eb3 gfp  (Addgene inc)

Journal: bioRxiv

Article Title: Microtubule networks in zebrafish hair cells facilitate presynapse transport and fusion during development

doi: 10.1101/2024.04.12.589161

Figure Lengend Snippet: EB3-GFP tracks show plus-ends of microtubules move to the cell base A) Example image of a neuromast at 2 dpf. The growing or plus-ends of microtubules are marked with EB3-GFP. In this example the apex of 8 developing cells is at the center of the image and the base of each cell is at the periphery. 2 example cells are outlined (dashed lines) and expanded in more detail in in C-D, and E-F . B) A 22 min timelapse was taken of the example in A . All EB3-tracks, indicated by magenta arrows (Tracked in Imaris) acquired during the timelapse are shown. C-D) Example time courses of EB3-GFP in hair cells over 21 s; the cell apex is towards the top of each image. In the final image, the 4 images for each example (0-21 s) were projected overtime as a pseudocolor image represented by the colormap. The pseudocolor images show that many EB3-GFP tracks move to the cell base. E-F) The magenta arrows in E and F show all the EB3-GFP tracks acquired in the example cells in C-D over the entire 22 min duration. Arrowheads indicate direction of travel. G) The schematic in G shows how EB3-GFP tracks were aligned to each hair cell. Tracks moving toward the apex have a track angle of 180°, while those moving to the base have an angle of 0°. This analysis revealed that the majority of EB3-GFP tracks (shaded domains) move toward the base of the cell (n = 7 neuromasts, 2597 tracks). Scale bars in A = 5 µm and in C-F = 2 µm.

Article Snippet: To quantify the direction of EB3-GFP tracks we used Imaris to perform 3D analyses to detect and create vectors of EB3-GFP tracks (see arrows; ,F).

Techniques:

Journal: bioRxiv

Article Title: Microtubule-dependent cell polarity regulates skin-resident macrophage phagocytosis and directed cell migration

doi: 10.1101/2025.03.13.642867

Figure Lengend Snippet: A. Illustration of adult zebrafish showing overlapping scales along the trunk skin. B. Confocal images of adult trunk skin expressing reporters for Langerhans cells [Tg( mpeg1:mCherry) ] and alpha-catenin [ Gt(ctnna1-Citrine) ], which labels epidermal junctions. C. Representative image of a Langerhans cell expressing Tg(mpeg1.1:mCherry;mpeg1.1:EMTB-3xGFP) showing EMTB-3xGFP+ dendrites (yellow arrowheads), EMTB-3xGFP-dendrites (white arrows), and the presumptive microtubule organizing center (cyan arrow). D. Violin plot showing percentage of EMTB+ dendrites (n = 12 cells from N = 5 scales). E. Representative still images from time-lapse confocal microscopy of a Tg(mpeg1.1:mCherry;mpeg1.1:EB3-GFP)+ Langerhans cell showing the perinuclear focus of EB3 signal (yellow arrowhead). Yellow box in left panel denotes region magnified in the panels at right. Red and cyan arrows track individual growing microtubules. F-H. Representative still images from time-lapse confocal microscopy depicting effects of vehicle (F), nocodazole (G) , or paclitaxel (H) treatment on Tg(mpeg1.1:YFP) + Langerhans cell morphology. Yellow arrowheads in (G) indicate elongated dendrites. I-K. Violin plots showing average dendrite number (I) , average dendrite length (J) , or maximum dendrite length (K) through 80 minutes of vehicle, nocodazole, or paclitaxel treatment. n = 15 cells tracked from N = 3 scales for vehicle control, n = 27 cells tracked from N = 5 scales for nocodazole, n = 13 cells tracked from N = 6 scales for paclitaxel. L. Sholl analysis of dendrite lengths in vehicle- and nocodazole-treated conditions. n = 698 dendrites tracked in 14 cells from N = 3 scales for vehicle control and n = 476 dendrites tracked in 11 cells from N = 3 scales for nocodazole. Statistical significance in (I, J, K) was determined using Mann-Whitney U test, statistical significance in (L) was determined using a chi-squared test with the raw number of dendrites. * = p < 0.05, **** = p < 0.0001. Timestamps denote mm:ss. Scale bars, 1 mm (B), 100 μm (B’), 10 μm (B’’, C, E, F’-H’) , 20 μm (F-H).

Article Snippet: To amplify EB3-GFP, the Addgene plasmid #190164 was used as template and amplified with the primers EB3-F and EB3-R. Our previously published mpeg1.1 promoter-containing plasmid was amplified using the primers mpeg1.1_V_F’ and mpeg1.1_V_R.

Techniques: Expressing, Confocal Microscopy, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting the acetyltransferase NAT10 corrects pathologies in human frontotemporal dementia neurons and extends lifespan in an in vivo Drosophila tauopathy model

doi: 10.1101/2024.10.19.619211

Figure Lengend Snippet: A-B. Representative images of microtubule track duration (cumulative over a 150 sec interval movie), visualised by live-imaging of an EB3-GFP fusion protein, overlaid on stills from GFP-EB3 live imaging (gray) of control and FTD-MAPT-neurons (120 DIV) after 24 hours treatment with Remodelin or vehicle (DMSO). C-F. Microtubule growth, reflected in EB3 track duration, is reduced by Remodelin treatment in FTD-neurons but not in control neurons. Significance was determined using Student’s t test (ns = not significant; *p < 0.05; ****p < 0.0001); error bar represents SD; n > 15 movies from 3 independent experiments, dots represent the average of one movie).

Article Snippet: Neurons were grown to 100 DIV in individual m-Dish 35 mm dishes (Ibidi) and transfected with a plasmid encoding for GFP-EB3 (gift from Michael Davidson; Addgene plasmid # 56474).

Techniques: Imaging, Control

Journal: bioRxiv

Article Title: Microtubule networks in zebrafish hair cells facilitate presynapse transport and fusion during development

doi: 10.1101/2024.04.12.589161

Figure Lengend Snippet: EB3-GFP tracks show plus-ends of microtubules move to the cell base A) Example image of a neuromast at 2 dpf. The growing or plus-ends of microtubules are marked with EB3-GFP. In this example the apex of 8 developing cells is at the center of the image and the base of each cell is at the periphery. 2 example cells are outlined (dashed lines) and expanded in more detail in in C-D, and E-F . B) A 22 min timelapse was taken of the example in A . All EB3-tracks, indicated by magenta arrows (Tracked in Imaris) acquired during the timelapse are shown. C-D) Example time courses of EB3-GFP in hair cells over 21 s; the cell apex is towards the top of each image. In the final image, the 4 images for each example (0-21 s) were projected overtime as a pseudocolor image represented by the colormap. The pseudocolor images show that many EB3-GFP tracks move to the cell base. E-F) The magenta arrows in E and F show all the EB3-GFP tracks acquired in the example cells in C-D over the entire 22 min duration. Arrowheads indicate direction of travel. G) The schematic in G shows how EB3-GFP tracks were aligned to each hair cell. Tracks moving toward the apex have a track angle of 180°, while those moving to the base have an angle of 0°. This analysis revealed that the majority of EB3-GFP tracks (shaded domains) move toward the base of the cell (n = 7 neuromasts, 2597 tracks). Scale bars in A = 5 µm and in C-F = 2 µm.

Article Snippet: The EB3-GFP timelapses were tracked in Imaris.

Techniques: