Journal: iScience

Article Title: Golgi-mediated microtubule nucleation is associated with initiation of vertebrate peripheral neuron regeneration

doi: 10.1016/j.isci.2025.113697

Figure Lengend Snippet: Golgi compaction is associated with emergence of microtubules from the Golgi and initiation of axon regeneration (A) Time-lapse sequence of dissociated DRG neuron expressing GFP-GPI initiating axon regeneration at 29 h post-injury (yellow arrowhead). (B) Immunostaining to detect β-III-tubulin and GM130 from 2 h up to 7 days post-injury, imaged using super-resolution STED microscopy. (B’) GM130 shown in grayscale, highlighting the changes in Golgi morphology from 2 h to 7 days post-injury. Yellow boxes in (B) outline zoomed regions in (B’’), where linear tracks of microtubules appear to emerge from the Golgi at 24 h (yellow arrowheads). (B’’’) Quantification of mean number of disconnected components in the GM130 channel: 173 ± 20.26 at 2 h ( n = 44 cells), 101.5 ± 13.94 at 16 h ( n = 50 cells), 70.94 ± 6.12 at 24 h ( n = 36 cells), 65.02 ± 6.72 at 48 h ( n = 49 cells) and 40.19 ± 1.97 at 7 days ( n = 43 cells). N = 3 animals throughout. p = 0.0003 2 vs. 16 h; p < 0.0001 2 h vs. 24 h, 48 h, and 7 days; p = 0.003 16 h vs. 7 days. (C) Time projections of dissociated DRG neuron at 36 h post-injury transfected with Galt7-NeonGreen and EB3-mScarlet-I. Cyan boxes outline zoomed regions 1, 2, and 3. Yellow arrowheads indicate sites of nucleation. Scale bars: 20 μm. All graphs displayed as mean ± SEM,∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ordinary one-way ANOVA and Tukey’s post hoc test used for statistical analyses.

Article Snippet: To perform analysis of Golgi disconnected components Imaris 9.2.1 software (Bitplane) was used.

Techniques: Sequencing, Expressing, Immunostaining, Microscopy, Transfection

Journal: iScience

Article Title: Golgi-mediated microtubule nucleation is associated with initiation of vertebrate peripheral neuron regeneration

doi: 10.1016/j.isci.2025.113697

Figure Lengend Snippet: Sequential recruitment of AKAP9 and γ-tubulin to the Golgi (A) Immunolabeling of dissociated DRG neurons 2 h to 7 days post-injury for GM130 and AKAP9. Yellow arrowheads: AKAP9 puncta decorating Golgi. Yellow boxes outline zoomed in regions. Cyan channel displays regions of colocalization between AKAP9 and GM130. (A’) PLA to determine interactions between GM130 and AKAP9 at 16 h to 7 days. (A’’) Quantification of PLA puncta: 127.3 ± 17.52 at 16 h ( n = 27 cells); 32.68 ± 4.29 at 24 h ( n = 40 cells); 59.55 ± 5.62 at 48 h ( n = 29 cells) and 61.76 ± 3.48 at 7 days ( n = 37 cells). N = 2 animals throughout. p < 0.0001 16 vs. 24 h, 48 h and 7 days; p = 0.0381 24 h vs. 7 days. (B) Immunolabeling of dissociated DRG neurons 2 h to 7 days post-injury for GM130 and γ-tubulin. Yellow arrowheads: γ-tubulin puncta decorating Golgi. Yellow boxes outline zoomed in regions. Cyan channel displays regions of colocalization between γ-tubulin and GM130. (B’) PLA to determine interactions between GM130 and γ-tubulin at 16 h to 7. (B’’) Quantification of PLA puncta: 85.81 ± 20.64 at 16 h ( n = 32 cells), 184.2 ± 37.75 at 24 h ( n = 25 cells), 44.48 ± 5.97 at 48 h ( n = 27 cells) and 44.96 ± 7.07 at 7 days ( n = 28 cells). N = 2 animals throughout. p = 0.0069 16 h vs. 24 h; p < 0.0001 24 h vs. 48 h and 7 days. Scale bars: 20 μm. All graphs display mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001; ordinary one-way ANOVA and Tukey’s post hoc test used for statistical analyses.

Article Snippet: To perform analysis of Golgi disconnected components Imaris 9.2.1 software (Bitplane) was used.

Techniques: Immunolabeling

Journal: iScience

Article Title: Golgi-mediated microtubule nucleation is associated with initiation of vertebrate peripheral neuron regeneration

doi: 10.1016/j.isci.2025.113697

Figure Lengend Snippet: The mechanisms promoting initiation of axon regeneration in vitro are conserved in vivo and in human peripheral neurons (A) Immunolabeling of tissue sections from L4/5 rat DRGs following sciatic nerve transection and subsequent recovery for 0 h, 24 h, 48 h, and 7 days for β-III-tubulin and GM130. Yellow dashed lines outline individual cells. (A’) GM130 shown in grayscale demonstrating fragmentation at 24 h post-injury followed by compaction to baseline (control) from 48 h to 7 days. Cyan boxes outline zoomed regions in the bottom panel. (A’’) Quantification of mean number of disconnected components in GM130 channel: 121 ± 11.95 at 0 h ( n = 48 cells), 184.6 ± 3.97 at 24 h ( n = 50 cells), 135.7 ± 11.94 at 48 h ( n = 44 cells) and 109.7 ± 9.95 at 7 days ( n = 46 cells). N = 3 animals. p = 0.0012 0 h vs. 24 h, p = 0.0251 24 h vs. 48 h, p = 0.0001 24 h vs. 7 days. (B) Dissociated human DRG labeled for β-III-tubulin and GM130. Yellow boxes outline zoomed regions in (B’) GM130 shown in grayscale, highlighting the changes in Golgi morphology from 24 h to 72 h post-injury. (B’’) Quantification of mean number of disconnected components in GM130 channel: 553.1 ± 60.07 at 24 h ( n = 21 cells), 75 ± 8.93 at 48 h ( n = 34 cells) and 80.33 ± 9.65 at 72 h ( n = 21 cells). N = 3 human donors. p=<0.0001 24 h vs. 48 h, p=<0.0001 24 h vs. 72 h. (C) Immunolabeling of L4/5 rat DRGs tissue sections following sciatic nerve transection and recovery for 0/24/48 h and 7 days for GM130 and AKAP9. Yellow arrowheads: AKAP9 puncta decorating Golgi. Zoomed in regions identified by yellow arrows/boxes. (C’) PLA to determine interactions between GM130 and AKAP9 at 0 h to 7 days post-injury. (C’’) Quantification of PLA puncta: 74.9 ± 16.13 at 0 h ( n = 20 cells), 22.67 ± 6.23 at 24 h ( n = 21 cells), 316.8 ± 53.37 at 48 h ( n = 23 cells) and 53.91 ± 6.13 at 7 days ( n = 23 cells). N = 2 animals. p < 0.0001 48 h vs. 0 h, 24 h and 7 days. (D) Immunolabelling of tissue sections for GM130 and γ-tubulin. Yellow arrowheads: γ-tubulin puncta decorating Golgi. (D’) PLA to determine interactions between GM130 and γ-tubulin at 0 h to 7 days post-injury. (D’’) Quantification of PLA puncta: 34.32 ± 6.16 at 0 h ( n = 22 cells), 1.6 ± 3.51 at 24 h ( n = 20 cells), 166.3 ± 23.09 at 48 h ( n = 28 cells) and 34.32 ± 6.16 at 7 days ( n = 22 cells). N = 2 animals. p < 0.0001 48 h vs. 0 h, 24 h and 7 days. (E) Dissociated human DRG fixed at 24/48/72 h post-injury, labeled for GM130 and AKAP9. Zoomed in regions enclosed in blue/yellow boxes. (F) Dissociated primary human DRG fixed at 24/48/72 h post-injury, labeled for GM130 and γ-tubulin. Zoomed in regions enclosed in blue/yellow boxes. Scale bars: 20 μm. All graphs display mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001; ordinary one-way ANOVA and Tukey’s post hoc test used for statistical analyses.

Article Snippet: To perform analysis of Golgi disconnected components Imaris 9.2.1 software (Bitplane) was used.

Techniques: In Vitro, In Vivo, Immunolabeling, Control, Labeling

Journal: iScience

Article Title: Golgi-mediated microtubule nucleation is associated with initiation of vertebrate peripheral neuron regeneration

doi: 10.1016/j.isci.2025.113697

Figure Lengend Snippet: Golgi compaction is required for initiation of axon regeneration (A) Immunostaining to detect GM130 and AKAP9 in dissociated DRG neurons cultured in medium containing DMSO (top panels) or BFA (bottom panels). Yellow arrowheads: AKAP9 puncta decorating Golgi. Blue boxes indicate zoomed in regions shown in right-hand panel. (B) Immunostaining to detect GM130 and γ-tubulin in dissociated DRG neurons cultured in medium containing DMSO (top panels) or BFA (bottom panels). Yellow arrowheads: γ-tubulin puncta decorating Golgi. Blue boxes indicate zoomed in regions shown in right-hand panel. (A’) PLA to determine interactions between GM130 and AKAP9 in dissociated DRG neurons treated with DMSO or BFA. (A’’) Quantification of PLA puncta: 87.06 ± 7.09 DMSO ( n = 32 cells) and 43.25 ± 6.1 BFA treated cells ( n = 20 cells). N = 2 animals. (B’) PLA to determine interactions between GM130 and γ-tubulin in dissociated DRG neurons treated with DMSO or BFA. B″) Quantification of PLA puncta: 53.56 ± 6.87 DMSO ( n = 34 cells) and 30.17 ± 4.29 BFA treated cells ( n = 24 cells). N = 2 animals. (C) Quantification of mean number of disconnected components in GM130 channel: 49.56 ± 5.13 in DMSO treated cells ( n = 30 cells) and 199.3 ± 21.92 in BFA treated cells ( n = 30 cells). N = 2 animals. p=<0.0001. (D) Time projections of 1-min timelapse sequence of cell expressing EB3-mScarlet-I and Galt7-NeonGreen following addition of BFA. Yellow boxes indicate zoomed in regions shown in bottom panels. (E) Timelapse sequences of cells expressing GFP-GPI imaged in medium containing DMSO (top panels), BFA (middle panels) and gatastatin G2 (bottom panels). (F) Immunolabeling of cells cultured in media containing DMSO (top panel), BFA (middle panel) or gatastatin G2 (bottom panel) and fixed at 40 h to detect β-III-tubulin and GM130. (F’) Quantification of mean number of disconnected components in GM130 channel: 38.87 ± 5.1 in DMSO treated cells ( n = 30 cells), 184 ± 35.9 in BFA treated cells ( n = 30 cells) and 63.23 ± 4.1 in gatastatin G2 treated cells ( n = 22 cells). N = 2 animals. p < 0.0001 DMSO vs. BFA treated cells; p = 0.0027 gatastatin G2 treated cells vs. BFA treated cells. (F’’) Quantification of the percentage of cells at observed stages of regeneration for each treatment. N = 2 animals. Scale bars: 20 μm. All graphs displayed as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ordinary one-way ANOVA and Tukey’s post hoc test and unpaired t test used for statistical analyses.

Article Snippet: To perform analysis of Golgi disconnected components Imaris 9.2.1 software (Bitplane) was used.

Techniques: Immunostaining, Cell Culture, Sequencing, Expressing, Immunolabeling

Journal: iScience

Article Title: Golgi-mediated microtubule nucleation is associated with initiation of vertebrate peripheral neuron regeneration

doi: 10.1016/j.isci.2025.113697

Figure Lengend Snippet: AKAP9 is required for recruitment of γ-tubulin to the Golgi and subsequent axon regeneration (A) Cells fixed at 24 h expressing cis/medial Golgi marker Giantin-mScarlet and AKAP9-dis and immunostained for endogenous AKAP9. Top panel shows control cells not expressing AKAP9-dis and bottom panel cells expressing AKAP9-dis. Yellow dashes outline the area in the cell occupied by the Golgi and cyan boxes outline zoomed in regions. Yellow arrowheads: AKAP9 puncta decorating Golgi. (A’) PLA to determine interactions between GM130 and AKAP9 at 24 h, with non-transfected control cells in the rightmost panel. (B) Cells fixed at 24 h expressing Giantin-mScarlet, AKAP9-dis, and immunostained for endogenous γ-tubulin. Top panel shows control cells not expressing AKAP9-dis and bottom panel cells expressing AKAP9-dis. Yellow dashes outline the area in the cell occupied by the Golgi and cyan boxes outline zoomed regions. Yellow arrowheads: γ-tubulin puncta decorating Golgi. (B’) PLA to determine interactions between GM130 and γ-tubulin at 24 h, with control cells in the rightmost panel. (A’’) Quantification of PLA puncta: 0.58 ± 0.205 in cells transfected with AKAP9-Golgi displacement ( n = 56 cells) and 32.85 ± 4.4 control non-transfected cells ( n = 88 cells). N = 2 animals. p < 0.0001 transfected vs. non-transfected controls. (B’’) Quantification of PLA puncta: 0.143 ± 0.078 in cells transfected with AKAP9-Golgi displacement ( n = 27 cells) and 184.2 ± 37.75 in control non-transfected cells ( n = 85 cells). N = 2 animals. p < 0.0001 transfected vs. non-transfected controls. (C) Timelapse sequences of cells expressing GFP-GPI not expressing AKAP9 knockout construct (top panels) and expressing AKAP9 knockout construct (magenta, bottom panels). (D) Cells transfected with AKAP9-Golgi displacement (green) and EB3-mScarlet (magenta) with cell bodies outlined with white dashed lines. Time projections show no evidence of microtubule nucleation compared with non-transfected controls (rightmost panel). All graphs displayed as mean ± SEM. ∗∗∗∗ p < 0.0001; unpaired t test used for statistical analysis. Scale bars 20 μm.

Article Snippet: To perform analysis of Golgi disconnected components Imaris 9.2.1 software (Bitplane) was used.

Techniques: Expressing, Marker, Control, Transfection, Knock-Out, Construct

Journal: International Journal of Molecular Sciences

Article Title: Shape Matters: The Utility and Analysis of Altered Yeast Mitochondrial Morphology in Health, Disease, and Biotechnology

doi: 10.3390/ijms26052152

Figure Lengend Snippet: Available bioanalysis tools. Table modified from A Hitchhiker’s guide through the bio-image analysis software universe [ 113 ].

Article Snippet: zen , Software and components for microscope control, image acquisition, visualization, and analysis, integrating image analysis. , (Zeiss AG, Oberkochen, Germany).

Techniques: Modification, Software, Imaging, Extraction, Microscopy, Single Particle, Tomography, Fluorescence, Electron Microscopy, Control, Transmission Assay, Super-Resolution Microscopy