Dataset DOI: 10.5061/dryad.zkh1893nt

Description of the data and file structure

This dataset contains 5 types of primary data, most of which are live imaging data of tadpole optic nerves. Most data are either time series(t-series) of single focal planes, or single-time sections spanning the thickness of the optic nerve (z-series); both data types were acquired using a spinning disc confocal microscope. There is one image acquired with a stereo-fluorescence microscope. There is one figure containing both a z-series and serial block-face scanning electron microscopy (SBEM)for the same nerve and including an x-ray image of the fixed and embedded tissue block, which was used in the registration of the fluorescence and SBEM data.

T- and z-series fluorescence data were collected using an Andor Dragonfly Spinning Disc Confocal microscope. T-series were collected sequentially for different channels.  In the z-series, different channel data were collected at each individual z-position before moving to a new z-position. In z-series with two colors, green fluorescence was acquired in channel 1 and red was acquired in channel 2. In the z-series with three colors, green fluorescence was acquired in channel 1, red fluorescence in channel 2, and far-red fluorescence in channel 3. In the z-series with four colors, blue fluorescence was acquired in channel 1, green fluorescence in channel 2, red fluorescence in channel 3, and far-red fluorescence in channel 4. T-series analyses were performed using custom kymograph scripts written for IPLab software. Z-series analyses were performed using Imaris software.

X-ray data were acquired with an X-ray microscope (XRM) instrument. SBEM data was collected with a 3View unit.

Files attached contain both the raw data supporting primary and supplementary figures, as well as the analyses for each of these. Given the large number and size of files, they had to be organized and compressed to be able to be uploaded to Dryad. The raw data corresponding to each figure are provided separately for each figure, either all in one compressed file or subdivided in parts. All analysis files, also organized by figures, are provided also compressed and in 5 parts.

Abbreviations

Files and variables

File: Figure_5_part_2.zip

Description: Raw Data Files for Figure 5, part 2.

File: Figure_5_part_3.zip

Description: Raw Data Files for Figure 5, part 3.

File: Figure_1.zip

Description: Raw Data Files for Figure 1.

File: Figure_2_part_1.zip

Description:  Raw Data Files of Figure 2, part 1.

File: JeongEtAl_2025_Analysis_part_1.zip

Description: Analysis Files, part 1. Analysis files for Fig. 1, 2 and first part of Fig. 3.  Contain Excel files with data, GraphPad Prism files with data, graphs and statistics, and Imaris files containing the measured surfaces.

File: JeongEtAl_2025_Analysis_part_2.zip

Description:  Analysis Files, part 2. Analysis files for the second part of Fig. 3. Contain Imaris files containing the measured surfaces.

File: JeongEtAl_2025_Analysis_part_3.zip

Description:  Analysis Files, part 3. Analysis files for the third part of Fig. 3. Contain Excel files with data, GraphPad Prism files with data, graphs and statistics, and Imaris files containing the measured surfaces.

File: JeongEtAl_2025_Analysis_part_4.zip

Description: Analysis Files, part 4. Analysis files for the first part of Fig. 4. Contain Excel files with data, GraphPad Prism files with data, graphs and statistics, and Imaris files containing the measured surfaces.

File: JeongEtAl_2025_Analysis_part_5.zip

Description: Analysis Files, part 5. Analysis files for the second part of Fig. 4, Fig. 5, Fig. 6 and Supplemental Figs. 1-4.  Contain mainly Excel files with data, GraphPad Prism files with data, graphs and statistics, but Fig. 4 also Imaris files containing the measured surfaces, and for Fig. 5 two .txt files containing coordinate data for the models.

File: Figure_2_part_2.zip

Description: Raw Data Files for Figure 2, part 2.

File: Figure_2_part_3.zip

Description: Raw Data Files for Figure 3, part 3.

File: Figure_4_part_1.zip

Description: Raw Data Files for Figure 4, part 1.

File: Figure_5_part_1.zip

Description: Raw Data Files for Figure 5, part 1.

File: Figure_3.zip

Description: Figure 3 Raw Files.

File: Movies.zip

Description: All movies.

File: Figure_4_part_2.zip

Description: Raw Data Files for Figure 4, part 2.

File: Figure_6.zip

Description: Figure 6 Raw Files.

File: Figure_4_part_3.zip

Description: Raw Data Files for Figure 4, part 3.

File: Sup_Figure_1.zip

Description: Raw Data Files for Supplementary Figure 1.

File: Sup_Figure_2_part_3.zip

Description: Raw Data Files for Supplementary Figure 2, part 3.

File: Sup_Figure_3_part_1.zip

Description: Raw Data Files for Supplementary Figure 3, part 1.

File: Sup_Figure_2_part_1.zip

Description: Raw Data Files for Supplementary Figure 2, part 1.

File: Sup_Figure_2_part_2.zip

Description: Raw Data Files for Supplementary Figure 2, part 2.

File: Sup_Figure_3_part_4.zip

Description: Raw Data Files for Supplementary Figure 3, part 4.

File: Sup_Figure_4_part_1.zip

Description: Raw Data Files for Supplementary Figure 4, part 1.

File: Sup_Figure_3_part_5.zip

Description: Raw Data Files for Figure 3, part 5.

File: Sup_Figure_2_part_4.zip

Description: Raw Data Files for Supplementary Figure 2, part 4.

File: Sup_Figure_3_part_2.zip

Description: Raw Data Files for Supplementary Figure 3, part 2.

File: Sup_Figure_3_part_3.zip

Description: Raw Data Files for Supplementary Figure 3, part 3.

File: Sup_Figure_4_part_2.zip

Description: Raw Data Files for Supplementary Figure 4, part 2.

File: JeongEtAl_2025_excel.csv

Description: Table with both the file names as they appear in Dryad and the corresponding original name that contains a time stamp.

Description of the data and file structure

We have provided our raw data corresponding to each figure or supplementary figure in parts, to comply with the size limit requirements of the Dryad depository and similarly, all analysis files in five separate folders, all compressed in .zip formats.

Raw data

Due to formatting requirements of the UC Davis Dryad data repository, some of the original Raw data in their original formats and naming schemes are not provided.  Rather, raw data is provided only in processed forms, as .tif formats. The processed files have been renamed to make explicit the Figure panel(s) for which they correspond. The naming convention list files as belonging to this study, by the prefix YJ, followed by the Figure and Panel to which they correspond, followed by the date the original data was acquired. An Image_Renaming table, YJ_rawdata_table.xlsx, is provided listing all image files in both their renamed formats as well as in their original formats; original data had unique identifier names which were based on date and time of acquisition. The data in their original formats and names are only available upon request. Some raw data files were used for multiple figures or panels, in which case the same original data raw file is presented duplicated with different processed names. In the Image_Renaming table, all such examples have been marked by a D in the 4th column.

Folders for Figures 1, S1, 2, S2, and S4 contain exclusively t-series data, with the exception of Figure 1, which also contains a lower resolution image of the setup acquired with a stereo-fluorescence microscope.

Folders for Figures 3 and 4 contain exclusively z-series data.

Folders for Figure S3 contain a combination of t-series and z-series data.

The folder for Figure 5 contains correlated light and SBEM. data. A lower resolution single channel image for the entire length of the optic nerve, presented as a single maximum intensity projection, was obtained by registering multiple overlapping z-series in 3-dimensions prior to the projection.  A higher resolution 4-color z-series was obtained for the same nerve, centered on a region of interest (ROI). An X-ray image for the fixed and embedded block containing the head of the same animal lived imaged was produced by rotating X-ray tomographic data to correspond with the rotation angle of the live imaging data, and is presented solely as a maximum intensity projection. The raw SBEM data for the region of the optic nerve is presented in .tif format, converted from its original .mrc formats using the IMOD mrc2tif command, as the original format was also not supported by the UCD dryad. It can be reconverted back to its original format by using the tif2mrc command in IMOD.

Folder for Figures 6 contains a t- and z-series.  That is, a ROI imaged as a z-series was reimaged as a t-series every 30 seconds.

Analysis

Time-Series (t-series) Analysis – Figures 1, S1, 2, S2, S3, and S4

To analyze time-lapse microscopy data, we developed a custom script in IPLab, which is included in this dataset. The analysis began with manual tracing of the optic nerve’s contour in each image. Once outlined, the script automatically divided the nerve into a series of evenly spaced segments along its length.

For each of these segments, a kymograph was generated. These images are powerful tools for visualizing the movement of particles over time. On each kymograph, we manually traced clear, continuous lines—each one corresponding to the path of a moving particle. From these traces, we extracted two key metrics: the proportion of stationary versus moving particles (in both directions), and the average velocity of those that moved.

In experiments with sparsely labeled axons, we adjusted the approach slightly. Instead of dividing the nerve into equal parts, we manually traced individual axons before generating kymographs. These variants of the script are also included.

While intermediate files such as kymographs and manual traces were generated during the analysis, they are not included here due to technical limitations. Many kymographs were processed using older software versions without standardized export options, and in some cases, tracing was performed live without generating separate files. However, all raw image sequences and the full analysis scripts are provided, allowing complete reproduction of the kymographs and quantitative analyses described in the manuscript.

The quantitative results from kymograph analysis were first averaged at the nerve level, combining data across all segments within a single optic nerve. These nerve-level values were then summarized per animal to generate a final set of metrics for statistical comparison. These results are saved in Excel spreadsheets named according to the figure and panel they correspond to—for example, YJ_Fig1E_table.xlsx. These summary tables were then imported into GraphPad Prism for statistical analysis and figure generation. The Prism project files are also included and follow a matching naming convention (e.g., YJ_Fig1E_graph.pzf).

Z-Series Analysis via Imaris – Figures 3, S3, and 4

For z-stack imaging data, we used Imaris software to perform 3D segmentation of particles within axons. Each dataset was first masked to isolate the optic nerve. Using fluorescent labeling, particles were segmented and their volumes calculated based on consistent processing parameters.

Each Imaris analysis produced two main outputs: first, structured Excel spreadsheets listing individual particles, their volumes, and group assignments (e.g., Wt vs E50K). These files follow a consistent naming format, such as YJ_Fig3B_table.xlsx. Second, the Imaris .ims files are included to ensure full reproducibility. These files preserve all processing parameters—including thresholds, segmentation settings, and masking details—and can be opened in ImageJ or Fiji using the Bio-Formats plugin. These files follow a naming format like YJ_Fig3A_3DImage.ims.

In some experiments, we distinguished particles located inside axons from those outside. This was achieved by creating additional masks for axons and for the nerve boundary within Imaris.

As with the time-series analysis, summary Excel files were imported into GraphPad Prism for statistics and figure creation. The corresponding .pzfx or .pzf files are included in the same structured format (e.g., YJ_Fig3B_graph.pzfx).

Electron Microscopy (EM) Analysis – Figure 5

Our EM analysis was primarily qualitative and focused on generating 3D models from correlated light EM datasets. These models were manually traced and represent individual astrocytes and axons. The included .txt files are converted versions of the original .mod models generated with IMOD software. These were created using the model2point command and can be converted back to .mod format using point2model. Once converted, they can be visualized alongside the corresponding .mrc files in IMOD.

Mito sphericity and fluorescence intensity analysis -Figure 6

Mitochondria sphericity in Figure 6C was determined using Imaris.

Figure 6D–F show changes in fluorescence intensity within axon protrusions and the axon of origin, as well as the minimum distance between protrusions and the parent axon, supporting the interpretation of active cargo loading and mitophagy. Fluorescence intensity and localization for each label were measured at defined ROIs over time using IPLab software with a custom script, which is included in the dataset. The resulting data were compiled into summary spreadsheets (e.g., YJ_Fig6E_table.xlsx) and imported into GraphPad Prism for analysis. Corresponding Prism project files (.pzfx or .pzf) are also included, following the same structured naming format (e.g., YJ_Fig6E_graph.pzfx).

Software

• .tif image files can be opened in most standard image viewers, including ImageJ and Fiji.
• .txt files are readable in Excel, Word, or any text editor.
• .pzfx and .pzf files require GraphPad Prism.
• .ims files can be opened in Imaris or in Fiji (ImageJ) with the Bio-Formats plugin.