DOI: 10.5061/dryad.tqjq2bw66
title: Microscopy and biophysical data for: Synthetic control of actin polymerization and symmetry breaking in active protocells

description: Non-linear biomolecular interactions on membranes drive membrane
 remodeling crucial for biological processes including chemotaxis,
 cytokinesis, and endocytosis. The complexity of biomolecular interactions,
 their redundancy, and the importance of spatiotemporal context in membrane
 organization impede understanding of the physical principles governing
 membrane mechanics. Developing a minimal in vitro system that mimics
 molecular signaling and membrane remodeling while maintaining
 physiological fidelity poses a significant challenge. Inspired by
 chemotaxis, we reconstructed chemically regulated actin polymerization
 inside vesicles, guiding membrane self-organization. External, undirected
 chemical inputs induced directed actin polymerization and membrane
 deformation uncorrelated with upstream biochemical cues, suggesting
 symmetry breaking. A biophysical model incorporating actin dynamics and
 membrane mechanics proposes that uneven actin distributions cause
 non-linear membrane deformations, consistent with experimental findings.
 This protocellular system illuminates the interplay between actin dynamics
 and membrane shape during symmetry breaking, offering insights into
 chemotaxis and other cell biological processes.
description: Experimental datasets were collected from confocal microscopy
 experiments and analyzed as described in the manuscript and supplementary
 materials. 
description: # Quantifying biophysical properties of giant unilamellar vesicles (GUVs)
 from microscopy data The files here contain input TIF files from
 microscopy data for **A) global and local rapamycin** and
 **B) supplemental** experiments with giant unilamellar vesicles (GUVs).
 Output files of segmentations and biophysical kymographs (1D GUV boundary
 quantities plotted as a function of time) are also provided. Details
 regarding image processing and analysis can be found in the Supplementary
 Analysis section of the primary article: Shiva Razavi et al., Synthetic
 control of actin polymerization and symmetry breaking in active
 protocells. *Science Advances* **10**, eadk9731 (2024).
 [DOI:10.1126/sciadv.adk9731](https://doi.org/10.1126/sciadv.adk9731) The
 general MATLAB code used to generate and conduct symmetry breaking
 analysis on these datasets can be found on Zenodo as well as GitHub:
 Zenodo repository:
 [https://zenodo.org/doi/10.5281/zenodo.10645538](https://zenodo.org/doi/10.5281/zenodo.10645538) GitHub repository: [https://github.com/basharif/GUV-symmetry-breaking-analysis](https://github.com/basharif/GUV-symmetry-breaking-analysis) ## Dataset Organization ### A. Global and Local Rapamycin Folders * GUVs were analyzed separately for the global rapamycin experiments (N=18) and local rapamycin experiments (N=3). Each one is labeled by "G"/"L" for global/local rapamycin, and a two-digit number (e.g. G01, G02, L01, L02 etc.). * The **globalguv.csv** and **localguv.csv** files provide a table of 5 analysis parameters for each GUV that were adjusted as appropriate (naming, number of frames to use, time of rapamycin addition, inner-outer mask size parameter, and center angle for kymographs). * For each analyzed GUV, there are two associated folders: an input and an output folder (e.g. **G01_input**, **G01_output**) #### Input Subfolders: "raw" and "processed" * Each input folder usually contains two subfolders. One folder contains raw microscopy TIF files (e.g. **G01_raw**). The other contains the processed TIF files (e.g. **G01_processed**), with smoothing and segmentation. **Note**: these processed TIF files were generated using "Code_1_Segmentation.m" found in the Zenodo/Github repository. * **"_raw"** folder: usually contains three TIF files separated by fluorescent channels for Actin (YFP), ActA (CFP), and membrane marker ("memb", mCherry). * **"_processed"** folder: contains processed versions of the same three TIF files in the "_raw" folder, as well as two mask files: a "mask_final" file which was used for segmentation, and a "mask_fused" file which overlays the mask boundary on the original TIF to check the quality. #### Output Subfolders: "Boundary" and "Boundary2Lumen" * Each output folder usually contains two subfolders, "**Boundary**" and "**Boundary2Lumen**", both containing kymographs for biochemical and physical boundary quantities. **Note**: the output kymographs and time-series tifs in these subfolders were generated using "Code_2_Kymographs.m" found in the Zenodo/Github repository. * Output subfolders labeled with "**Boundary**" in the folder name: contain biophysical kymographs computed only using boundary quantities, without normalization to signal in the lumen of the GUV, nor normalization to membrane marker. These kymographs are the standard ones used in the research article cited, especially for correlation analysis. * Output subfolders labeled with "**Boundary2Lumen**" in the folder name: contain biophysical kymographs based on boundary:lumen ratios, without any normalization to the membrane marker, as used specifically for PCA analysis. * Within each of the two output subfolders, the following file types can be found:  * "**.mat**" files: saved MATLAB variables for biophysical quantities * "**.png**" files: image files for biophysical kymographs. These include "**_fig_biochemkymos_Fix.png**", "**_fig_biochemkymos_Var.png**", "**_fig_physkymos_Fix.png**", "**_fig_physkymos_Var.png**", and "**_fig_macrosummary.png**" * Files with "biochemkymos" in the name are kymographs of fluorescent biochemical signals (Actin, ActA, membrane marker). Files with "physkymos" in the name are kymographs of physical quantities (velocity, cumulative displacement, and curvature). * File names containing "Var" refer to biophysical kymographs with varying number of boundary points (to track changes in GUV size). * File names containing "Fix" refer to biophysical kymographs with a fixed number of boundary points.  * "**.fig**" files: MATLAB-based figure files from which the above ".png" files were obtained. These include "**_figsout0.fig**", "**_figsoutFix.fig**" and "**_figsoutVar.fig**" * "**_tseries_mask**" **.tif** files: These are time-series of inner/outer masks used to segment each GUV and compute boundary quantities. The masks were all based on the membrane marker channel (mCherry), but they are overlayed on the signal from each fluorescent channel (labeled in the file name) to check for accuracy in estimating relevant biophysical boundary quantities. Regions in and around each GUV were demarcated as follows: * Green zone indicates the region outside the GUV; * Red zone indicates the lumen region inside the GUV; * Space between the dashed cyan and dashed red lines indicates the boundary region of the GUV from which biochemical quantities are computed; and * Dashed white line indicates the boundary from which physical quantities are computed. ### B. Supplemental Folder * This folder contains data described in the Supplementary Materials section of the research article. * Input and output subfolders are organized as described above for each GUV experiment. * The following GUV negative controls described in Supplementary Figures 6 and 7 are found in this folder, with **negcontrolguv.csv** containing the GUV analysis parameters: * "**Supp_Nctrl_DMSO**": DMSO (No rapamcyin) * "**Supp_Nctrl_NoActA**": No ActA * "**Supp_Nctrl_NoArp**": No Arp2/3 * The following global and local rapamycin supplemental results are also found in this folder, with **extrasuppguv.csv** containing the GUV analysis parameters: * "**Supp_Global1_cap3uM**" and "**Supp_Global2_cap100nM**": Supplementary Figure 15a and 15b, respectively * "**Supp_Global3_LatruncBegin**" and "**Supp_Global4_LatruncMid**": Supplementary Figure 5a and 5b, respectively * "**Supp_Global5_DPPC**": DPPC-based GUV with global rapamycin; Supplementary Figure 10, 14 * "**Supp_Local1_NoArp23**": Supplementary Figure 11a * "**Supp_Local2_CapCofilinDPPC**" and "**Supp_Local3_CapCofilinDPPC**": Supplementary Figure 11b and 11c * "**Supp_Local4_POPC**": Supplementary Figure 14