Data for: The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)

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.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.Funding provided by: National Institute of General Medical SciencesCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000057Award Number: New Innovator Award 1DP2GM123493Funding provided by: Pew Scholars Program in the Biomedical Sciences*Crossref Funder Registry ID: Award Number: 00027340Funding provided by: David and Lucile Packard FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000008Award Number: 2014–40376Funding provided by: Howard Hughes Medical InstituteCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000011Award Number: Gilliam Graduate Student Fellowship AwardFunding provided by: National Science FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000001Award Number: Graduate Research FellowshipShort description: The archive contains the following: 1) raw microscopy images, videos collected via total internal reflection fluorescence (TIRF) for the following: 1a) Xenopus laevis 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: > 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: > 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.