DOI: 10.5061/dryad.f1vhhmh3v
title: In vivo timelapse imaging and analysis of Golgi satellite organelle distribution and movement in the neural progenitor cells of the brain

description: The dividing stem cells of the developing brain are the radial glial
 neural progenitor cells (NPCs), multifunctional cells that proliferate to
 generate all of the cells of the brain, but also act as scaffolds for
 their migrating neuron progeny, guideposts for pathfinding growing axons
 and regulators of synaptic activity. These remarkable cells perform these
 very different activities while remaining in contact with the inner and
 outer surface of the ever-growing brain. NPCs synthesize proteins locally
 to support the compartmentalized protein expression required for the cells
 to perform their specialized functions, but it is not clear how the
 necessary processing that normally occurs in the Golgi apparatus is
 achieved at locations far from the cell body. Golgi satellites, motile
 organelles, and members of the protein maturation machinery, control
 protein glycosylation and maturation in polarized cells like neurons. To
 investigate whether NPCs also rely on Golgi satellites, we expressed a
 fluorescent reporter to label Golgi satellites in the NPCs in the intact
 brains of Xenopus laevis tadpoles. Quantitative analysis of in vivo
 timelapse images revealed dynamic, motile Golgi satellites that distribute
 throughout the cell, suggesting that NPCs have local proteostasis to
 support their diverse functions.
description: Confocal images acquired with Slidebook 2023 software (3i. Inc.)
 were exported as .tifs. 
description: # In vivo timelapse imaging and analysis of Golgi satellite organelle
 distribution and movement in the neural progenitor cells of the brain
 [https://doi.org/10.5061/dryad.f1vhhmh3v](https://doi.org/10.5061/dryad.f1vhhmh3v) 3D timelapse confocal images and the files generated by FIJI/ImageJ to complete the morphometric analyses. ## Description of the data and file structure Description of the Image Acquisition Equipment: Confocal z-stacks of labeled cells ≤1 µm z-step intervals from the dorsal surface to ~150 µm depths using Slidebook image acquisition software (3i, Inc.) with the VIVO spinning disc confocal system composed of a Yokogawa CSU-X1 spinning disk confocal and a Photometrics Prime 95B sCMOS camera mounted on a Zeiss Axio Examiner microscope equipped with a C-APO 63x/1.15 objective. The 561 nm laser was used to excite the mCherry-GOLT3 and the 488 nm laser excited EGFP. Signals were distinguished with single bandpass Semrock 617/73 nm and 525/30 nm filters for the mCherry and EGFP signals. Details of the microscope system are available here: [FPBase](https://www.fpbase.org/microscope/UWYt8if6d6LKMJ3seq8NxA/) (Lambert, 2019). Image analysis was conducted using the raw, 16 bit image stacks using FIJI/Image J (Schindelin et al., 2012). ·        “straightened” images were generated using the FIJI/ImageJ tool, Simple Neurite Tracer (SNT; Arsgradi et al., 2021) to reconstruct the cell from the 3D confocal stack and then generate the straightened process image. ·        The Multikymograph tool was used to generate the “kymograph” images collected in this dataset.   Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82. Arshadi C, Gunther U, Eddison M, Harrington KIS, Ferreira TA. SNT: a unifying toolbox for quantification of neuronal anatomy. Nat Methods. 2021;18(4):374-7.     Channels 488:  488nm laser, emission filter: 525/30 – for the EGFP signal 561: 561 nm laser excitation, emission filter: filter: 617/73 for mCherry   0.187745 or 0.295699 microns/pixel Zstep sizes 0.33 to 1 micron (noted with each 3D file) Timelapse images are repeated 3D confocal stacks that are acquired only at 1-micron z-steps and at 30 second intervals. File #: ID number of the dryad file Filename: name of the file in dryad. Figs: the figures of the submitted manuscript that are made from this data. Channels: the 488 or 561 channels (or both) in the image Parameter: image parameters. The files are 3D confocal stacks with or without timelapse (TL). Some files are 2D projections of the 3D files. Some files (.mp4 and .gif) are alternate versions of some files or rendered movies of the cells rotating in 3D. Kymograph images are as described above. Straightened images are as described above.   | **File #** | **Filename** | **Figs** | **Channels** | **Parameters** | | :--------- | :---------------------------------------------- | :------- | :----------- | :-------------------------------------------------------------------------------------------------------------------------------------------------- | | 1 | Fig1d\_2channel\_stack.tif | 1,3 | 488/561 | 0.187745 micron/pixel. 0.38 micron z-step, 238 z planes | | 2 | Fig1d\_3d\_rotation.mp4 | 1 | 488/561 | Rendered 3D rotation movie. Grid = 10 micron | | 3 | Fig1e\_GFP.tif | 1 | 488 | The greyscale projection of Fig 1D’s eGFP channel.  0.187745 micron/pixel. | | 4 | Fig1f\_mCh.tif | 1,3 | 561 | The Greyscale projection of Fig 1D’s mCherry-GOLT3 channel.  0.187745 micron/pixel. | | 5 | Fig1f\_Straightened\_Process.tif | 1,3 | 561 | Straightened process of Fig1. | | 6 | Fig1\_Kymograph.tif | 3 | 561 | Kymograph rendering of the cell in Figure 1 | | 7 | Fig1\_TL\_max\_projection.tif | 3 | 561 | Assembled 2D projection of the 3D timelapse images  Fig1\_TL\_T00  to \_T19 | | 8 | Fig1\_TL\_T00.tif | 3 | 561 | 0 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 9 | Fig1\_TL\_T01.tif | 3 | 561 | 30 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 10 | Fig1\_TL\_T02.tif | 3 | 561 | 60 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 11 | Fig1\_TL\_T03.tif | 3 | 561 | 90 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 12 | Fig1\_TL\_T04.tif | 3 | 561 | 120 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 13 | Fig1\_TL\_T05.tif | 3 | 561 | 150 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 14 | Fig1\_TL\_T06.tif | 3 | 561 | 180 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 15 | Fig1\_TL\_T07.tif | 3 | 561 | 210 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 16 | Fig1\_TL\_T08.tif | 3 | 561 | 240 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 17 | Fig1\_TL\_T09.tif | 3 | 561 | 270 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 18 | Fig1\_TL\_T10.tif | 3 | 561 | 300 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 19 | Fig1\_TL\_T11.tif | 3 | 561 | 330 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 20 | Fig1\_TL\_T12.tif | 3 | 561 | 360 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 21 | Fig1\_TL\_T13.tif | 3 | 561 | 390 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 22 | Fig1\_TL\_T14.tif | 3 | 561 | 420 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 23 | Fig1\_TL\_T15.tif | 3 | 561 | 450 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 24 | Fig1\_TL\_T16.tif | 3 | 561 | 480 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 25 | Fig1\_TL\_T17.tif | 3 | 561 | 510 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 26 | Fig1\_TL\_T18.tif | 3 | 561 | 540 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 27 | Fig1\_TL\_T19.tif | 3 | 561 | 570 sec. 0.187745 micron/pixel, 91 planes, z step = 1 micron | | 28 | Fig1\_30sec\_Interval\_TL\_projection.tif | 3 | 561 | Assembled 2D projection of the 3D timelapse data in files 8-27 | | 29 | Fig1\_3D\_timelapse\_stack.tif | 3 | 561 | Assembled 3D timelapse of the data in files 8-27 | | 30 | Fig2a.tif | 2 | 488/561 | 2D projection of the 3D confocal stack. A 2 channel image. | | 31 | Fig2d-g\_3D\_timelapse\_stacks.tif | 2,3 | 561 | The 3D timelapse image stack. 0.295699 microns/pixel, 80 1 micron z slices 30 sec. interval time intervals. | | 32 | Fig2d-g\_Maxprojection.tif | 2.3 | 561 | 2D projection of the 30 timelapse images. 30 sec intervals. 0.295699 microns/pixel. | | 33 | Fig2d-g\_timelapse.avi | 2 | 561 | Movie version of the timelapse. | | 34 | Fig2d-g\_timelapse.tif | 2 | 561 | 2D projection of the 30 timelapse images. 30 sec intervals. 0.295699 microns/pixel. Greyscale inverted. | | 35 | Fig2H\_straightened.tif | 2,3 | 561 |  The straightened stack of the Fig2 cell. | | 36 | Figure\_2H\_Kymograph.tif | 2,3 | 561 | Kymograph of the cell in figure 2. | | 37 | Figure2\_rotation.mp4 | 2 | 488/561 | Rendered movie of 3D rotating cell from figure 2 | | 38 | Figure2\_rotation2.mp4 | 2 | 488/561 | Rendered movie of 3D rotating cell from figure 2 | | 39 | TL1.gif | 3 | 561 | GIF timelapse version of TL1.tif | | 40 | TL1.tif | 3 | 561 | Original 16 bit .tif of the timelapse image. 0.187745 microns/pixel. 30 sec intervals. 65 1 micron z step stack. | | 41 | TL1\_Kymograph.tif | 3 | 561 | Kymograph of TL1 timelapse. | | 42 | TL1\_lowmagstack.wmv | 3 | 488/561 | Fluorescent images + brightfield of the image stack | | 43 | stk\_TL1\_0001\_.tif | 3 | 561 | 0 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 44 | stk\_TL1\_0002\_.tif | 3 | 561 | 30 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 45 | stk\_TL1\_0003\_.tif | 3 | 561 | 60 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 46 | stk\_TL1\_0004\_.tif | 3 | 561 | 90 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 47 | stk\_TL1\_0005\_.tif | 3 | 561 | 120 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 48 | stk\_TL1\_0006\_.tif | 3 | 561 | 150 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 49 | stk\_TL1\_0007\_.tif | 3 | 561 | 180 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 50 | stk\_TL1\_0008\_.tif | 3 | 561 | 210 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 51 | stk\_TL1\_0009\_.tif | 3 | 561 | 240 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 52 | stk\_TL1\_0010\_.tif | 3 | 561 | 270 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 53 | stk\_TL1\_0011\_.tif | 3 | 561 | 300 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 54 | stk\_TL1\_0012\_.tif | 3 | 561 | 330 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 55 | stk\_TL1\_0013\_.tif | 3 | 561 | 360 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 56 | stk\_TL1\_0014\_.tif | 3 | 561 | 390 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 57 | stk\_TL1\_0015\_.tif | 3 | 561 | 420 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 58 | stk\_TL1\_0016\_.tif | 3 | 561 | 450 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 59 | stk\_TL1\_0017\_.tif | 3 | 561 | 480 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 60 | stk\_TL1\_0018\_.tif | 3 | 561 | 510 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 61 | stk\_TL1\_0019\_.tif | 3 | 561 | 540 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 62 | stk\_TL1\_0020\_.tif | 3 | 561 | 570 sec. 0.187745 micron/pixel, 65 planes, z step = 1 micron | | 63 | TL2\_3D\_timelapse\_Max\_projection.tif | 3 | 561 | 30 sec interval 2D timelapse. Projection of 75 micron stack. 0.295699 micron/pixel. | | 64 | TL2\_kymograph.tif | 3 | 561 | Kymograph of the TL2\_3D timelapse | | 65 | TL2-3\_TL\_projection.tif | 3 | 561 | 30 sec interval 2D timelapse. Projection of 75 micron stack. 0.295699 micron/pixel. Contains TL2 and TL3 files. File contains cells in TL2 and TL3. | | 66 | TL2-3 TL projection\_2channel\_2Dprojection.tif | 3 | 488/561 | 2D projection of the 3D confocal stack (TL2-3 TL projection\_2channel\_stack.tif) 0.295699 micron/pixel, 0.33 micron z-step size, 456 z steps. | | 67 | TL2-3 TL projection\_2channel\_Stack.tif | 3 | 488/561 | confocal stack captured at 0.295699 micron/pixel, 0.33 micron z-step size, 456 z steps. | | 68 | TL3\_kymograph.tif | 3 | 561 | Kymograph of TL3\_TL projection file, TL3\_max\_timelapse | | 69 | TL3\_max\_timelapse.tif | 3 | 561 | | | 70 | 30secTL2-3\_T00.tif | 3 | 561 | 0 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 71 | 30secTL2-3\_T01.tif | 3 | 561 | 30 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 72 | 30secTL2-3\_T02.tif | 3 | 561 | 60 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 73 | 30secTL2-3\_T03.tif | 3 | 561 | 90 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 74 | 30secTL2-3\_T04.tif | 3 | 561 | 120 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 75 | 30secTL2-3\_T05.tif | 3 | 561 | 150 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 76 | 30secTL2-3\_T06.tif | 3 | 561 | 180 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 77 | 30secTL2-3\_T07.tif | 3 | 561 | 210 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 78 | 30secTL2-3\_T08.tif | 3 | 561 | 240 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 79 | 30secTL2-3\_T09.tif | 3 | 561 | 270 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 80 | 30secTL2-3\_T10.tif | 3 | 561 | 300 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 81 | 30secTL2-3\_T11.tif | 3 | 561 | 330 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 82 | 30secTL2-3\_T12.tif | 3 | 561 | 360 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 83 | 30secTL2-3\_T13.tif | 3 | 561 | 390 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 84 | 30secTL2-3\_T14.tif | 3 | 561 | 420 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 85 | 30secTL2-3\_T15.tif | 3 | 561 | 450 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 86 | 30secTL2-3\_T16.tif | 3 | 561 | 480 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 87 | 30secTL2-3\_T17.tif | 3 | 561 | 510 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 88 | 30secTL2-3\_T18.tif | 3 | 561 | 540 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 89 | 30secTL2-3\_T19.tif | 3 | 561 | 570 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 90 | 30secTL2-3\_T20.tif | 3 | 561 | 600 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 91 | 30secTL2-3\_T21.tif | 3 | 561 | 630 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 92 | 30secTL2-3\_T22.tif | 3 | 561 | 660 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 93 | 30secTL2-3\_T23.tif | 3 | 561 | 690 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 94 | 30secTL2-3\_T24.tif | 3 | 561 | 720 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 95 | 30secTL2-3\_T25.tif | 3 | 561 | 750 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 96 | 30secTL2-3\_T26.tif | 3 | 561 | 780 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 97 | 30secTL2-3\_T27.tif | 3 | 561 | 810 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 98 | 30secTL2-3\_T28.tif | 3 | 561 | 840 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron | | 99 | 30secTL2-3\_T29.tif | 3 | 561 | 970 sec. 0.295699 micron/pixel, 75 planes, z step = 1 micron |   ## Sharing/Access information not applicable ## Code/Software not applicable