DOI: 10.5061/dryad.8sf7m0d01
title: Data for: Doublecortin reinforces microtubules to promote growth cone advance in soft environments

description: Doublecortin (DCX) is a microtubule (MT)-associated protein in immature
 neurons. DCX is essential for early brain development, and DCX mutations
 account for nearly a quarter of all cases of lissencephaly-spectrum brain
 malformations that arise from a neuronal migration failure through the
 developing cortex. By analyzing pathogenic DCX missense mutations in
 non-neuronal cells, we show that disruption of MT-binding is central to
 DCX pathology. In human induced pluripotent stem cell (hiPSC)-derived
 cortical i3Neurons genome-edited to express DCX-mEmerald from the
 endogenous locus, DCX-MT interactions polarize very early during neuron
 morphogenesis. DCX interacts with MTs through two conserved DCX domains
 that bind between protofilaments and adjacent tubulin dimers, a site that
 changes conformation during GTP hydrolysis. Consequently and consistent
 with our previous results, DCX specifically binds straight growth cone MTs
 and is excluded from the GTP/GDP-Pi cap recognized by EB1. Comparing
 MT-bound DCX fluorescence to mEmerald-tagged nanocage standards, we
 measure approximately one hundred DCX molecules per micrometer growth cone
 MT. DCX is required for i3Neuron growth cone advance in soft
 microenvironments that mimic the viscoelasticity of brain tissue, and
 using high resolution traction force microscopy, we find that growth cones
 produce comparatively small and transient traction forces. Given our
 finding that DCX stabilizes MTs in the growth cone periphery by inhibiting
 MT depolymerization, we propose that DCX contributes to growth cone
 biomechanics and reinforces the growth cone cytoskeleton to counteract
 actomyosin generated contractile forces in soft physiological environments
 in which weak and transient adhesion-mediated traction may be insufficient
 for productive growth cone advance.
description: This dataset contains raw data and analysis used to produce
 figures and conclusions associated with the referenced Current Biology
 manuscript. Detailed description of the data collection and analysis
 methodology can be found in the associated manuscript.
 Raw microscopy data are provided as 16-bit Nikon ND2 files that
 are native to Nikon Elements microscopy software or TIFF files. ND2 files
 can also be opened with ImageJ/Fiji with the Bioformats plugin (Note:
 metadata may not be imported correctly into ImageJ), or with a freely
 available Nikon Elements Viewer. Data analysis on which
 manuscript figures are based are provided as Microsoft Excel
 spreadsheets.
description: # Data for: Doublecortin reinforces microtubules to promote growth cone
 advance in soft environments
 [https://doi.org/10.5061/dryad.8sf7m0d01](https://doi.org/10.5061/dryad.8sf7m0d01) **Corresponding investigator:** Torsten Wittmann [torsten.wittmann@ucsf.edu](mailto:torsten.wittmann@ucsf.edu) University of California, San Francisco **Co-Investigators:** Alessandro Dema (current address: Helmholtz Zentrum München, Germany) Rabab A Charafeddine Jeffrey van Haren (Erasmus MC, Rotterdam, The Netherlands) Shima Rahgozar Giulia Viola Kyle L Jacobs Matthew L Kutys University of California, San Francisco #### Description This dataset contains raw data and analysis used to produce figures and conclusions associated with the associated Current Biology paper (will add link when published). Detailed description of the data and analysis methodology can be found in the associated manuscript. Raw 16-bit microscopy data are provided as Nikon ND2 files that are native to Nikon Elements microscopy software. ND2 files can also be opened with ImageJ/Fiji with the Bioformats plugin (Note: microscopy metadata may not be imported correctly into ImageJ), or with a freely available Nikon Elements Viewer. Data analysis on which manuscript figures are based are provided as Microsoft Excel spreadsheets. Data are organized by figure panel in the manuscript with .ZIP files containing both raw data and analysis for each figure. See manuscript for full experimental detail on each figure. Outlined below is where to find which kind of data with additional explanation on Excel spreadsheets containing all data used to generate the corresponding figure panels. --- #### Figure 1 ***Microscopy Data Folders*** **Figure_1A**: Time-lapse data of DCX-mEmerald and SPY555-tubulin dynamics in an i3Neuron growth cone. **Figure_1C**: Time-lapse data of DCX-mEmerald dynamics in a paclitaxel-treated i3Neuron growth cone. **Figure_1D**: Time-lapse data of DCX-mEmerald and EB3-tagRFP dynamics in an i3Neuron growth cone. **Figure_1E**: Z-stacks of DCX-mEmerald and SPY555-tubulin in an i3Neuron. These images were used to generate the composite image shown in 1E. **Figure_1F_G**: Images of transfected RPE cells used to generate the I3-01 nanocage fluorescence intensity standard curve in 1F (Folders mEmerald_I3 and mEmerald_mEmerald_I3), and DCX-mEmerald i3Neuron growth cone images used to measure DCX molecule number in 1G (Folder: i3Neurons). These data are also related to Figure S4. **Figure_1H_I**: Images of DCX-mEmerald and SPY555-tubulin i3Neuron growth cones used to estimate neurite shaft MT number (1H) and DCX growth cone enrichment (1I). ***Data Analysis*** **Figure_1F_1G_nanocages_DCX.xlsx** Tab 'Nanocage analysis in RPEs (1F)'contains all nanocage 2D Gaussian fit parameters and fluorescence intensity measurements with ten nanocages per cell and ten cells each. Tab 2 'DCX on growth cone MTs (1G)' contains all DCX_mEmerald fluorescence measurements along i3Neuron growth cone MTs. Corresponding measurements from straight and curved MT segments are in the same rows. However, fewer curved MT segments were measured resulting in empty cells. **Figure_1H_GCvsShaft.xlsx** Tab 'SPY555tub_DCX_analysis' contains SPY555-tubulin and DCX-mEmerald measurements from individual growth cone microtubules and the neurite shaft. Tab 'Shaft MT number' are the estimates of neurite shaft microtubule number from this and a similar experiments in fixed cells with the colors indicating three independent experiments. **DCX_mEmerald_genomicPCR_sequence.gbk** is an annotated Genebank file of the genomic PCR sequencing result from DCX-mEmerald i3Neurons showing the in-frame mEmerald insertion at the DCX exon 7 3' end. --- #### Figure 2 ***Microscopy Data Folders*** **Figure_2F_G**: This folder contains three subfolders corresponding to the different genotypes each containing time-lapse SiR-lysosome microscopy data used to track lysosome velocities. Folders for individual i3Neurons also contain the ROIs used to differentiate neurite shaft and growth cone as well as all the output generated by the MATLAB-based u-track tracking algorithm. Details are given in the paper. ***Data Analysis*** **Figure_2B_Day1_neurite_length.xlsx** contains neurite lengths measurements in μm for the indicated conditions. **Figure_2E_neurite_numbers.xlsx** contains neurite numbers extending into the channels with or without BDNF. **Figure_2G_lysosome_tracking_data.xlsx** contains lysosome tracking data in the indicated conditions. --- #### Figure 3 ***Microscopy Data Folders*** **Figure_3D_E** This folder contains three subfolders corresponding to the different genotypes each containing SPY555-tubulin time-lapse sequences used to quantify growth cone microtubule polymerization dynamics. ***Data Analysis*** **Figure_3B_MT_distance_GC_edge.xlsx** contains distance measurements of the longest three pioneer microtubules to the growth cone edge in the indicated conditions. **Figure_3D_E_MT_dynamics.xlsx** Tab 'All MT track rates' contains the frame-to-frame growth and shortening rate measurements organized by cell and MT. Note that each track has different lengths. Hence, there are many empty fields. Tab 'All frame-to-frame rates' contains these data reorganized by condition corresponding to the violin plots in Figure 3D. Tab 'Phases' contains the precentage of time each tracked microtubule spent in growth, shortening, or pause in the different conditions. Tab 'Averages per cell' contains all data averaged per cell corresponding to the box plots in Figure 3D and 3E. --- #### Figure 4 ***Microscopy Data Folders*** **Figure_4A_B** This folder contains three subfolders corresponding to the different genotypes each containing SPY555-FastAct time-lapse sequences used to quantify growth cone F-actin retrograde flow rates. **Figure_4C_to_G** This folder contains four subflolders. 'DCX_null' and 'WT' contain the fluorescent bead and CellMASK time-lapse sequences used to determine traction stress fields. 'DCX_null_TFM_analyzed' and 'WT_TFM_analyzed' are the downsampled images with the traction stress field overlaid. Note that these files are floating point images and may not open properly in ImageJ/Fiji. These files also contain the ROIs used to define the growth cone area to calculate average traction stress (4F). ***Data Analysis*** **Figure_4A_B_actin retrograde flow.xlsx** contains F-actin retrograde flow measurements in the indicated conditions. **Figure_4F_G_traction_stress.xlsx** Tab 'GC_forces (4F)' summarizes traction stress and strain energy measurements for WT and DCX -/Y (A6) i3Neuron growth cones. The 'WT_filo_forces (4G)' and 'A6_filo_forces (4G)' tabs contain the total force measurements generated by transient traction peaks along filopodia as well as the Gaussian fit results of these profiles over time. --- #### suppl\_Figures ***Microscopy Data Folders*** **Figure_S1_S2** This folder contains images of RPE cells transfected with the indicated DCX missense mutation constructs. **Figure_SF_H** contains long-term timelapse data of DCX-mEmerald and SPY555-FastAct. ***Data Analysis*** **Figure_S1_S2_RPE_DCX_mutation_MT_binding.xlsx** Tab 'RPE_DCX_MT_binding' contains all microtubule to cytoplasm ratio measurements of the indicated missense mutations for all quantified cells. Tab 'Normalized_data' are the average per experiment normalized to the binding ratio of the WT construct.