in acetone; resin embedding in Eponate 12<b>Contributions</b>: 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).<b>Acquisition ID</b>: aic desmosome-2<b>Voxel size (nm)</b>: 8 x 8 x 8 (X, Y, Z)<b>Data dimensions (um)</b>: 42.3 x 10.4 × 52.5 (X, Y, Z)<b>Imaging start date</b>: 2021-01-27<b>Dataset URL</b>: S3://janelia-cosem-datasets/aic_desmosome-2/aic_desmosome-2.n5/labels/<b>EM DOI</b>: https://doi.org/10.25378/janelia.22670176<b>Visualization Website</b>: https://openorganelle.janelia.org/datasets/aic desmosome-2<b>Publication</b>: "Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle" by Bharathan et al., 2022<b>Imaging duration (days)</b>: 4<b>Landing energy (eV)</b>: 1200<b>Imaging current (nA)</b>: 2.0<b>Scanning speed ( MHz)</b>: 0.500

<b>样本</b>：稳定表达桥粒斑蛋白-增强绿色荧光蛋白（Desmoplakin-eGFP，Addgene #32227）与mApple-囊泡相关膜蛋白结合蛋白B（mApple-VAPB）的培养A431细胞（ATCC CRL-1555），经MitoView650染色。
<b>样本描述</b>：内质网（endoplasmic reticulum, ER）是一类动态膜网络结构，可与其他细胞内膜结构相互接触，以此调控细胞应激反应、钙信号传导及脂质转运过程。本研究借助高分辨率体积电子显微镜成像技术，发现内质网此前未被报道的与角蛋白中间丝及桥粒细胞间连接的关联现象：外周内质网可在桥粒位点形成镜像样排布结构，并与角蛋白中间丝及桥粒胞质斑块处于纳米级邻近距离；内质网管泡可与桥粒形成稳定结合，而桥粒或角蛋白中间丝的扰动会改变内质网的组织形式与运动特性。上述结果表明，桥粒与角蛋白细胞骨架可塑造内质网网络的空间分布格局。综上，本研究揭示了一种此前未被发现的亚细胞结构，其核心特征为内质网管泡与上皮细胞间连接的结构整合。
<b>实验方案</b>：高压冷冻、冷冻替代树脂包埋：采用含2%四氧化锇（OsO₄）、0.1%乙酸铀（UA）、3%过氧化氢（H₂O₂）的丙酮溶液；使用Eponate 12树脂进行包埋。
<b>贡献人员</b>：样本制备：Jesse Aaron与Satya Khuon（贾内利亚研究园区AI中心，AIC Janelia）；染色与树脂包埋：Nirmala Iyer（霍华德·休斯医学研究所/贾内利亚研究园区，HHMI/Janelia）；样品修整与成像：Jesse Aaron与Satya Khoun（AIC Janelia）；后处理：Eric Trautman与Stephan Preibisch（HHMI/Janelia）；人工图像分割：Kowalczyk实验室（宾夕法尼亚州立大学医学院）。
<b>采集标识</b>：aic_desmosome-2
<b>体素尺寸（纳米）</b>：8×8×8（X、Y、Z轴）
<b>数据维度（微米）</b>：42.3×10.4×52.5（X、Y、Z轴）
<b>成像起始日期</b>：2021-01-27
<b>数据集链接</b>：S3://janelia-cosem-datasets/aic_desmosome-2/aic_desmosome-2.n5/labels/
<b>电子显微镜数据集DOI</b>：https://doi.org/10.25378/janelia.22670176
<b>可视化网站</b>：https://openorganelle.janelia.org/datasets/aic_desmosome-2
<b>相关论文</b>：Bharathan等人于2022年发表的《新型桥粒-内质网细胞器的结构与动态》（"Architecture and dynamics of a novel desmosome-endoplasmic reticulum organelle"）
<b>成像时长（天）</b>：4
<b>着陆能量（电子伏特）</b>：1200
<b>成像电流（纳安）</b>：2.0
<b>扫描速度（兆赫兹）</b>：0.500