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Journal Articles

3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells

In Special Collection:
Structural Biology 2021 , The Year in Cell Biology: 2021
Andreas Müller, Deborah Schmidt, C. Shan Xu, Song Pang, Joyson Verner D’Costa, Susanne Kretschmar, Carla Münster, Thomas Kurth, Florian Jug, Martin Weigert, Harald F. Hess, Michele Solimena
Journal: Journal of Cell Biology
J Cell Biol (2020) 220 (2): e202010039.
https://doi.org/10.1083/jcb.202010039
Published: 16 December 2020
View article titled, 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells
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Images
FIB-SEM volumes of pancreatic β cells and 3D segmentation of microtubules and organelles. (A) Full FIB-SEM volume of a pancreatic islet (left), one of which was acquired for low- and high-glucose conditions containing three (low) and four (high) complete β cells (right). Shown are the lateral (x-y) and axial (x-z) views of a small crop, highlighting the quasi-isotropic resolution of the FIB-SEM volumes. The arrowhead indicates a microtubule, the asterisk an insulin SG. Scale bar, 500 nm. (B) Raw lateral images with segmentation overlay for insulin SGs, mitochondria, Golgi apparatus, nucleus (of the identical region), and microtubules and centrioles (of different regions). Below the overlays, we show 3D renderings of the corresponding organelles of one whole cell (high-glucose condition) accompanied by a transparent rendering of the plasma membrane. Centrioles are magnified in the last panel. Scale bars, 500 nm. (C) 3D rendering of one cell containing all segmented organelles. The plasma membrane and insulin SGs were removed in the left half of the cell to help visualize its inner parts.

FIB-SEM volumes of pancreatic β cells and 3D segmentation of microtubules a...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure 1. FIB-SEM volumes of pancreatic β cells and 3D segmentation of microtubules and organelles. (A) Full FIB-SEM volume of a pancreatic islet (left), one of which was acquired for low- and high-glucose conditions containing three (low) and More about this image found in FIB-SEM volumes of pancreatic β cells and 3D segmentation of microtubules a...
Images
Raw FIB-SEM data and workflow for sample preparation, imaging, segmentation, and data integration within BetaSeg Viewer. (A) Snapshots of samples prepared according to the old and new freeze substitution protocol. Arrowhead, microtubule. Scale bar, 500 nm. (B) Full raw volume of the low-glucose dataset with pixel and micrometer dimensions. (C) Detailed views of microtubules with arrowheads pointing to microtubule ends. (D) Snapshots of ultrastructural details: insulin SGs, mitochondria, nucleus, centrioles, ER, and Golgi apparatus. Scale bar, 500 nm. (E) Workflow for sample preparation, imaging, segmentation, and data integration within BetaSeg Viewer: isotropic volumes of cryo-immobilized, freeze-substituted, and resin-embedded pancreatic islets were acquired with FIB-SEM followed by manual and machine learning segmentation, 3D data analysis, integration into BetaSeg Viewer, and 3D visualization. (F) Screenshot of BetaSeg Viewer with a slice through an overlay of the raw volume of one β cell with the corresponding segmentation masks, a table depicting quantitative data, and a plot showing the distance of insulin SGs to the plasma membrane generated with BetaSeg Viewer. ML, machine learning; Res., resolution.

Raw FIB-SEM data and workflow for sample preparation, imaging, segmentation...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure S1. Raw FIB-SEM data and workflow for sample preparation, imaging, segmentation, and data integration within BetaSeg Viewer. (A) Snapshots of samples prepared according to the old and new freeze substitution protocol. Arrowhead, More about this image found in Raw FIB-SEM data and workflow for sample preparation, imaging, segmentation...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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Images
3D renderings of all β cells and organelles/organelle subtypes analyzed in this study. Color-coded are microtubule-associated and –not associated SGs, mitochondria, nuclei, microtubules, centrioles, centrosomal microtubules, Golgi apparati, and Golgi microtubules. Cubes on the right of each cell have a side length of 1 µm for scaling. assoc., associated; MT, microtubule; centr., centrosomal.

3D renderings of all β cells and organelles/organelle subtypes analyzed in ...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure S2. 3D renderings of all β cells and organelles/organelle subtypes analyzed in this study. Color-coded are microtubule-associated and –not associated SGs, mitochondria, nuclei, microtubules, centrioles, centrosomal microtubules, Golgi More about this image found in 3D renderings of all β cells and organelles/organelle subtypes analyzed in ...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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Images
Microtubule network properties and distance distributions. (A) Fully reconstructed microtubule network of one β cell with microtubules in red, centrioles in purple, Golgi apparatus in green, and plasma membrane (PM) in gray transparent. Scale, cube with a side length of 1 µm. (B) Microtubule (MT) length and tortuosity distribution of all seven analyzed cells (blue, low-glucose cells; orange, high-glucose cells). Horizontal and vertical lines signify the mean and the interquartile range, respectively. (C) Rendering of only centriole-connected (centrosomal) microtubules of the same cell as in A. The plots show the distributions of distances of MT ends to centrioles for all analyzed cells. Scale, cube with a side length of 1 µm. (D) Rendering of only Golgi-connected microtubules of the same cell as in A. Scale, cube with a side length of 1 µm. The plots show the distributions of distances of the microtubule ends to Golgi for all analyzed cells. (E) Distribution of the distance of microtubule ends to centrioles for one representative low-glucose (blue) and one high-glucose (orange) cell with random distributions ρCent represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. (F) Distribution of the distance of microtubule ends to Golgi membranes for the same cells as in E with random distributions ρGolgi represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. (G) Distribution of the distance of microtubule ends to the plasma membrane for the same cells as in E with random distributions ρPM represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. (H) Distribution of the distance of microtubule pixels to the plasma membrane for the same cells as in E with random distributions ρPM represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively.

Microtubule network properties and distance distributions. (A) Fully recon...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure 2. Microtubule network properties and distance distributions. (A) Fully reconstructed microtubule network of one β cell with microtubules in red, centrioles in purple, Golgi apparatus in green, and plasma membrane (PM) in gray More about this image found in Microtubule network properties and distance distributions. (A) Fully recon...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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Images
Microtubule and SG analysis for all cells. (A) Distance of microtubule ends to the nucleus. (B) Distance of microtubule ends to Golgi (bin 20 nm). (C) Distance of microtubule ends to Golgi (bin 200 nm). (D) Distance of microtubule ends to plasma membrane. (E) Distance of microtubule pixels to plasma membrane. (F) Surface areas of SGs. (G) Sphericity of SGs. (H) Distance of SGs to plasma membrane. (I) Distance of SGs to nucleus. (J) Distance of SGs to Golgi apparatus. (K) Distance of SGs to microtubules. Black lines in all distance distribution plots show the respective random distributions. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. MT, microtubule; PM, plasma membrane.

Microtubule and SG analysis for all cells . (A) Distance of microtubule e...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure S3. Microtubule and SG analysis for all cells . (A) Distance of microtubule ends to the nucleus. (B) Distance of microtubule ends to Golgi (bin 20 nm). (C) Distance of microtubule ends to Golgi (bin 200 nm). (D) Distance of More about this image found in Microtubule and SG analysis for all cells . (A) Distance of microtubule e...
Images
Insulin SG properties and distance distributions. (A) Volume fraction (percentage) of segmented organelles for all analyzed cells. (B) 3D rendering of one β cell with plasma membrane (transparent gray), insulin SGs (orange), Golgi apparatus (green), and nucleus (yellow). Scale: cube with a side length of 1 µm. (C) Diameter and volume distributions of insulin SGs of all seven analyzed cells (blue, low-glucose cells; orange, high-glucose cells). Horizontal and vertical lines signify the mean and the interquartile range, respectively. (D) Distribution of the distance of insulin SGs to the plasma membrane (PM) for one representative low-glucose (blue) and one high-glucose (orange) cell with random distributions ρPM represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. (E) Distribution of the distance of insulin SGs to the nucleus for one representative low-glucose (blue) and one high-glucose (orange) cell with random distributions ρNuc represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. (F) Distribution of the distance of insulin SGs to the Golgi apparatus for one representative low-glucose (blue) and one high-glucose (orange) cell with random distributions ρGolgi represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively.

Insulin SG properties and distance distributions. (A) Volume fraction (per...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure 3. Insulin SG properties and distance distributions. (A) Volume fraction (percentage) of segmented organelles for all analyzed cells. (B) 3D rendering of one β cell with plasma membrane (transparent gray), insulin SGs (orange), Golgi More about this image found in Insulin SG properties and distance distributions. (A) Volume fraction (per...
Images
Spatial association between microtubules and insulin SGs. (A) 3D rendering of a cell with plasma membrane (transparent gray), microtubules (red), and insulin SGs (orange). Inset shows a magnified region. Scale: cube with a side length of 1 µm. (B) Distribution of the distance of insulin SGs to microtubules (MTs) for one representative low-glucose (blue) and one high-glucose (orange) cell with random distributions ρMT represented by a black line. Red dotted and black dotted lines represent actual and random cumulative distributions, respectively. (C) 3D rendering of a cell with plasma membrane, microtubule-associated SGs (orange), not associated SGs (light gray), and microtubules (red). Scale: cube with a side length of 1 µm. (D) Violin plots depicting the distance of associated and not associated insulin SGs to the plasma membrane (PM). (E) Violin plots depicting the distance of associated and not associated insulin SGs to the nucleus. (F) Violin plots depicting the distance of associated and not associated insulin SGs to the Golgi apparatus. assoc., associated.

Spatial association between microtubules and insulin SGs. (A) 3D rendering...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
Figure 4. Spatial association between microtubules and insulin SGs. (A) 3D rendering of a cell with plasma membrane (transparent gray), microtubules (red), and insulin SGs (orange). Inset shows a magnified region. Scale: cube with a side length More about this image found in Spatial association between microtubules and insulin SGs. (A) 3D rendering...

in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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in 3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells > Journal of Cell Biology
Published: 16 December 2020
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Journal Articles

Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia

In Special Collection:
Structural Biology 2021
Landi Sun, Lihong Cui, Zhen Liu, Qixuan Wang, Zhaoyu Xue, Menghua Wu, Tianhui Sun, Decai Mao, Jianquan Ni, José Carlos Pastor-Pareja, Xin Liang
Journal: Journal of Cell Biology
J Cell Biol (2020) 220 (1): e202004184.
https://doi.org/10.1083/jcb.202004184
Published: 02 December 2020
View article titled, Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia
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Images
Ultrastructural organization of microtubules in the outer segment of haltere campaniform receptors. (A) Cartoon schematic of the sensory neuron in haltere receptors. BB, basal body. (B) The localizations of NompC-GFP (MO), Mks1-GFP (transition zone [TZ]), and GFP-Cnn1 (BB) in haltere receptors (top view). Upper: nompC-gfp-KI. Middle: Mks1-gfp. Lower: uas-gfp-cnn1/+; +/+; dcx-emap-gal4/+. Scale bar, 5 µm. (C–F) Cross-sectional views of the MO (C), neck (D), TB (E), and cilium (F). (G) Lateral view of the cilium. Black arrowhead, a doublet. (H) Lateral view of the mother centriole (MC), daughter centriole (DC), and cilium. Scale bars (C–H), 250 nm. C–H are ET slice images. (I) Reconstructed model of microtubules in the outer segment. Each microtubule was shown as a rod. White, microtubule in the outer segment. Red (tubule-A) and green (tubule-B), doublet. Yellow, microtubule in the inner segment. Scale bar, 1 µm. Also see Video 1.

Ultrastructural organization of microtubules in the outer segment of halter...

in Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia > Journal of Cell Biology
Published: 02 December 2020
Figure 1. Ultrastructural organization of microtubules in the outer segment of haltere campaniform receptors. (A) Cartoon schematic of the sensory neuron in haltere receptors. BB, basal body. (B) The localizations of NompC-GFP (MO), Mks1-GFP More about this image found in Ultrastructural organization of microtubules in the outer segment of halter...
Images
Ultrastructural organization of microtubules in the outer segment of leg campaniform receptors. (A) Cartoon schematic of the sensory neuron in leg receptors. (B) The localizations of NompC-GFP (MO), Mks1-GFP (transition zone [TZ]), and GFP-Cnn1 (basal body [BB]) in leg receptors (lateral view). Upper: nompC-gfp-KI; dcx-emap-gal4, uas-cd4-tdTom/+. Middle: +/Mks1-gfp; +/dcx-emap-gal4, uas-cd4-tdTom. Lower: uas-gfp-cnn1/+; +/+; dcx-emap-gal4, uas-cd4-tdTom/+. Scale bar, 5 µm. (C) Lateral view (ET slice image) of the outer segment. Red arrowhead, a TB microtubule. (D) Reconstructed model of microtubules in the outer segment. Blue, MO microtubule. White, TB microtubule. Red (tubule-A) + green (tubule-B), doublet. (E) Spatial distribution of microtubule ends in the outer segment. Every spot represented one end. Green, distal end. Red, proximal end. The yellow line (yellow arrowhead) indicates the distal tip. Scale bars (C–E), 500 nm. (F) The distribution of the distance between each microtubule end to the distal tip (representative of data from five cells). Red, proximal end. Green, distal end. The pie chart shows the percentage of MO microtubules that had the proximal end (p-end) in the TB. MT, microtubule; mem, membrane.

Ultrastructural organization of microtubules in the outer segment of leg ca...

in Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia > Journal of Cell Biology
Published: 02 December 2020
Figure 2. Ultrastructural organization of microtubules in the outer segment of leg campaniform receptors. (A) Cartoon schematic of the sensory neuron in leg receptors. (B) The localizations of NompC-GFP (MO), Mks1-GFP (transition zone [TZ]), More about this image found in Ultrastructural organization of microtubules in the outer segment of leg ca...
Images
KI strains and live-cell imaging of leg campaniform mechanoreceptors. (A) Cartoon schematic for the NompC-GFP KI strain (nompC-gfp-KI). The insertion site of GFP was indicated. (B) Localization of NompC-GFP (nompC-gfp-KI) in fly bristle mechanoreceptors (left panel) and haltere campaniform mechanoreceptors (middle and right panels). These observations were consistent with the immunostaining data obtained using a NompC monoclonal antibody (Liang et al., 2011). Scale bar, 20 µm. (C) A decapitated fly was kept in a wet chamber, and the locations of leg campaniform mechanoreceptors are indicated (red arrowheads). During imaging, the fly was immobilized on a cover glass using double-sided tape. The three legs on the same body side were covered by the second coverslip to facilitate imaging using an inverted spinning-disk confocal microscope. s3, the third segment. s5, the fifth segment. (D and E) Campaniform mechanoreceptors at the third (s3) and fifth (s5) segments of a fly leg (genotype, dcx-emap-gal4/uas-cd4-tdTom). The outer segment in these cells is indicated by a white arrow. Scale bar, 5 µm. (F) Cartoon schematic for the Patronin-RFP KI strain (patronin-rfp-KI). The insertion site of RFP is indicated. Based on the genome annotation in Flybase, all Patronin isoforms were tagged. (G) Using an endogenously tagged Shot-GFP (Sun et al., 2019b) as a marker, we showed that Shot-GFP and Patronin-RFP together formed puncta signals in epidermal cells (left and middle panels) and campaniform mechanoreceptors (right panel) in fly legs (genotype, Shot-GFP/patronin-rfp-KI). In these puncta, Shot and Patronin showed either contiguous or partially overlapping localizations (white arrowheads, two regions enlarged in the insets, right panel), consistent with the Shot-Patronin foci observed in the fly embryo (Nashchekin et al., 2016). Yellow arrowheads (right) indicate two dot-shaped Shot-Patronin signals that were in the thecogen cell. Scale bars, 1 µm. (H) Cross-sectional view (ET slice image) of the cilium and its surrounding structures in a haltere receptor. Scale bar, 250 nm. (I) Lateral view (ET slice image) of the structures near the cilium in a leg receptor. Scale bar, 250 nm. In H and I, microtubules in the thecogen cells are indicated by red arrowheads.

KI strains and live-cell imaging of leg campaniform mechanoreceptors. (A) ...

in Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia > Journal of Cell Biology
Published: 02 December 2020
Figure S1. KI strains and live-cell imaging of leg campaniform mechanoreceptors. (A) Cartoon schematic for the NompC-GFP KI strain (nompC-gfp-KI). The insertion site of GFP was indicated. (B) Localization of NompC-GFP (nompC-gfp-KI) in fly More about this image found in KI strains and live-cell imaging of leg campaniform mechanoreceptors. (A) ...

in Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia > Journal of Cell Biology
Published: 02 December 2020
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