Research ArticleSTRUCTURAL BIOLOGY

Visualizing subcellular rearrangements in intact β cells using soft x-ray tomography

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Science Advances  09 Dec 2020:
Vol. 6, no. 50, eabc8262
DOI: 10.1126/sciadv.abc8262
  • Fig. 1 Representative 3D reconstruction of INS-1E cells and the effect of stimulation.

    (A) Representative orthoslice through the 3D reconstruction of a cell, where indicated LAC values are from a representative voxel and (B) 3D volumetric images of segmentation. To illustrate the segmentation process, (B) depicts before (left) and after (right) organelle segmentation (scale bars, 350 nm). (C) Comparison of insulin secretion with cells in suspension measured in triplicate using enzyme-link immunosorbent assay (ELISA; Mercodia). Statistical differences are shown, where **P = 0.0049 and ***P < 0.0001 (Tukey’s multiple comparison test). (D) Plot of mitochondria/cytosol volume ratios, where *P = 0.042 (Dunnett’s multiple comparison test). (E) Number of insulin vesicles normalized by cell volume and statistical differences, where ***P < 0.0001, **P = 0.0062, and *P = 0.0436 (Tukey’s multiple comparison test). (F) Mean insulin vesicle LAC value for each condition with a difference between unstimulated and glucose + Ex-4 conditions (*P = 0.0106; Dunnett’s multiple comparison test). Error bars in all panels represent SDs. n values in figure correspond to (D) to (F).

  • Fig. 2 Organization and distributions of insulin vesicles by phenotype within multiple cellular neighborhoods.

    Four major phenotypes of insulin vesicle organization were observed: (A) docking and (B) budding outward from the PM, (C) clustered, and (D) in chains in the cell interior. Four orthoslices are shown for each phenotype from representative cells. (E) Distance distribution plots of a representative cell from each stimulation condition. Insulin vesicles dock along the PM in unstimulated INS-1E cells (left) and in large clusters, causing a unique shape to the distribution pattern in those treated with glucose (center). The second smaller peak here (highlighted by the dotted line) corresponds to four insulin vesicle clusters within this cell. In contrast, cells treated with glucose + Ex-4 tended toward a diffuse insulin vesicle distribution (right). Scale bars, 2 μm. (F) Three classes of random distribution. Yellow regions represent the allowed volume of random vesicle placement. (G) Bhattacharyya distance plots comparing random versus observed distribution of insulin vesicles. For each cell, the observed distribution of insulin vesicles was compared with 50 randomly distributed points shown here as scatter plots. Cell nomenclature refers to capillary number (first number) and cell position within that capillary (number after hyphen).

  • Fig. 3 Dynamics of cell topology rearrangements during insulin secretion.

    (A) The number of insulin vesicles normalized by cell volume showing statistical differences between conditions (*P = 0.0118 and **P = 0.0007; Sidak’s multiple comparison test). (B) Mean molecular density or LAC value of insulin vesicles in each condition showing statistical differences (*P = 0.0240 and **P = 0.0029; Sidak’s multiple comparison test). (C) Plots of the number of insulin vesicles distributed from the PM at both 5- and 30-min time points. (D) Plot of the mitochondrial/cytosol volume ratio. Relative to the unstimulated condition, mitochondrial volume was larger for glucose-stimulated cells at 5 min (*P = 0.0252), and the glucose + Ex-4 at 5 and 30 min condition (***P = 0.0007 and *P = 0.0325) (Holm-Sidak’s multiple comparison test). (E) Mean mitochondria-insulin vesicle distance for each condition. Relative to the unstimulated condition, there is a smaller distance between insulin vesicles and mitochondria in the glucose-stimulated 5- and 30-min time points (*P = 0.0099 and ****P = 0.0001) and the glucose + Ex-4–stimulated 30-min time point (**P = 0.0040; Dunnett’s multiple comparison test).

  • Fig. 4 Condition clustering and cell-to-cell heterogeneity in topology.

    Mean insulin vesicle LAC value versus numbers of insulin vesicles normalized by cell volume for each cell. Each point represents one cell as indicated in the key. Representative orthoslices from the 3D reconstructions for the cells at the extremes of the plots are shown for reference. Diamonds in the box and whisker plots represent points outside the SD.

  • Fig. 5 SXT mesoscale maps will be a foundation for future of whole-cell modeling efforts.

    (A) Representative electron tomograms of INS-1E cells depict the different subcellular neighborhood environments in the periphery and center of the cell as indicated. (B) Representative SXT orthoslice depicting whole-cell architecture. (C) 3D molecular model of an INS-1E cell generated using cellPACK (38) from segmented SXT data. Nucleus is shown in green, insulin vesicles are shown in blue, and core of insulin vesicles is shown in yellow. The mesh of each structure is textured with lipids, and the zoom views depict the atomic details of protein packing. The black widow is a rendering of the segmented vesicle mask used to generate the model. Image courtesy of L. Autin and A. Olson.

Supplementary Materials

  • Supplementary Materials

    Visualizing subcellular rearrangements in intact β cells using soft x-ray tomography

    Kate L. White, Jitin Singla, Valentina Loconte, Jian-Hua Chen, Axel Ekman, Liping Sun, Xianjun Zhang, John Paul Francis, Angdi Li, Wen Lin, Kaylee Tseng, Gerry McDermott, Frank Alber, Andrej Sali, Carolyn Larabell, Raymond C. Stevens

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    • Figs. S1 to S8
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