DOI: 10.5061/dryad.gb5mkkwt3
title: Data for: The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)

description: To establish the microtubule cytoskeleton, the cell must tightly regulate
 when and where microtubules are nucleated. This regulation involves
 controlling the initial nucleation template, the γ-tubulin ring complex
 (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity
 is restricted to specific sites including the centrosome and Golgi. The
 well-conserved γ-tubulin nucleation activator (γTuNA) domain has been
 reported to increase the number of microtubules (MTs) generated by γTuRCs.
 However, previously we and others observed that γTuNA had a minimal effect
 on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et
 al., eLife, 2020; Liu et al., 2020). Here we instead report, based on
 improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time
 observation that γTuNA directly increases γTuRC activity in vitro, which
 is thus a bona fide γTuRC activator. We further validate this effect in
 Xenopus egg extract. Via mutation analysis, we find that γTuNA is an
 obligate dimer. Moreover, efficient dimerization as well as γTuNA's
 L70, F75, and L77 residues are required for binding to and activation of
 γTuRC. Finally, we find that γTuNA's activating effect opposes
 inhibitory regulation by stathmin. In sum, our improved assays prove that
 direct γTuNA binding strongly activates γTuRCs, explaining previously
 observed effects of γTuNA expression in cells and illuminating how
 γTuRC-mediated microtubule nucleation is regulated.
description: Short description: The archive contains the
 following:  1) raw microscopy images, videos collected
 via total internal reflection fluorescence (TIRF) for the
 following:     1a) <em>Xenopus laevis</em>
 egg extracts treated with gTuNA (with fluorescent end-binding protein 1
 marking growing microtubule plus-ends);     1b) in
 vitro nucleation assays with purified gTuRC and gTuNA at 15 µM tubulin
 (with fluorescent tubulin to visualize microtubules);  
   1c) in vitro assays with purified gTuNA bound to beads (post-extract
 pulldown);     1d) in vitro nucleation assays with
 purified gTuRC and gTuNA at 7 µM tubulin (critical concentration for gTuRC
 activity);     1e) in vitro nucleation assays with
 purified gTuRC, gTuNA, and stathmin at 15 µM tubulin;
 2) images of purified gTuRCs generated via negative-stain
 electron microscopy; 3) size-exclusion chromatography
 files generated by GE Unicorn software (for Akta Pure Systems);
 4) mass spectrometry data to identify co-precipitants of gTuNA
 from extract pulldowns; 5) supplemental videos of
 processed data.  The included raw microscopy data was
 binarized using Otsu's thresholding method (ImageJ/FIJI) to remove
 background signal. These binarized movies were then quantified either
 using fully automated analysis (for total signal intensity) or a
 semi-automated pipeline for counting individual microtubules in
 movies. The archive is structured first by the
 data's relation to specific figures in our manuscript, with a short
 title describing whether it was taken from Xenopus egg extract or in vitro
 assays, and what specific assay it contains. Inside those folders will be
 either the raw data or another internal file structure with each number
 representing the steps in our processing with raw data first, processed
 data second, the tabulated data and/or the MATLAB script used to generate
 plots.   Detailed metadata
 (from README.md in dataset download) :  
 Reference Information
 =====================  
 Provenance for this README
 -------------------------- * File name:
 README.txt (also README.md) * Authors: Michael J.
 Rale * Other contributors: Brianna Romer, Brian P.
 Mahon, Sophie M. Travis, and Sabine Petry * Date
 created: 2022-12-14 * Date modified:
 2022-01-19   Dataset Version and
 Release History
 ----------------------------------- * Current
 Version:   * Number: 1.0.2   * Date:
 2023-01-23   * Persistent identifier: DOI:
 10.5061/dryad.gb5mkkwt3   * Summary of changes: New
 README with expanded details on each folder and sub-folder in the
 archive. * Embargo Provenance: n/a  
 * Scope of embargo: n/a   * Embargo period:
 n/a   Dataset Attribution and
 Usage -----------------------------
 * Dataset Title: "Data for: The conserved centrosomin motif,
 γTuNA, forms a dimer that directly activates microtubule nucleation by the
 γ-tubulin ring complex" * Persistent Identifier:
 https://doi.org/10.5061/dryad.gb5mkkwt3 * Dataset
 Contributors:   * Creators: Michael J. Rale, Brianna
 Romer, Brian P. Mahon, Sophie M. Travis, and Sabine Petry
 * Date of Issue: 2022-10-17 * Publisher:
 Princeton University * License: Use of these data is
 covered by the following license:   * Title: CC0 1.0
 Universal (CC0 1.0)   * Specification:
 https://creativecommons.org/publicdomain/zero/1.0/ *
 Suggested Citations:   * Dataset citation:
     &gt; Rale, Michael et al. (2022), Data for: The conserved
 centrosomin motif, γTuNA, forms a dimer that directly activates
 microtubule nucleation by the γ-tubulin ring complex, Dryad, Dataset,
 https://doi.org/10.5061/dryad.gb5mkkwt3   *
 Corresponding publication:    &gt; Michael J Rale,
 Brianna Romer, Brian P Mahon, Sophie M Travis, Sabine Petry (2022) The
 conserved centrosomin motif, γTuNA, forms a dimer that directly activates
 microtubule nucleation by the γ-tubulin ring complex (γTuRC). eLife
 11:e80053 https://doi.org/10.7554/eLife.80053
   Contact Information
 -------------------   * Name: Michael J.
 Rale   * Affiliations: Department of Molecular Biology,
 Princeton University; Department of Cell Biology, Harvard Medical
 School   * ORCID ID: 0000-0003-1426-6611
   * Email: michael_rale@hms.harvard.edu (current)
   * Alternate Email: mrale@princeton.edu   *
 Alternate Email 2: n/a   * Address: Dept. of Cell
 Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA
 02115   * Alternative Contact:
 Corresponding Author   * Name: Sabine Petry
   * Affiliations: Department of Molecular Biology, Princeton
 University   * ORCID ID: 0000-0002-8537-9763
   * Email: spetry@princeton.edu   * Address:
 401 Schultz Laboratory, Princeton University, Princeton, NJ
 08544   * Contributor ORCID
 IDs:   * Michael J. Rale:
 https://orcid.org/0000-0003-1426-6611   * Brianna
 Romer: https://orcid.org/0000-0003-1772-4243   * Brian
 P. Mahon: https://orcid.org/0000-0002-5571-8058   *
 Sophie M. Travis: https://orcid.org/0000-0002-1728-1705
   * Sabine Petry: https://orcid.org/0000-0002-8537-9763
 - - - Additional Dataset Metadata
 ===========================
 Acknowledgements ----------------
 * Funding sources: National Institutes of Health, Award: New
 Innovator Award 1DP2GM123493 to SP; Pew Scholars Program in the Biomedical
 Sciences, Award: 00027340 to SP; David and Lucile Packard Foundation,
 Award: 2014–40376 to SP; Howard Hughes Medical Institute, Award: Gilliam
 Graduate Student Fellowship Award to MR; National Science Foundation,
 Award: Graduate Research Fellowship to MR.  
 Methodological Information
 ========================== * Methods of data
 collection/generation: see manuscript
 (https://doi.org/10.7554/eLife.80053) for details - -
 -   Data and File Overview
 ====================== Summary
 Metrics --------------- * File
 count: 11 main folders containing 4909 files (counted using "find .
 -type f | wc -l" in Terminal, Mac OS 12) * Total
 file size: 9.42 GB unzipped, 6.26 GB zipped * Range of
 individual file sizes: 4 KB - 381 MB * File formats:
 .csv, .nd2, .tif, .tiff, .txt, .xlsx, .png, .ai, .zip, .mp4, .ijm, .m,
 .mat, .svg, .pzfx    Naming
 Conventions ------------------ *
 File naming scheme: see each folder's description for specific file
 naming schemes. Table of Contents
 ----------------- *
 1_Fig1_Extract_gTuNA_WT_vs_F75Amutant *
 2_Fig2_InVitro_gTuNABeadAssay *
 3_Fig2_Negative-Stain_EM_gTuRC_Prep_Examples *
 4_Fig3_Size-Exclusion-Chromatography *
 5_Fig4_Extract_gTuNA_DimerMutData_MTcounts *
 6_Fig5_InVitro_TIRF_gTuRC_gTuNA *
 7_Fig6_Extract_Stathmin_gTuNA *
 8_Fig6_InVitro_TIRF_gTuRC_Stathmin_gTuNA *
 9_Fig6_InVitro_TIRF_gTuRC_CritConcTubulin_gTuNA *
 10_MassSpectrometry_gTuNA_IP_From_Extract *
 11_SupplementalVideos  
 Setup ----- * Unpacking
 instructions: n/a * Relationships between
 files/folders: Each main folder corresponds to a figure in our related
 manuscript (eLife80053) and is labeled as "folder number_figure
 number_short title of experiment." Each subfolder or file is data
 presented within that figure. More details on each subfolder are provided
 below. * Recommended software/tools: ImageJ (NIH) or
 preferably its FIJI implementation
 (https://imagej.net/software/fiji/downloads) with Bio-formats Plugin
 enabled for Nikon ".nd2" files (also used for .ijm macros);
 text-editors (e.g. TextEdit Mac OS); Microsoft Excel 2004 or higher (for
 .csv, .xlsx); MathWorks MATLAB R2019b or higher (for use with .m, .mat,
 .csv, .txt). GNU-Octave can be used to open and view the MATLAB scripts,
 although some plotting functions might not be available in Octave. For
 viewing raw chromatography traces, use GE UNICORN software. For figure
 files using .ai, use Adobe Illustrator. The .ai files have embedded .PNG
 files that can be viewed without this. - - -
   File/Folder Details
 =================== Details for:
 1_Fig1_Extract_gTuNA_WT_vs_F75Amutant
 --------------------------------------------------
 * Description: a folder containing raw, processed, and quantified
 total internal reflection (TIRF) microscopy data concerning the effect of
 adding wildtype or F75A mutant gamma-TuNA to Xenopus laevis egg extract
 (assessed by EB1 spots as a proxy for microtubule number). This folder
 contains three subfolders with the following structure:
 * Format(s): .nd2, .tif, .csv, .pzfx, .ai, .tif, .tiff
 * Size: 901.4 MB * Subfolder Tree:
   * 0_NikonND2Images --1_gTuNA_WT
 ---Set1 ----50 um window (described
 below) ----six raw ".nd2" files
 ----LUTs_Auto_5umbar_800wind (described below)
 ----Result (described below)
 ----Semi-Auto_Counts_Yen_optimized LUTs_800wind (described
 below) ---Set2 ----50micron
 windows ----six raw ".nd2" files
 ----LUTs_Auto_5umbar_800wind
 ----Result ---Set3 ----50
 um window ----six raw ".nd2" files
 ----LUTs_Auto_5umbar_800wind
 ----Result ---Set4
 ----Auto_Counts_LUTs_105_132_800wind ----six
 raw ".nd2" files
 --2_gTuNA_F75Amutant ---Set1
 ----50 um window ----six raw ".nd2"
 files ----LUTs_Auto_5umbar_800wind
 ----Result
 ----Semi-Auto_counts_800wind ---Set2
 ----50 um window ----six raw ".nd2"
 files ----LUTs_Auto_5umbar_800wind
 ----Semi-Auto_counts_800wind ---Set3
 ----50 um window ----six raw ".nd2"
 files ----LUTs_Auto_5umbar_800wind
 ----Semi-Auto_counts_800wind   *
 1_CropsUsedInFigure
 --1_gTuNA_WT_Auto_5umbar_800wind ----six
 cropped Tiff images of the wildtype gTuNA experiment shown in
 Fig.1 --2_gTuNA_F75A_Auto_5umbar_800wind
 ----six cropped Tiff images of the F75A gTuNA experiment shown in
 Fig.1   * 2_Quantification_Prism7
 --gTuNAinExtract_MTnucleationEB1counts.pzfx
   * Naming convention for images:
   * Raw .nd2 images and processed images: All raw microscopy
 images in this folder note first which Xenopus laevis egg extract was used
 that day (designated by a number or letter), whether the protein Ran was
 added, the final concentration of gTuNA protein tested, and the time in
 minutes (since placed on scope) at which the image was captured.
 *Example for reading file name:
 "ex4_noRan_0p5uM_gTuNA_WT_7m_001.nd2": 
 * ex4 = on the day of the experiment, we used *extract #4* as it
 passed a quality control test where branching MT nucleation was
 recapitulated (by adding Ran or the protein TPX2).  *
 noRan/minRan = To observe just single MT nucleation events, we did not add
 Ran (denoted by noRan or minRan for minus Ran). All files will show either
 noRan or the equivalent, minRan. * 0p5uM = final
 concentration of gTuNA added to extract, read as 0.5 µM, where p = decimal
 point. * gTuNA_WT = wildtype gTuNA (if mutant then
 gTuNA_Mut or gTuNA_F75A, these are equivalent in this folder)
 * 7m = image taken at 7 minutes post-addition of gTuNA to
 extract. If not a whole number of minutes passed, we used 6p5min as 6.5
 min for example. * _001 or three-digit number (_XXX)
 = appended by Nikon Elements program during acquisition (can be ignored by
 user as this number is incremented even for test acquisitions during laser
 power, TIRF angle, or focus adjustments and thus do not correspond to any
 experimental order). * Description of
 sub-folders contained within "0_NikonND2Images": 
   This folder contains the raw .nd2 files for
 this set of experiments, as well as cropped .nd2 files, result figures
 (.ai), and final counts of MT number contained with .csv files.
   -Please refer to the tree above to locate
 each file referred to here.   *
 "Set" = one experiment set (Set1, Set 2, Set3, Set4).  The
 wildtype and mutant gTuNA folders both have Set1, Set2, and Set3 folders,
 meaning they come from the same experiment. In these cases, both forms of
 gTuNA were run in parallel with the same extract source. Wildtype gTuNA
 has an additional experiment (Set4) without an accompanying gTuNA mutant
 titration.  * "50 um window" or "50
 micron windows" = folder containing a duplicate set of the raw .nd2
 images for non-destructive cropping to a size of 50 micron by 50 micron
 (contained in its subfolder
 "LUTs_Auto_5umbar_387wind"). *
 "LUTs_Auto_5umbar_800wind" = a folder containing tiff images
 with automatic LUT contrast adjustment by FIJI (LUTs_Auto), with added 5
 micron scale bar (5umbar) at a crop of 800 pixels by 800 pixels (~50
 micron by 50 micron). * "Result" = folder
 containing Adobe Illustrator figures showing the processed images for all
 conditions side-by-side (for that particular experiment) and any .tif or
 .tiff exports of this figure. This is shown for easier interpretation by
 the user. The exact crops used for Figure 1 are found in folder
 "1_CropsUsedInFigure."   *
 "Semi-Auto_Counts_Yen_optimized LUTs_800wind" or "Auto
 Segmentation_LUTs101_160_macro800x800wind" or
 "Auto_Counts_102_146_800wind" or "Semi-automated
 version_optimized LUTS" = a folder containing .CSV files recording
 the number of EB1 spots counted within the 50 um by 50 um crops from
 "LUTs_Auto_5umbar_800wind."   
 -- Description of Variables contained within each .csv (name will
 match its source .nd2 file): * 1st column =
 blank title = the specific spot measured (the last number in this column
 represents the total count for the image). This total count is what is
 used in Figure 1 to assess MT number in each condition.
 * 2nd column = "Area" = area of the spot in
 pixels * 3rd column = "IntDen" = integrated
 density or the averaged pixel values in the total area of the spot (not
 used in our analysis). * 4th column =
 "RawIntDen" = total, unaveraged density or pixel intensities in
 the area of the spot (not used in our analysis). 
   * Description of sub-folders contained
 within "1_CropsUsedInFigure": This folder
 contains the cropped, RGB images used in Figure 1. These images both come
 from the same set, same extract (Set1, extract 4).
   *
 "1_gTuNA_WT_Auto_5umbar_800wind" = folder containing
 wildtype-gTuNA titration cropped images (RGB mode) as .tif
 files. -the images use the same naming
 scheme described at the beginning of this section.
   *
 "2_gTuNA_F75A_Auto_5umbar_800wind" = folder containing F75A
 gTuNA mutant titration cropped images (RGB mode) as .tif
 files.  -the images use the same naming
 scheme described at the beginning of this section.
   * Description of files contained within
 "2_Quantification_Prism7":  This folder
 contains the Prism 7 file used to construct the graph in Figure
 1. *
 "gTuNAinExtract_MTnucleationEB1counts.pzfx" = Prism 7 file
 containing the normalized MT counts used in Figure 1. 
 *Data 1 = a sheet containing the Set1 data as an example. It has
 an accompanying graph also called Data 1. *Data 2 = a
 sheet containing the normalized data from all Sets. It has an accompanying
 graph also called Data 2, which was used in Figure 1.
   Details
 for: 2_Fig2_InVitro_gTuNABeadAssay
 ------------------------------------------ *
 Description: A folder containing the raw .nd2 time-lapse of an attached
 wild-type gTuNA bead nucleating a microtubule aster. *
 Format(s): .nd2 * Size: 380.8 MB
   * Detail for
 "gTuNaWT_bead_1to1000dil_Mzt1antiDDS_2m_.nd2":
 * "gTuNaWT_bead_1to1000dil_Mzt1antiDDS_2m_.nd2" = raw
 .nd2 time-series of Cy5-tubulin signal from an attached wildtype gTuNA
 bead on glass.    Details for:
 3_Fig2_Negative-Stain_EM_gTuRC_Prep_Examples
 --------------------------------------------------------- * Description: A folder containing raw negative-stain electron microscopy micrographs (images) of gTuRCs purified via the Halo-gTuNA method described in our study in Figure 2. These images were taken on a Philips CM100 transmission electron microscope at 80kV, at 64,000x magnification. See our materials and methods for more information. This folder also contains a prep example where we used a modified form of human gTuNA containing a small AU1 epitope, which we used to follow gTuNA throughout our prep and determine the residual concentration of gTuNA present in our preps. This folder also contains an Excel file describing how we calculated the final concentration of gTuRCs resulting from our preps. * Format(s): .tif, .xlsx * Size: 89.2 MB * Subfolder tree:   *3_Fig2_Negative-Stain_EM_gTuRC_Prep_Examples -- 1_Example1_Of_standard_Halo-gTuNA_gTuRC_prep ---ten .tif negative-stain EM images of Halo-gTuNA gTuRC prep from 10/13/21. -- 2_Example2_Of_standard_Halo-gTuNA_gTuRC_prep ---ten .tif negative-stain EM images of Halo-gTuNA gTuRC prep from 10/22/21. -- 3_Example3_Of_standard_Halo-gTuNA_gTuRC_prep ---ten .tif negative-stain EM images of Halo-gTuNA gTuRC prep from 02/04/21. -- 4_Example_Of_Halo-AU1-gTuNA_gTuRC_Prep ---eleven .tif  negative-stain EM images of a Halo-AU1-gTUNA gTuRC prep from 03/04/21. -- 5_Analysis_of_gTuRC_concentration ---DetermininggTuRCconc_GCP4standard.xlsx   Naming scheme for negative-stain EM images:    Example: "MR210113_HalogTuRCno3_fr7full_64kx_1.tif"   *"MR211013" = experiment/purification date of 10/13/21 *"HalogTuRCno3" = Halo-gTuNA prep number 3 with that batch of human Halo-gTuNA or "HaloAU1gTuNAgTuRC" to indicate that this is the modified version of the prep with Halo-AU1-gTuNA to track the concentration of gTuNA in the final yield. *"fr7full" = sample is from Fraction 7 of the sucrose gradient step. This was the peak as determined via Western. *"_64kx" = 64,000x magnification. *"_1" = image number   * Description of "DetermininggTuRCconc_GCP4standard.xlsx":An Excel file containing a single sheet with quantifications of the  concentration of gTuRC from five standard Halo-gTuNA gTuRC preps. This file also includes an embedded image of the blot used  for the quantifications. This blot contains a standard titration of recombinant GCP4 (a gTuRC subunit)   Details for: 4_Fig3_Size-Exclusion-Chromatography ------------------------------------------------- * Description: A folder containing size-exclusion chromatography data for wildtype and mutant versions of gTuNA. This data is used in Figure 3, which demonstrates that Halo-tagged wildtype gTUNA is a dimer, with certain types of mutations causing loss of dimerization to different degrees. This data is in the form of .zip files containing the raw chromatography data generated by the GE Unicorn software used with our Akta Pure chromatography system. GE UNICORN is required to open the files, however we have also included an Excel file with the normalized chromatogram curves for all the experiments contained here. * Format(s): .xlsx, .zip (containing GE UNICORN files and metadata for each version of gTuNA tested) * Size: 23.1 MB * Description of files contained:   *"Figure 3- Source Data.xlsx": an Excel file containing the normalized chromatography curves (absorbance at 280 nm light) for wildtype or mutant versions of gTuNA protein (human and Xenopus as designated). This file is a copy of the Figure 3- Source Data file included in our eLife manuscript. -Sheets contained within: *"Figure 3B-SEC": normalized chromatography traces (volume since start of run VS the absorbance value at 280 nm  light, normalized by the highest absorbance value for that particular run).  *"Figure 3D_3F" : Quantifications of Western Blots probing for gTuRC pulldown by wildtype and mutant versions  of gTuNA (both human and Xenopus). *"Fiugre 3E_3G" : Quantifications of Western Blots probing for gTuRC pulldown by wildtype and mutant versions  of gTuNA (all except one Xenopus).   *Seventeen (17) raw chromatography files generated by GE UNICORN software during the experiments for each version of gTuNA  tested. * Naming scheme for files: -Example: "MR210616_HumgTuNA_WT_S200 increase 10 300 001.zip" -"MR210616" =  experiment ID/date of 06/16/2021 -"HumgTuNA_WT" = Halo-tagged human gTuNA wildtype -If Xenopus gTuNA wildype = "XengTuNAWT" or "SHT-Halo-Xen_gTuNA_WT" -If mutant forms, will be followed by description: *F75A = single alanine mutant at position F75 *I67A = single alanine mutant at position I67 *L70A = single alanine mutant at position L70 *L77A = single alanine mutant at position L77 *I67D/L70D = double aspartate mutant at positions I67 and L70 *2xA-I-L = double alanine mutant at positions I67 and L70. *2xD-I67D-L77D = double aspartate mutant at positions I67D and L77D. *3xD-167D-L70D-L77D = triple aspartate mutant at positions I67, L70, and L77. *4xA-F-I-L-L = quadruple alanine mutant at positions F63, I67, L70, and L77. *2xDimerMutant = double aspartate mutants at position I67 and L70.   -"S200 increase 10 300" = column used; same for all: GE Superdex 200 increase 10 300 GL column.       Details for: 5_Fig4_Extract_gTuNA_DimerMutData_MTcounts ------------------------------------------------------- * Description: a folder containing raw and processed TIRF microscopy data assessing mutations affecting dimerization of gTuNA and whether these impair its ability to increase microtubule number in Xenopus egg extract. This pertains to Figure 4 in our study. As with the experiments in Figure 1 (see 1_Fig1_Extract_gTuNA_WT_vs_F75Amutant), the raw .nd2 files contain snapshots of EB1 spots, used as proxies for microtubule number. In addition, this folder contains FIJI/ImageJ macros and MATLAB scripts used to process this data. * Format(s): .nd2, .ijm, .csv, .m * Size: 1.85 GB * Subfolder tree: *1_Data --ExpSet1 ---four .nd2 multi-position (TIRF images) of three channel slides, labeled "ExpSet1"  --ExpSet2 ---six .nd2 multi-position (TIRF images) of three channel slides (contains an internal replicate denoted by "_2"), labeled "ExpSet2" --ExpSet3 ---four .nd2 multi-position (TIRF images) of three channel slides, labeled "ExpSet3" *2_Fiji Macros --gTuNAMutinExtractv2.ijm --SpotCounter.ijm --SUPERBatch_Crop_RGB_5umbar_Tiffs.ijm --"Used for processing" ---LUTsandCrop.ijm ---NewEB1counter.ijm *3_ImageJ_Output --3 min --5 min --MR220127_CollatedMTcounts_gTuNADimerMutantsinExtract.csv   *4_Matlab Processing --CollateDatafromImageJ_gTuNAdimerExt.m --MR220130_NormActExt_gTuNAdimerMut.png --MR220130_NormActExt_gTuNAdimerMut.svg   Details for: 6_Fig5_InVitro_TIRF_gTuRC_gTuNA -------------------------------------------- * Description: a folder containing in vitro TIRF microscopy data assessing the impact of adding wildtype or mutant gTuNA to purified gTuRCs on microtubule nucleation. This pertains to Figure 5 in our study. This folder inlcudes .nd2 files, ImageJ macros (and their output .txt or .csv files), Matlab scripts used to make the charts in Figure 5.  * Format(s): .nd2, .tif, .ijm, .txt, .m * Size: 2.4 GB * Subfolder tree: *1_Mass_Intensity_Analysis -0_ImageJ_Macros --Batch_SetOtsuThreshold.ijm --ManualThresholdOtsu.ijm -1_Videos_and_MassIntensities --Buffer --F75A --L77A --Wildtype -2_CSVs_InputToMatlab --twenty-two (22) .csv files recording the intensity per frame (MT mass) of the videos. -3_Matlab_Analysis --MassAnalysis.m *2_Kymograph_Analysis -0_ImageJ Macro --Crop_TIFFsave_150secRxns.ijm --Crop_TIFFsave.ijm --IJ_StackRectif.ijm --KymographTime_v8.ijm --ProcessKymograph_v5.ijm -1_Videos_Stack_Rectified_whole_time_series --twenty-two (22) .tif time-lapses of purified gTuRCs nucleating MTs in the presence of buffer or gTuNA; these  have been corrected for slight translational drift of the microscope stage (using the ImageJ plugin StackReg,  see materials and methods for more details) -2_Videos_Cropped_and_onlyfirst75frames --Cropped_UpToFrame75 ---twenty-two (22) .tif movies (to be opened in FIJI/ImageJ) that are generated by taking only the  first 75 frames from the drift corrected .tif files in the folder "1_Videos_Stack_Rectified_whole_time_series." -3_Kymographs --Control ---Kymographs_frame75 ----Reactions (named according to naming scheme below) -----Kymographs -----a variable number of space-time plots for each MT present in the movies ( manually selected with the help of our ImageJ macro "KymographTime_v8.ijm" -----Map_and_Result -----MAP+nameOfCurrentReaction.tif -----Result --------a .txt which is the output of the processed kymograph using the "ProcessKymograph_v5.ijm" macro. --F75A ---same structure as the Control folder --L77A ---same structure as the Control folder --WT ---same structure as the Control folder -4_Processed --TXT_inputForMatlab_KymographAnalysisScript ---twenty-two (22) .txt files -5_MatlabAnalysis --5-1_Kymograph_Analysis ---SingleMolecule_KymographAnalysis.m --5-2_Simulation_Code ---Rale_NumericalMTSimulation.m ---Rale_NumericalMTsimulation.mat ---Rale_VisualMTSimulation.m * Description of "MassAnalysis.m": a MATLAB script used to collate tubulin signal intensity per frame stored in the .csv files in "2_CSVs_InputToMatlab". This script is commented to describe what is happening at each step, but to summarize: the script grabs the .CSV files from the user-directed folder and extracts the data and file names. It then averages the intensities per frame and calculates the standard error. These are then normalized by the buffer control. These values are then plotted as mean normalized intensity (MT mass) over time with error clouds. The outputs are .png and .svg files of the plot. * Description of "KymographTime_v8.ijm": an IMAGEJ macro that helps a user manually select individual microtubules (MT) from each movie, with a line tool, for "re-slicing" into a space-time plot, or kymograph, visualizing that MT over time. Before using this macro, the user must first make and save a mean intensity Z projection of the movie (.nd2) time series to make a single "map" image of all MTs present throughout the time frame. Then the user opens both the "Map" image side-by-side with the source movie. The macro will ask for an output directory. The macro will then ask the user to select each window when prompted in order to automate switching between them. It will also ask for the last recorded MT number. When this is the first time running the macro this should be set to zero. If coming back to a movie, the last MT number should be entered so that the macro can properly label the next MT. The user should next be prompted to draw a line over a previously unprocessed MT present in the movie. The user can move back and forth through the movie to determine the maximum length this MT achieves, before drawing the line from just outside the origin point to just outside the endpoint of the MT. The macro will then reslice this MT into a single kymograph image, save it as "MT_#.tif", mark its location on the map, save the map, and close the generated kymograph. This will repeat for a maximum of 500 loops, or until the user holds down the space bar and clicks OK during the "Draw a line on MT." prompt. The output folder will contain all processed MT kymographs as .tif files. These will be passed as input to "ProcessKymograph_v5.ijm" to manually extract parameters like growth speed, max MT length, etc. * Description of "ProcessKymograph_v5.ijm": an IMAGEJ macro that takes kymographs (as .tifs) generated by "KymographTime_v8.ijm" and assists the user in batch processing them to extract whether it was nucleated by gTURC (no growth on one end = gTuRC origin), the frame (= time as y position) at which the MT was nucleated,  the growth speed of the MT (as slope of line along growing edge), and the maximum length the MT achieved during its growth (distance of line). This macro will output a single .txt file for each batch with the following columns: * Column 1 = file name (e.g. MT_1.tif) * Column 2 = y or frame at which MT nucleated (gTuRC origin point) * Column 3 = whether MT grew from one end (gTURC nucleated = 1) or from both ends (spontaneous = 0) * Column 4 = x1 = x-coordinate of first end of line along growing edge of MT (used for growth speed calculation) * Column 5 = frame1 = y-coordinate of first end of line along growing edge of MT (used for growth speed calculation) * Column 6 = x2 = x-coordinate of other end of line along growing edge of MT (used for growth speed calculation) * Column 7 = frame2 = y-coordinate of other end of line along growing edge of MT (used for growth speed calculation) * Column 8 = x3 = x-coordinate of origin end of line along max extent of MT (used for max length calculation) * Column 9 = x4 = x-coordinate of other end of line along max extent of MT (used for max length calculation) * Column 11 = maxlength = calculated max length of MT in microns * Column 12 = growthspeed = calculated growth speed of MT in micron per minute. * Description of "SingleMolecule_KymographAnalysis.m" : a MATLAB script used to collate and plot data stored in .txt files generated by "ProcessKymograph_v5.ijm." This script is commented to describe what is happening at each step. To summarize, this script will extract the file names, columns 2, 3, 11, 12 in the tab-delimited .txt files generated by "ProcessKymograph_v5.ijm." These will then be filtered to remove any MTs that were spontaneously generated (Column 3 = 0). It will then count how many MTs were generated at each timepoint (binned by frame, converted into time with 2.02 sec per frame conversion). It will then plot number of MTs over time for all individual movies (for user to see prior to averaging). The script will then average the number of MTs over time for each condition (wildtype gTuNA, buffer, mutant F75A or mutant L77A gTuNA) and calculate the standard error. These will then be normalized by the buffer, plotted with averages number of MTs over time as a trendline surrounded by error clouds for each normalized average. These plots are output as .png or .svg and saved. Next it will measure the slopes of the lines in the first 12 seconds to generate the nucleation rate (k) at the initial state. Next it will measure the slope of the line at the saturation stage to generate the late-stage nucleation rate. It will also plot the average maximum MT length, the growth speeds as both bar charts or violin plots (requires the Statistics Toolbox Add-On for MATLAB R2019). These are all saved as .png and .svg images. * Description of "Rale_NumericalMTSimulation.m" : a MATLAB script that runs a numerical simulation of the number of MTs over time given a constant growth speed and the mean measured nucleation rates from "SingleMolecule_KymographAnalysis.m" at the initial stage. These are then used in a equation (EQUATION #2 in Methods) to calculate MT number over time for each condition. These values are stored in a .mat file, here stored as "Rale_NumericalMTsimulation.mat." This .mat is used by "Rale_VisualMTSimulation.m" as input to generate simulated frames of MTs growing at each time point. * Description of "Rale_VisualMTSimulation.m": a MATLAB script that takes as input the output of "Rale_NumericalMTSimulation.m" (Rale_NumericalMTsimulation.mat), to generate simulated frames of MTs growing at each time point under buffer, wildtype gTuNA, or mutant gTuNA conditions. It will calculate how many MTs are nucleated per frame, plot these new and existing MTs per frame, and increment all MTs by a constant growth speed at each new frame. Individual frames are then saved as .png files in a folder for each condition, which can then be combined in ImageJ or Apple QuickTime to make a single movie (based on sequential numbering in each frame's title). An example of the simulated movies can be seen in the folder "11_SupplementalVideos", Video-3.   Details for: 7_Fig6_Extract_Stathmin_gTuNA ------------------------------------------ * Description: a folder containing TIRF microscopy images tracking fluorescent EB1 spots in Xenopus egg extract in the presence of stathmin and gTuNA (used for Figure 6). This folder contains four data sets of raw images and their related cropped images. Dataset 1's folder has a different structure than the other three datasets (divided by presence of gTuNA), as noted below.  * Format(s): .nd2, .tif * Size: 442.9 MB * Subfolder tree:   *Dataset 1 -Stathmin_2p3uMgTuNA --sixty (60) .nd2 images of fluorescent EB1 spots in extract titrated with stathmin with 2.3 µM wildtype gTuNA added (individual files are timepoints from 3 min to 22 min) -Stathmin_NogTuNA --same as above, except no gTuNA is added. Titration of stathmin in extract. *Dataset 2 -twenty-two (22) .nd2 TIRF images of fluorescent EB1 spots with stathmin or gTuNA or control buffer (see naming scheme below) -Output_CenterCrop_512x512 --twenty two (22) .tif crops of the .nd2 files above (crops showing only the center of field of view) --Data2_EB1counts ---twenty two (22) binarized/thresholded images .tif images showing the EB1 spots counted ---twenty two (22) .csv files recording the EB1 spots counted in each accompanying .tif image above ---Dataset2 ----final .csv files from roughly the same timepoint (~7 min) used for figure *Dataset 3 -same structure as Dataset 2 *Dataset 4 -same structure as Dataset 2   *Naming scheme for .nd2 files (and related processed files):   Example: "Data2_1p35Stath_gTuNA_3min004.nd2" - "Data2_" = experiment dataset number - "1p35Stath_" = 1.35 µM exogenous concentration of purified stathmin present in extract - "0p45Stath" = 0.45 µM exogenous concentration of purified stathmin present in extract (note that endogenous stathmin is ~1.5 µM, so 0.45 µM additional brings extract to 1.3x concentration.)  - "2p7Stath" = 2.7 µM exogenous concentration of purified stathmin present in extract - "_gTuNA" = purified gTUNA present at 2.3 µM final concentration - "_3min" = image acquired 3min post addition to slide - "004" = can be ignored as it is added by Nikon Elements program sequentially to every new aquisition file name, even for TIRF angle or focus adjustment related acquisitions that were not saved.   Details for: 8_Fig6_InVitro_TIRF_gTuRC_Stathmin_gTuNA ----------------------------------------------------- * Description: a folder containing in vitro TIRF microscopy time-lapses recording fluorescent tubulin signal (MT nucleation and polymerization) in the presence of stathmin and/or gTuNA. These experiments are performed in the same manner as in Figure 5 ("single molecule" TIRF set up with passivated and functionalized coverslip glass to bind biotin-tagged gTURC), except now with a titration of stathmin. This data relates to Figure 6C/D.  * Format(s): .ijm, .nd2, .tiff, .csv, .m * Size: 2.44 GB *Subfolder tree:   *0_Fiji Macros -Batch_SetOtsuThreshold.ijm -ManualThresholdOtsu.ijm *1_Videos -twenty one (21) .nd2 TIRF time-lapses of microtubules nucleating and polymerizing (tubulin signal) with or without stathmin or gTuNA present (naming scheme below) *2_OtsuThreshold -twenty (20) binarized/thresholded .tif images as a result of processing the .nd2 files in "1_Videos" to remove background (same naming scheme as "1_Videos" files from which they are derived) *3_CSV_InputToMatlab -tabulated mass signal (intensity per frame) as .csv format for each binarized/thresholded image above (same naming scheme as "2_OtsuThreshold" files from which they are derived) *4_MatlabAnalysis -OtsuMassIntensityAnalysis.m : MatLab script used to collect the data from the .csv files above, output is plot of intensity at the end of 250 seconds.   *Naming scheme for .nd2 files in "1_Videos": Example: "6_220319_4Stath_gTuNA_16_1min_.nd2" - "6_" = experiment condition set (common to all replicates of same condition) - "220319" = experiment ID, date of March 19, 2022 - "4Stath" = 4 µM final concentration of purified stathmin present - If "2p5Stath" = 2.5 µM final concentration of stathmin - "_gTuNA" = purified gTUNA present at 3.3 µM final concentration - "_16" = reaction ID - "_1min" = this is common to all files, indicates this was captured 1 min after adding cold reaction mix to slide - All reactions have gTuRC present. In the case of gTuRC alone or gTuRC and gTuNA, the file name will have "gTuRC."   * Description of "OtsuMassIntensityAnalysis.m" : a MATLAB script used to collate all the MT mass intensities per frame for each video. As input it will expect the .CSVs in the folder "3_CSV_InputToMatlab." The script is commented to indicate what is happening at each step, but to summarize: the intensity values are combined and averaged by condition (e.g. gTuRC + gTuNA + Stathmin), standard error is calculated, and then the final values at 250 seconds are plotted into two bar charts (one for conditions with gTuNA and one chart without gTuNA). These bar charts are saved as both .png or .svg in the current working folder.   Details for: 9_Fig6_InVitro_TIRF_gTuRC_CritConcTubulin_gTuNA ------------------------------------------------------------ * Description: a folder containing in vitro TIRF microscopy time-lapses recording microtubule nucleation and polymerization at gTuRC's critical tubulin concentration (7 µM tubulin) in the presence or absence of gTuNA. Please note that due to a broken filter in the light-path of the camera, part of the field of view is identically obscured in each .nd2 file/image. For processing, we analyzed the largest unobstructed area as a cropped image. The outputs were max projection images of all microtubules nucleated and polymerized during a five minute window. We present the data as max projections due to the increased dynamic instability and short life-time of individual MTs as a result of being at the lower 7 µM tubulin concentration (compared to 15 µM normally used in this study). * Format(s): .nd2, .tif, .ijm, .png, .csv, .xlsx * Size: 857.1 MB * Subfolder tree and descriptions: *1_NikonFiles_InVitro_gTuRC_gTuNA_at7uMTubulin -five (5) .nd2 TIRF microscopy videos recording microtubule nucleation and polymerization (fluorescent tubulin signal) at gTuRC's critical tubulin concentration (7µM) either without gTuNA present ("Buffer") or with wildypte gTuNA added ("WT"); see naming scheme below. *2_Full_Size_Vids_and_MaxZProjections -1_FullSize_Vids_Tiffs -- five (5) .tif files, identical to the files above except saved as .tif format -2_MaxZProjections_Tiffs -- five (5) .tif max intensity-projections of the .tif files in "1_FullSize_Vids_Tiffs" -- ProcessinggTuRCCritConcFullVids.ijm (ImageJ macro used to generate the max intensity-projections using built-in ImageJ functions) *3_Cropped_Vids_and_MaxZProjections - 0_Cropped_Vids_Tiffs -- five (5) cropped .tif time-lapse files (field of view cropped to exclude the areas occluded by broken filter) - 1_MaxZProjectionsOfTimeSeries_Tiffs -- five (5) max intensity projections of the cropped files above -- ProcessinggTuRCCritConc.ijm (ImageJ script used to generate the cropped max intensity projections.) - 2_MTCounts -- AuthResponseFig1B_quant.png: image of quantification presented in Fig 6E.  -- CountingMTs.xlsx: a single sheet Excel file with collated MT counts *4_Gray Max Projection_tiffs - two representative max projections (Buffer Rxn 6 and WT Rxn 2) presented in Figure 6E. Each was converted into RGB color, and the Buffer reaction had a scale bar added. In total there are five (5) .tif images in this folder.   *Naming scheme for videos: -Example: Buffer_Rxn5_1min_.nd2 or WT_Rxn1_1min_.nd2 * "Buffer" = control reaction at 7 µM tubulin with only Tris-control buffer present * "WT" = reaction with 7 µM tubulin with wildtype gTuNA domain present * "Rxn #" = reaction ID (each was a separate experiment, run one at a time, with the same source frozen gTuRC). Wildtype gTuNA reactions will have either Rxn 1, 2, or 3 as IDs, Buffer will be Rxn 5 or Rxn 6.  * "_1min" = all reactions were captured at 1 min post-addition of reaction mix to chamber and placement on scope. * For any derived max intensity projections, the same naming scheme applies, except the tag "MAX_" is added to the name to indicate it is the max projection. Details for: 10_MassSpectrometry_gTuNA_IP_From_Extract ------------------------------------------------------ * Description: a folder containing mass spectrometry data identifying Xenopus egg extract proteins that co-precipitate with wildtype or mutant gTuNA beads. This folder contains the raw Excel dataset produced by the ThermoFischer Center for Mutliplexed Proteomics (TCMP) at Harvard Medical School, Boston, MA. It also includes an Excel file with the volcano plot data used in our supplement to identify significant co-precipitants/interactors of gTuNA.  * Format(s): .xslx * Size: 532 KB * Subfolder tree:   *1_RawDataset --TCMP_831_ProtQuant.xslx *AnalysisofHarvardMassSpec_Logp-Values_VolcanoPlotData.xslx   * Description of TCMP_831_ProtQuant.xslx: an Excel file containing the SEQUEST matched mass spectrometry data identifying proteins present on wildtype or mutant gTuNA beads pulled down from Xenopus egg extract. -The file contains sheet "protein_quant_2512 (2)" with the following column headers: *"Protein id" = the Uniprot protein id *"Gene symbol" = if present, notes the common gene designation *"Description" = longer form title of protein *"Group ID" = group number containing associated peptides leading to overall protein id *"Number of peptides" = number of unique peptides belonging to that particular protein id *"Collapsed" = all peptides are collapsed to single protein group = C *"Parsimony" = all "UR" *"Hum_gTuNA1" or "Hum_gTuNA2" or "Xen_gTuNAWT1" or "Xen_gTuNAWT2" = results of two independent pulldowns for either human or Xenopus wildtype gTuNA (higher value = higher number of spectral counts detected for that peptide") -The F75A gTuNA pulldowns are designated by "Xen_gTUNA Mt1" or "Xen_gTuNA Mt2". * Description of AnalysisofHarvardMassSpec_Logp-Values_VolcanoPlotData.xslx: an Excel file containing the log(p-values) and fold-change data used to generate volcano plots and identify the most significantly enriched proteins present in wildtype but not mutant gTuNA pulldowns.  --The file contains:   * sheet "HumWTvsMut_1_0.1" with the following column headers: -This sheet compares the mass spectrometry identified proteins co-precipitated by human wildtype gTuNA against F75A Xenopus gTuNA. Proteins are listed in order of the number of peptides detected for that unique protein. P-values are comparing the wildtype over mutant (described as fold-change or FC) and the significance statement (i.e. "Down") refers to lack of this protein in the mutant pulldown. -"Num" = rank of protein in list based on fold change (FC) -"UniprotID" = the Uniprot designation for the identified protein -"GeneSymbol" = common gene symbol or abbreviation for identified protein -"Description" = longer form title or description of protein -"FC" = fold-change (wildtype/mutant) -"PVal" = p-value of fold-change -"log2FC" = the log base-2 of the fold-change (processing for volcano plot) -"neglogPVal"= negative log of the p-value (processing for volcano plot) -"NumPeps" = the number of peptides associated with that protein's identification -"Significant" = all "DOWN", referring to enrichment of protein in wildtype bin, less of that protein in mutant bin. * sheet "XenWTvsMut_1_0.1" with the following same column headers as above. -This sheet compares the mass spectrometry identified proteins co-precipitated by Xenopus wildtype gTuNA against F75A Xenopus gTuNA. Proteins are listed in order of the number of peptides detected for that unique protein. P-values are comparing the wildtype over mutant (described as fold-change or FC) and the significance statement (i.e. "Down") refers to lack of this protein in the mutant pulldown. *sheet "Present in Both Hum_Xen" with the same meaning for column headers as above, except certain columns have been removed to focus on the gene/protein information and number of peptides behind identification. -This sheet contains the common set of proteins that are present in both prior sheets "HumWTvsMut_1_0.1" and "XenWTvsMut_1_0.1," meaning both wildtype versions of gTuNA enriched for these same proteins. Highlighted proteins are known gamma-tubulin ring complex components (except delta-tubulin, please disregard).   Details for: 11_SupplementalVideos ---------------------------------- * Description: a folder containing copies of the three supplemental videos included in our study.  * Format(s): .mp4 * Size: 41.1 MB * Subfolder tree and descriptions:   *Video-1_compressed.mp4: compressed video of a wildtype gTuNA bead nucleating microtubule asters in vitro after pulldown from Xenopus egg extract. *Video-2.mp4: video comparing gTuRC microtubule nucleation activity in vitro in the presence of buffer or wildtype Xenopus gTuNA with quantification of the number of microtubules over time (mean +/- standard deviation) *Video-3.mp4: video comparing both gTuRC activity in vitro with buffer or wildtype or mutant gTuNAs and the results of simulated gTuRC activity. - - - END OF README
description: The microscopy data (either Nikon ".nd2" or
 ".tif" extensions) can be opened using the open-source program
 ImageJ/FIJI (with the BioFormats plugin installed and the Nikon
 ".nd2" library enabled). This same program can open the
 ".ijm" extension macros. CSV and TXT files can be opened with
 most text-based editors, although we used Microsoft Excel or MATLAB to
 view or process them. MATLAB code requires access to MathWorks MATLAB
 environment, although an open-source alternative, GNU-Octave, can be used.
 However, GNU-Octave does not contain all the required dependencies for all
 the scripts included here.