Case_Science_2019_Data_README.txt THIS README FILE IS A SINLGE SUMMARY FOR ALL OF THE UPLOADED FILES IN THE DATA PACKAGE. THIS DATA PACKAGE ACCOMPANIES THE PAPER: Case, L.B., Zhang, Z., Ditlev, J.A., and Rosen, M.K. "Stoichiometry controls activity of phase separated clusters of actin signaling proteins." Science. 2019. It contains all of the unprocessed microscopy data and simulation data used in the paper. **************************************CONTENT INFORMATION**************************************** Microscopy data was acquired using a Windows operating system running Metamorph Software. TIRF images were captured using a TIRF/iLAS2 TIRF/FRAP module (Biovision) mounted on a Leica DMI6000 microscope base equipped with a 100 X 1.49 NA objective and a 405/488/561/647nm Laser Quad Band Set filter cube for TIRF applications (Chroma). Illumination was provided by an integrated laser engine equipped with multiple laser lines (405nm-100mw/445nm-75mw/488nm-150mw/514nm-40mw/561nm-150mw/637nm-140mw/730nm-40mw, Spectral). Confocal images were captured using a Yokogawa spinning disk and a 405/488/561/647nm Laser Quad Band Set filter cube (Chroma). Images were acquired using a Hamamatsu ImagEMX2 EM-CCD camera. Microscopy data analysis was performed using ImageJ (https://fiji.sc/) and MATLAB run on a Mac operating system as described in the supplemental materials of the manuscript. Computational simulation was performed using SpringSaLaD (http://vcell.org/ssalad) on the UT Southwestern Linux-based computer cluster. Datasets were organized and compressed using the UT Southwestern Linux-based computer cluster. File Roller 2.28.2 (Archive Manager for GNOME) was used to compress data using either tar.gz or .zip formats. Test Datasets were successfully transfered and decompressed successfully on bot both Mac and Linux operating systems. Most microscopy data were aquired using the Multidimensional Acquisition feature in Metamorph. These data often contain multiple channels, multiple stage positions, and multiple timepoints. Individual images are stored as TIF files. Both the images and acquisition Metadata can be best accessed using the .nd files. The datasets are best used by importing the .nd file into ImageJ/FIJI using File--> Import--> Bio-Formats. Data that was not acquired using Multidimensional Acquisition are stored as single TIF files or TIF stacks that can be opened in ImageJ. Files are organized according to the figures in the manuscript. For example, all of the data used to generate the graph in figure 1d can be found in the archived file Figure1_d.tar.gz In additon to data, each archived .tar.gz file also contains a README.txt file describing the data in more detail. Some folders have been additionally grouped and compressed into .zip files, and the contents of each .zip file or folder is described in detail within the README.txt files. For example, Figure1_d.tar.gz this information file is called Figure1d_README.txt. Figures that contain no data, such as cartoon schematics or cartoon summaries, do not have folders in the dataset. Naming convention notes: In the manuscript we primarily discuss three molecules (Nck, Nephrin and N-WASP). Some of the files contained in this data have names containin Nck and BPVCA. BPVCA is another name for N-WASP, so any files with BPVCA in the name are refering the N-WASP. Detailed information about specific experimental conditions in each figure can be found in both the relevant figure legends and the supplemental information accompanying the manuscript. *******************************************FILES*************************************************** The dataset consists of the following files: Figure1_b-c.tar.gz Figure1_d.tar.gz Figure1_e.tar.gz Figure2_b.tar.gz Figure2_c.tar.gz Figure2_e.tar.gz Figure2_f.tar.gz Figure3_a.tar.gz Figure3_c.tar.gz Figure3_d.tar.gz Figure3_e.tar.gz Figure3_f.tar.gz Figure3_g.tar.gz Figure3_h-i.tar.gz Figure3_j.tar.gz Figure4_a.tar.gz Figure4_b-c.tar.gz Figure4_e.tar.gz Figure4_f.tar.gz Figure4_g.tar.gz Supplemental_Figures_1-4.tar.gz Supplemental_Figures_5-13.tar.gz *******************************************CONTACT*************************************************** Corresponding Author: Michael Rosen, Ph.D. Department of Biophysics and Howard Hughes Medical Institute UT Southwestern Medical Center Dallas, TX, USA Email: michael.rosen@UTSouthwestern.edu ******************************************ACKNOWLEDGEMENTS******************************************** We thank Ethan Garner (Harvard) for suggesting addition of CapZ to the assays to focus quantification on actin nucleation rather than filament elongation, Les Loew (Uconn Health) for helpful advice about the SpringSaLaD software, and Jay Groves and William Huang for discussions on dwell time and technical advice. FUNDING: L.B.C. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation (DRG-2249-16). This work was supported by a grant from the Welch Foundation (I-1544 to M.K.R.), a National Research Service Award from NIDDK (F32 DK101188 to J.A.D.), and a Howard Hughes Medical Institute Collaborative Innovation Award. AUTHOR CONTRIBUTIONS: L.B.C., J.A.D. and X.Z. prepared reagents. L.B.C. performed the experiments and analyzed data. L.B.C. and M.K.R. wrote the manuscript. All authors commented on the manuscript. L.B.C organized and uploaded data to DRYAD.