Data from: The mechanisms by which RhoA activity and associated synaptic effects are controlled by the DISC1 scaffolding-like protein
Data files
Dec 16, 2025 version files 193.15 MB
-
Fig._4.zip
129.36 MB
-
Fig._5.zip
63.53 MB
-
README.md
20.03 KB
-
Table_of_Raw_data_for_all_figures_of_the_source_manuscript.xlsx
237.79 KB
Abstract
This dataset contains the raw data for the manuscript entitled "The mechanisms by which RhoA activity and associated synaptic effects are controlled by the DISC1 scaffolding-like protein." The raw data corresponding to each main figure and supplementary figure of the article are shown in a spreadsheet. The spreadsheet tabs clearly indicate which figure of the paper the dataset corresponds to. Also, the microscopy dendritic spine images and dendrite tree images used for quantification are compiled and shared. The raw microscopy images are from primary neuronal cultures transfected with full-length PDZ-RhoGEF (i.e, wild-type PDZ-RhoGEF) or PDZ-RhoGEF containing deletions of the enzymatic domain of the PDZ domain. The quantification of the microscopy images demonstrates the effects of these PDZ-RhoGEF constructs on dendritic spine density, dendritic spine morphology, and the length and branching of the apical and basal dendrite trees.
Dataset DOI: 10.5061/dryad.jwstqjqq2
Description of the data and file structure
The dataset spreadsheet is the raw data in an Excel file for the above-named manuscript. Each tab detailed below corresponds to the raw data for the indicated figure number in the above-named manuscript. For each tab, a brief overview of the experiment, the abbreviation used, and the processing of the raw data is described.
The interaction between the DISC1 protein and active RhoA and PDZ-RhoGEF was examined using overexpression in HEK293T cells. The effects of PDZ-RhoGEF deletion constructs on active RhoA levels were also assessed, as were the effects of DISC expression on the membrane vs. cytosolic localization of PDZ-RhoGEF in cells using a fractionation procedure. Finally, the effects of PDZ-RhoGEF deletion constructs on dendritic spine density and morphology in cultured cortical neurons, as well as on dendrite length and branching were also assessed. The raw dendritic spine images and images for dendrite branching are compiled and shared in the Fig. 4 and Fig. 5 zipped folders.
Files and variables
File: Table_of_Raw_data_for_all_figures_of_the_source_manuscript.xlsx
Description: The dataset in this spreadsheet is the raw data in an excel file for the previously named source manuscript. Each tab detailed below corresponds to the raw data for the indicated figure number in the above named manuscript. For each tab, a brief overview of the experiment, the abbreviation used, and the processing of the raw data are described.
The interaction between the DISC1 protein and active RhoA and PDZ-RhoGEF was examined using overexpression in HEK293T cells. The effects of PDZ-RhoGEF deletion constructs on active RhoA levels were also assessed as was the effects of DISC expression on the membrane vs. cytosolic localization of PDZ-RhoGEF in cells using a fractionation procedure. Finally, the effects PDZ-RhoGEF deletion constructs on dendritic spine density and morphology in cultured cortical neurons as well as on dendrite length and branching were also assessed.
Variables
Figure 2C tab.
HEK293T cells were transfected with HA-DISC1 in combination with Myc-tagged PDZ-RhoGEF constructs. HA-DISC1 was immunoprecipitated, and the pull down probed for Myc to determine the recovery of PDZ-RhoGEF. The data sheet shows the raw data for this protein analysis organized per experiment. The raw intensity of each PDZ-RhoGEF signal was normalized to that of the corresponding DISC1. These values were then normalized to that of full-length PDZ-RhoGEF. WT = Full-length PDZ-RhoGEF, 128-1522 = delta PDZ PDZ-RhoGEF, 1-735 = delta DH-PH PDZ-RhoGEF; delta 435-1522 = delta PDZ and RGS PDZ-RhoGEF; delta 25 = delta actin PDZ-RhoGEF, 1-960 = delta PH PDZ-RhoGEF. Column A show the transfection condition per sample. Column B is the intensity of the Myc signal. Column C is the Myc signal of column B that has been background subtracted. For each experiment, the PRG/DISC1 column is the PRG intensity divided by that of DISC1. For each experiment, the (PRG/DISC1)/PRG input column is the PRG intensity divided by that of DISC1 that is then normalized the amount of PRG in the input lanes. For experiment 1, column S; experiment 2, column Q, and experiment 3 column P, the (PRG/DISC1/PRG column was normalized to that experiments WT data.
Figure 3C FL-PRG mem to cyt tab.
HEK293T cells were transfected with full-length Myc-PDZ-RhoGEF alone or in combination with DISC1. Cells were then subcellularly fractionated into membrane and cytosol fractions, and the amount of PDZ-RhoGEF in each fraction quantified. The data sheet shows the raw data for the amount of PDZ-RhoGEF per condition in the membrane and cytosolic fractions organized by experiment. For each sample, the ratio of PDZ-RhoGEF levels in the membrane fraction was divided by that in the corresponding cytosolic fraction (membrane fraction/cytosolic fraction). Column A show the condition for each sample (WT = wildtype/full-length PDZ-RhoGEF; C = cytosolic fraction; MF = membrane fraction; the number is the cell plate number). Column B shows the Myc-PDZ-RhoGEF protein intensity. Column C shows the background level per sample. Column D is the Myc signal minus the background. The background subtracted data are then shown again in column G. Column J is the Myc membrane to cytosol ratio per cell for cells not transfected with DISC1, whereas column K is the level of the Myc membrane to cytosol ratio per cell for cells transfected with DISC1. Columns M and N show the same data as columns J and K, but are normalized to average values. Columns R and S show the values for columns M and N for all experiments complied.
Figure 3C delta DH-PH mem to cyt tab.
HEK293T cells were transfected with Myc-PDZ-RhoGEF lacking the DH-PH domain (delta DH-PH) alone or in combination with DISC1. Cells were then subcellularly fractionated into membrane and cytosol fractions, and the amount of Myc-delta DH-PH in each fraction quantified. The data sheet shows the raw data for the amount of delta DH-PH per condition in the membrane and cytosolic fractions organized by experiment. For each sample, the ratio of delta DH-PH level in the membrane fraction was divided by that in the corresponding cytosolic fraction (membrane fraction/cytosolic fraction). Column A show the condition for each sample (1-735 = Myc-delta DH-PH PDZ-RhoGEF; C = cytosolic fraction; MF = membrane fraction; the number is the cell plate number). Column B shows the Myc-PDZ-RhoGEF protein intensity. Column C shows the background level per sample. Column D is the Myc signal minus the background. The background subtracted data are then shown again in column G. Column I is the Myc membrane to cytosol ratio per cell for cells not transfected with DISC1, whereas column J is the level of the Myc membrane to cytosol ratio per cell for cells transfected with DISC1. Columns L and < show the same data as columns I and J, but are normalized to average values. Columns Q and R show the values for columns L and M for all experiments complied.
Figure 3C delta PDZ mem to cyt tab.
HEK293T cells were transfected with full-length Myc-PDZ-RhoGEF lacking the PDZ domain (delta PDZ) alone or in combination with DISC1. Cells were then subcellularly fractionated into membrane and cytosol fractions, and the amount of PDZ-RhoGEF in each fraction quantified. The data sheet shows the raw data for the amount of delta PDZ per condition in the membrane and cytosolic fractions organized by experiment. For each sample, the ratio of delta PDZ levels in the membrane fraction was divided by that in the corresponding cytosolic fraction (membrane fraction/cytosolic fraction). Column A show the condition for each sample (128 = delta PDZ PDZ-RhoGEF; the number is the cell plate number). Rows 3-15 of columns A-G are the membrane fraction data, whereas rows 20-32 of columns A-G are the cytosolic fraction data. Column B shows the Myc-delta PDZ PDZ-RhoGEF protein intensity. Column C shows the background level per sample. Column D is the Myc signal minus the background. The background subtracted data are then shown again in column G. Column J is the Myc membrane to cytosol ratio per cell for cells not transfected with DISC1, whereas column K is the level of the Myc membrane to cytosol ratio per cell for cells transfected with DISC1. Columns M and N show the same data as columns J and K, but are normalized to average values.
Figure 3F tab.
Full-length PDZ-RhoGEF, or PDZ-RhoGEF deletion constructs lacking the PDZ or DH-PH domain were transfected into HEK293T cells, and active RhoA isolated using a commercial kit. The data sheet shows the raw data for the amount of active RhoA per sample normalized to the corresponding level of total RhoA. Raw data is shown per experiment. Per experiment, the ration of active to total RhoA was normalized to the full-length PDZ-RhoGEF condition. Column A and B show the transfection condition per sample (128 = delta PDZ, 1-735 = delta DH-PH; FL-PRG = full length PDZ-RhoGEF). Column C show levels of active RhoA. Column D shows levels of active RhoA that are background subtracted. Column E show levels of total RhoA, and column F is total RhoA data that is background subtracted. Column G is the background subtracted active RhoA divided by the background subtracted total RhoA. Column H is the data of column G that has been normalized to the full-length PDZ-RhoGEF condition.
Figure 4B-E tab.
Primary neuronal cultures were transfected with GFP alone or GFP in combination with full-length (FL) PDZ-RhoGEF, PDZ-RhoGEF lacking the PDZ domain, or PDZ-RhoGEF lacking the DH-PH domain. The effects on total dendritic spine density and that of the thin, stubby, and mushroom spine subtypes was analyzed. The data sheet shows the raw data per cell for each of the transfection conditions. Shown per cell, is the number of mushroom spines, the number of stubby spines, the number of thin spines, and the number of total spines. These spine numbers were then divided by the indicated total dendrite length (abbreviated TDL in the data sheet).
Figure 4F, H, J tab
Data described for the Figure 4B-E tab was analyzed and presented in a different way. Here the head diameter (in micron) of all thin spines, for the GFP only condition, the full length PDZ-RhoGEF condition (FL-PRG), the delta DH-PH, and delta PDZ conditions are shown. The ID of each cell and the corresponding thin spines for that cell are indicated.
Figure 4G, I, K tab
Data described for the Figure 4B-E tab was analyzed and presented in a different way. Here the head diameter (in micron) of all mushroom spines, for the GFP only condition, the full length PDZ-RhoGEF condition (FL-PRG), the delta DH-PH, and delta PDZ conditions are shown. The ID of each cell and the corresponding mushroom spines for that cell are indicated.
Figure 5B tab
Primary cultured cortical neurons were transfected with GFP alone or in combination with full-length PDZ-RhoGEF, delta DH-PH PDZ-RhoGEF, or delta PDZ PDZ-RhoGEF. Sholl analysis on the basal dendrite tree was performed. Column B of the data sheet is the distance from the soma (in micron). The numbers in the adjacent columns are the number of intersections. The ID and transfection of each condition in indicated in Row 1. WT is the full length PDZ-RhoGEF condition, 1-735 is the delta DH-PH condition, and 128 is the delta PDZ condition.
Figure 5C, D tab
Primary cultured cortical neurons were transfected with GFP alone or in combination with full-length PDZ-RhoGEF, delta DH-PH PDZ-RhoGEF, or delta PDZ PDZ-RhoGEF tab. The data sheet shows raw data for each cell indicated in column A. The total length of the basal dendrite tree is shown in column B (in microns). The number of branches per cell in indicated in column C. Colum D show the sholl ramification per cell. WT is the full length PDZ-RhoGEF condition, 1-735 is the delta DH-PH condition, and 128 is the delta PDZ condition.
Figure 5E tab.
Primary cultured cortical neurons were transfected with GFP alone or in combination with full-length PDZ-RhoGEF, delta DH-PH PDZ-RhoGEF, or delta PDZ PDZ-RhoGEF. Sholl analysis on the apical dendrite tree was performed. Column B of the data sheet is the distance from the soma (in micron). The numbers in the adjacent columns are the number of intersections. The ID and transfection of each condition in indicated in Row 2. WT is the full length PDZ-RhoGEF condition, 1-735 is the delta DH-PH condition, and 128 is the delta PDZ condition.
Figure 5F, G tab.
Primary cultured cortical neurons were transfected with GFP alone or in combination with full-length PDZ-RhoGEF, delta DH-PH PDZ-RhoGEF, or delta PDZ PDZ-RhoGEF tab. The data sheet shows raw data for each cell indicated in column A. The total length of the apical dendrite tree is shown in column B (in microns). The number of branches per cell in indicated in column C. Colum D show the sholl ramification per cell. WT is the full length PDZ-RhoGEF condition, 1-735 is the delta DH-PH condition, and 128 is the delta PDZ condition.
Suppl Fig 2B 95 kDA DISC human tab
Brain homogenates from control subjects, subjects with bipolar disorder, or subject with schizophrenia were quantified for the levels of the 95 kDA DISC1 signal. The data for 5 separate blots are shown. The diagnosis of each sample is indicated in column B. Whether the subject had or did not have psychosis is indicated in column C. The sex of the subjects in indicated in column D. The order of each sample is indicated in column E. The mean intensity of the 95 kDA DISC1 band is shown in column G. The blot background is shown in column H. The intensity of the DISC1 band minus the background is shown in column I. The intensity of the corresponding GAPDH bank is shown in column Q, and the background intensity subtracted intensity of GAPDH shown in column R. Column J show the background subtracted DISC1 signal divided by that of the background subtracted GAPDH signal. Column K is the data from column J normalized to the male control samples.
Suppl Fig 2C 110 kDA DISC human tab
Brain homogenates from control subjects, subjects with bipolar disorder, or subject with schizophrenia were quantified for the levels of the 110 kDA DISC1 signal. The data for 5 separate blots are shown. The diagnosis of each sample is indicated in column B. Whether the subject had or did not have psychosis is indicated in column C. The sex of the subjects in indicated in column D. The order of each sample is indicated in column E. The mean intensity of the 110 kDA DISC1 band is shown in column G. The blot background is shown in column H. The intensity of the DISC1 band minus the background is shown in column I. The intensity of the corresponding GAPDH bank is shown in column Q, and the background intensity subtracted intensity of GAPDH shown in column R. Column J show the background subtracted DISC1 signal divided by that of the background subtracted GAPDH signal. Column K is the data from column J normalized to the male control samples.
Suppl Fig 2D 250 kDA DISC human tab
Brain homogenates from control subjects, subjects with bipolar disorder, or subject with schizophrenia were quantified for the levels of the 250 kDA DISC1 signal. The data for 5 separate blots are shown. The diagnosis of each sample is indicated in column B. Whether the subject had or did not have psychosis is indicated in column C. The sex of the subjects in indicated in column D. The order of each sample is indicated in column E. The mean intensity of the 250 kDA DISC1 band is shown in column G. The blot background is shown in column H. The intensity of the DISC1 band minus the background is shown in column I. The intensity of the corresponding GAPDH bank is shown in column Q, and the background intensity subtracted intensity of GAPDH shown in column R. Column J show the background subtracted DISC1 signal divided by that of the background subtracted GAPDH signal. Column K is the data from column J normalized to the male control samples.
Suppl Fig 2E > 250 kDA DISC human tab
Brain homogenates from control subjects, subjects with bipolar disorder, or subject with schizophrenia were quantified for the levels of the > 250 kDA DISC1 signal. The data for 5 separate blots are shown. The diagnosis of each sample is indicated in column B. Whether the subject had or did not have psychosis is indicated in column C. The sex of the subjects in indicated in column D. The order of each sample is indicated in column E. The mean intensity of the > 250 kDA DISC1 band is shown in column G. The blot background is shown in column H. The intensity of the DISC1 band minus the background is shown in column I. The intensity of the corresponding GAPDH bank is shown in column Q, and the background intensity subtracted intensity of GAPDH shown in column R. Column J show the background subtracted DISC1 signal divided by that of the background subtracted GAPDH signal. Column K is the data from column J normalized to the male control samples.
File: Fig._4.zip
Description: Images of neurons transfected with wildtype (WT)/full-length (FL PDZ) PDZ-RhoGEF, PDZ-RhoGEF lacking the PDZ domain (delta PDZ), or PDZ-RhoGEF lacking the GEF domain (delta GEF). A GFP only condition was used for baseline comparisons. A Keyence BZ-X700E scanning microscope was used to image dendrites and dendritic spines. Each folder in the main folder contains images that correspond to the above indicated transfection conditions. These images show dendritic spines from neurons that were used for quantification that comprises Figure 4 of the source manuscript.
Sub-folders: There is a folder for each condition (DeltaGEF, DeltaPDZ, FL PRG, GFP) which contains TIF image files labeled by cell #, apical #, and dendrite # where appropriate. Note that DeltaGEF is also labeled as Delta DH-PH condition in raw data files.
DeltaGEF (A):
cell 1 apical 1 dendrite 1 and 2.tif
cell 4 apical 1.tif
cell 4 apical 2.tif
cell 6 apical 1.tif
cell 6 apical 2.tif
cell 7 apical 1.tif
cell 7 apical 2.tif
cell 8 apical 1.tif
cell 8 apical 2.tif
DeltaPDZ (B):
cell 1 apical 2 dendrites 1 and 2.tif
cell 2 apical 1.tif
cell 2 apical 2.tif
cell 5 apical 2.tif
cell 6 apical 1.tif
cell 6 apical 2.tif
cell 7 apical 2.tif
cell 8 apical 1.tif
cell 8 apical 2.tif
cell 9 apical 1.tif
cell 10 apical 1.tif
cell 10 apical 2.tif
cell 11 apical 1.tif
cell 11 apical 2.tif
cell 12 apical 1.tif
cell 12 apical 2.tif
cell 14 apical 2.tif
cell 15 apical 1.tif
cell 15 apical 2.tif
FL PRG (E&F):
cell 1 apical 1.tif
cell 1 apical 2.tif
cell 2 apical 1.tif
cell 4 apical 1.tif
cell 4 apical 2.tif
cell 3 apical 1.tif
cell 2 (F) apical 1.tif
cell 4 (F) apical 1.tif
cell 5 apical 1.tif
cell 5 apical 2.tif
cell 6 apical 1.tif
cell 9 apical 1.tif
cell 9 apical 2.tif
cell 10 apical 1.tif
cell 11 apical 1.tif
cell 11 apical 2.tif
GFP (C&D):
cell 1 apical 1.tif
cell 1 apical 2.tif
cell 3 apical 1 and 2.tif
cell 7 apical 1.tif
cell 7 apical 2.tif
cell 8 apical 1.tif
cell 9 apical 1.tif
cell 9 apical 2.tif
cell 10 apical 1.tif
cell 10 apical 2.tif
cell 5 apical 1.tif
cell 5 apical 2.tif
File: Fig._5.zip
Description: Images of neurons transfected with wildtype (WT)/full-length (FL PDZ) PDZ-RhoGEF, PDZ-RhoGEF lacking the PDZ domain (delta PDZ), or PDZ-RhoGEF lacking the GEF domain (delta GEF). A GFP only condition was used for baseline comparisons. Each folder in the main folder contains images that correspond to the above indicated transfection conditions. A Keyence BZ-X700E scanning microscope was used to image neurons. These images show apical and basal dendrites from neurons that were used for quantification that comprises Figure 5 of the source manuscript.
Sub-folders: A folder for each condition (DeltaGEF, DeltaPDZ, FL PRG, GFP) contains TIF image files labeled as FF_cell #. The raw data file refer to the PDZ-RhoGEF constructs by the missing kd, for example DeltaGEF refers to the same condition as 1-735, 128 refers to DeltaPDZ, and FL PRG is also referred to as WT.
DeltaGEF (1-735):
FF_cell 1.tif
FF_cell 2.tif
FF_cell 3.tif
FF_cell 4.tif
FF_cell 5.tif
FF_cell 6.tif
FF_cell 5.1.tif
FF_cell 7.tif
FF_cell 8.tif
DeltaPDZ (128):
FF_cell 1.tif
FF_cell 2.tif
FF_cell 5.tif
FF_cell 9.tif
FF_cell 15.tif
FF_cell 17.tif
FF_cell 19.tif
FL PRG (WT):
FF_cell 2.tif
FF_cell 4.tif
FF_cell 5.tif
FF_cell 7.tif
FF_cell 8.tif
FF_cell 10.tif
GFP:
FF_cell 3.tif
FF_cell 9.tif
FF_cell 11.tif
FF_cell 12.tif
FF_cell 13.tif
FF_cell 14.tif
FF_cell 15.tif
FF_cell 16.tif
Code/software
Images are Tiff files and can be opened with most photo programs. The spreadsheet of raw data is provided as an Excel spreadsheet.
Primary Neuronal Transfection:
Primary cortical rat neurons were transfected with cDNA between DIV21-DIV23 with Lipofectamine 2000. cDNA of Myc-tagged PDZ-RhoGEF constructs in combination with GFP were mixed with Lipofectamine in DMEM containing 10 % fetal bovine serum and incubated at 37 °C for 30 minutes, then 100 µl of this combination was added to the cells. 4 hours later, the cell media was removed, and 500 µl of old neurobasal media that was not exposed to Lipofectamine (removed just prior to the addition of the cDNA+Lipofectamine) and 500 µl of fresh neurobasal media supplemented with B27 (Gibco), DL-AP5 (Tocris), penicillin/streptomycin, and L-glutamine was added to each culture dish.
Cell culture fixation and immunocytochemistry for dendritic spine and Sholl analysis:
Five days following transfection with cDNA, primary cortical neurons were fixed (between DIV26-DIV28) by the application of a combination of 3.7 % formaldehyde and 4 % sucrose for 10 minutes at room temperature. The fixative was rinsed off with PBS, followed by incubation for 10 minutes with pre-chilled methanol. Cells were then rinsed with PBS containing 0.3 % Triton X-100, and blocked in this PBS-Triton containing 3 % normal donkey serum for 1 hour at room temperature. A 1:300 dilution of chicken anti-GFP antibody was used to enhance the GFP signal and the labeling of PDZ-RhoGEF constructs was accomplished via a mouse anti-Myc-Tag antibody.
Cells were incubated in primary antibody overnight at room temperature. Cells were then washed with PBS, and incubated with Donkey anti-chicken AlexaFluor 488 in PBS containing donkey serum for 3 hours at room temperature. Cells were then rinsed three times with PBS, and coverslipped with VectaShield hardset anti-fade mounting media.
Dendrite and dendritic spine imaging and analysis
A Keyence BZ-X700E scanning microscope was used to image dendrites and dendritic spines. For dendrite images of pyramidal neurons, a 20x objective lens was used with a 0.3 µm step size for Z-stack collection. For images of dendritic spines, a 100x objective lens was used with a 0.1 µm step size for Z-stack collection. Spine images were all obtained for apical tree secondary dendrites, with each dendrite segment at least 50 µm long. BZ analyzer software was used to collapse and deconvolve images.
NeuronStudio software was used to analyze dendritic length and branching and to analyze dendritic spine density and morphology. For spine quantification, all spines from a given dendrite were manually selected, upon which the program places a hollow ellipse around the head of the spine. The diameter of the ellipse is indicative of the diameter of the spine head. In instances in which the ellipse is greater or less than the true size of the spine head, manual adjustments are made. Spines were then classified as thin, stubby, or mushroom. Briefly, spine that lack a clear neck region were always classified as stubby. Spines with a neck region and spine diameter larger than 0.5 µm were classified as mushroom spines. Spines with a neck region and a head diameter less than 0.5 µm were classified as thin. All dendrite and spine imaging and analysis was done blinded to experimental conditions.