DOI: 10.5061/dryad.dr7sqvb8b
title: Data from: Activity-dependent regulation of Cdc42 by Ephexin5 drives synapse growth and stabilization

description: Synaptic RhoGEFs play vital roles in regulating the activity-dependent
 neuronal plasticity critical for learning. Ephexin5, a RhoGEF implicated
 in the etiology of Alzheimer’s disease and Angelman Syndrome, was
 originally reported in neurons as a RhoA-specific GEF that negatively
 regulates spine synapse density. Here, we show that Ephexin5 activates
 both RhoA and Cdc42 in brain. Furthermore, using live imaging of GTPase
 biosensors, we demonstrate that Ephexin5 regulates activity-dependent
 Cdc42, but not RhoA, signaling at single synapses. The selectivity of
 Ephexin5 for Cdc42 activation is regulated by tyrosine phosphorylation,
 which is regulated by euronal activity. Finally, in contrast to Ephexin5’s
 role in negatively regulating synapse density, we show that downstream of
 neuronal activity Ephexin5 positively regulates synaptic growth and
 stabilization. Our results support a model in which plasticity-inducing
 neuronal activity regulates Ephexin5 tyrosine phosphorylation, driving
 Ephexin5-mediated activation of Cdc42 and the spine structural growth and
 stabilization vital for learning.
description: # Data from: Activity-dependent regulation of Cdc42 by Ephexin5 drives
 synapse growth and stabilization [Access Dataset on
 Dryad](https://doi.org/10.5061/dryad.dr7sqvb8b) # Overview This dataset
 contains 4 types of data: two-photon imaging (2-photon) fluorescence data,
 Forster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy
 (FRET-FLIM) data, Western blot data, and immunohistochemistry (IHC)
 fluorescence data. 2-photon fluorescence data was collected using a custom
 2-photon microscope. Green fluorescence was acquired in channel 1 and red
 was acquired in channel 2. Analysis was performed using custom MATLAB
 software. FRET-FLIM data was collected using a custom-built 2-photon
 microscope with time correlated single photon counting. Lifetime data is
 stored in channel 1 and the fluorescence intensity from a red cell fill is
 stored in channel 2. Data was analyzed using custom MATLAB software. IHC
 fluorescence data was collected using a Zeiss Apotome 3 structured
 illumination microscope. Data was captured from blue, green, and red
 wavelength channels and stored in a single z-stack image. Data was
 deconvolved in Zeiss ZenPro software. Western blot data was collected
 using a LI-COR Odyssey CLx blot imager. Fluorescence data was acquired
 from 700nm and 800nm wavelength channels and stored separately. ##
 Description of the data and file structure We have provided our raw data
 in the "PetshowEtAl_2025_RawData.zip" file and our analysis
 files in the "PetshowEtAl_2025_Analysis.zip" file. These two
 folders are sub-divided into files for each individual figure. ### Raw
 data: Folders for Figures 4 & 5 contain data from 2-photon imaging
 experiments. These Figure folders are separated into folders containing
 individual cells folders, under ‘Cells.’ Cell folders are named with the
 following convention: [initials, cell number]. Each cell folder contains
 all of the images for that cell with the image name convention: [initials,
 cell number, dendrite identifier (a or b)]. Z-stacks and maximum
 projection images are both included and stored in .tif format. A zoomed
 out image of the whole cell is included in .tif format with the name ‘m’
 in place of the dendrite identifier. Cell folders may also contain files
 with microscope stage position data (.pos files) and screenshots taken
 during the experiment (.bmp or .jpg files). Folders for Figures 1, 3, 4,
 & 5 contain data from 2-photon FRET-FLIM experiments. These Figure
 folders are separated into folders (‘Cells’) containing individual cells
 folders with the naming convention: [initials, cell number]. The cell
 folder contains the raw fluorescence data from channel 2 (red) and a
 sub-folder named "spc" which contains the raw single photon
 counting data per pixel. Both are stored in .tif format and use the naming
 convention [initials, cell number, dendrite identifier (a or b)]. The
 "spc" folders also include .mat files used to determine whether
 enough photons were collected. Cell folders may also contain files with
 microscope stage position data (.pos files) and screenshots taken during
 the experiment (.bmp or .jpg files). Folders for Figures 1, 2, & 3
 contain data from Western blot experiments. These Figure folders are
 separated into folders containing individual blots, under ‘Blots.’ Blot
 folders are named with the following convention: [date of acquisition,
 experiment/stain used]. Each blot folder contains a single raw image of
 the blot, stored in .tif format, with the naming convention [arbitrary
 number string, wavelength of acquisition (700 or 800)]. Blot folders also
 contain a .txt file with metadata on the instrument settings used to
 acquire the image, and low-resolution summary image of the blot as a .jpg.
 Folders for Figures 3 & 4 contain data from IHC experiments. These
 Figure folders are separated into folders containing IHC experiments,
 under ‘IHC.’ IHC folders are named with the following convention: [date of
 acquisition, experiment/stain used]. Each IHC folder contains a single raw
 images taken from a single experiment, stored in .czi format, with the
 naming convention [experiment, stain, cell number]. ### Analysis: Folders
 for Figures 4 & 5 contain analysis files for 2-photon imaging
 experiments. These Figure folders contain a ‘Cell ANNs’ folder and a ‘Cell
 Excels’ folder. Cell ANNs folders contain .ann files and .csv files. The
 .ann files store integrated fluorescence data for green and red
 fluorescence (channel 1 and channel 2, respectively) and the positions of
 the analyzed ROIs. These have the naming convention [initials, cell
 number, dendrite identifier (a or b)]. The .csv files store the integrated
 background subtracted fluorescence for the analyzed spines with the naming
 convention [initials, cell number, dendrite identifier (a or b)]. Their
 data structure is: individual ROIs are separated by row, and for each
 row/ROI, column entries for the mean background pixel value, integrated
 pixel density, and pixel area of the ROI ('Background,
 'Integral' 'IntegralPixelCount') exist in triplets for
 each timepoint/image in the series. The Cell Excels folders contain .xls
 files which organize and display the .ann and .csv data for each cell in a
 user-friendly way. Their data structure is: rows display the normalized
 fluorescence values for target ROIs or dendrites, columns display the
 timepoint. These files contain figures not necessary for reanalysis of the
 data. They have the naming convention [initials, cell number, dendrite
 identifier (a or b)]. Folders for Figures 1, 3, 4 & 5 contain analysis
 files for FRET-FLIM experiments. Figure folders contain a ‘Cell MATs’
 folder and a ‘Cell Excels’ folder. Cell MATs folders contain .MAT files
 which store data detailing the ROI position, the average lifetime (tau_m)
 and the red fluorescence intensity (int_int2). These .MAT files are named
 with the following convention:["A_", initials, cell number,
 dendrite identifier (a or b), "_ROI2"]. The Cell Excels folders
 contain .xls files which organize and display the .MAT data for each cell
 in a user-friendly way. Their data structure is: time points are separated
 by row, columns indicate values obtained from an ROI at each timepoint,
 including background subtracted red fluorescence intensity, green
 fluorescence intensity, calculated bound fraction of the FRET sensor, and
 fluorescence lifetime ('red_int2,' 'int_int2,'
 'fraction2,' 'tau_m'). These files contain figures not
 necessary for reanalysis of the data. They have the naming convention
 [initials, cell number, dendrite identifier (a or b)]. All Figure folders
 contain summary .xlsx files where normalization and averaging of spine
 size or average lifetime across cells were performed, or averaging of
 western blot band intensity, or quantification of IHC data. Their
 structure varies but is labeled in header information. These files contain
 figures not necessary for reanalysis of the data. All figure folders
 contain .prism files where statistical testing was performed on the
 datasets contained within the summary .xlsx files ## Usage Notes .tif and
 .czi images can be opened using most image analysis softwares (e.g.
 ImageJ) .MAT and .ann files can be opened using MATLAB .xls and .xlsx
 files can be opened using Microsoft Excel .prism files must be opened with
 GraphPad Prism