DOI: 10.5061/dryad.jwstqjqq2
title: Data from: The mechanisms by which RhoA activity and associated synaptic effects are controlled by the DISC1 scaffolding-like protein

description: 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.
description: <strong>Primary Neuronal
 Transfection:</strong> 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. <strong>Cell culture fixation and
 immunocytochemistry for dendritic spine and Sholl
 analysis:</strong>  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.
 <strong>Dendrite and dendritic spine imaging and
 analysis</strong>  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.
description: # Data from: The mechanisms by which RhoA activity and associated synaptic
 effects are controlled by the DISC1 scaffolding-like protein Dataset DOI:
 [10.5061/dryad.jwstqjqq2](https://doi.org/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 &lt; 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 &gt; 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 &gt; 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 &gt; 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.