DOI: 10.5061/dryad.ttdz08m8g
title: Data from: Expression, subcellular localization, and mechanistic analysis of intellectual disability syndrome protein ABBA

description: A missense mutation in the MTSS2 gene, which encodes the I-BAR domain
 protein ABBA (Mtss1l/Mtss2), has been linked to an intellectual disability
 syndrome. To better understand the MTSS2 mutation-related effect in the
 brain, we elucidated the cells expressing ABBA and the localization of
 ABBA in these cells to get insights into which cells and processes might
 be dysfunctional in mutation-carrying patients. As a novel discovery, we
 found that ABBA was highly expressed in GABAergic inhibitory neurons, such
 as parvalbumin-positive interneurons in the hippocampus. At the
 subcellular level, ABBA localizes to the edges of membrane protrusions in
 various cells in the brain, suggesting a role in cell migration and
 spinogenesis. Overexpression of ABBA in pyramidal excitatory and
 inhibitory neurons increased dendritic spine density. Through live-cell
 imaging, we demonstrated that ABBA facilitates spine initiation by
 clustering on the plasma membrane before a new filopodium appears.
 However, our live cell imaging data also revealed that ABBA localized not
 only to small focal points, typical for filopodia formation, on the plasma
 membrane, but also more broadly on the edge of lamellipodial structures.
 Compared to its close homolog MIM, ABBA appears to be a more general
 facilitator of protrusion formation, from dendritic filopodia to
 lamellipodial structures. Altogether, our findings provide insights into
 ABBA expression, localization, and functional mechanisms, advancing our
 understanding of its role in neurodevelopmental processes and disorders.
description: <strong>Animals</strong> In the
 present study, we have used wild-type and transgenic Tg(Thy1-EGFP)MJrs/J
 mice (Jackson Laboratory) on a C57Bl/6J background. Tg(Thy1-EGFP)MJrs/J
 mice express EGFP in sparse subsets of neurons within specific
 populations. All experimental procedures were carried out according to
 Finnish laws and ethics under the EU directive 2010/63/EU (licenses:
 ESAVI/7404/2021, ESAVI/30031/2024, and GMO 3/S/12). Animals were kept in
 cages as groups of 2-4 mice in a controlled environment (temperature
 21±1°C, humidity 50±10%, light period 06:00 AM to 6:00 PM) and supplied
 with food and water ad libitum. For tissue collection,
 animals were deeply anesthetized with pentobarbital solution (200 mg/kg)
 containing lidocaine and transcardially perfused using cold PBS. The
 brains were dissected and kept in 4% PFA for 24 hours and then
 cryoprotected in 20% sucrose solution for at least 48 hours before
 cryo-sectioning. The Thy1-GFP brains were cut sagittally into 50 µm
 sections with a cryostat (Leica) and stored in cryoprotectant solution
 (30% ethylene glycol, 20% glycerol in PBS) at -20°C until further
 processing. The wild-type mice's left-brain
 hemisphere was fixed with 4% PFA for 3 days, followed by 70% ethanol for 3
 days (daily refreshing solutions), and then paraffinized during a 10-hour
 adult mouse brain program. Then, 5 μm sections were cut for staining. The
 right brain hemisphere was dissected into the cortex, hippocampus, and
 cerebellum, which were fast frozen in liquid nitrogen and stored for
 Western blot at -80°C. <strong>Organotypic
 slices</strong> RccHan: WIST (Inotiv) rat pups
 were used for organotypic brain slices. At postnatal days 5–10, rat pups
 were first anesthetized by using isoflurane and then euthanized by
 decapitation. Both hippocampi were dissected and sliced transversely into
 400 μm cross-sections using a tissue chopper. The slices were placed onto
 sections of nitrocellulose membrane ~5 mm<sup>2</sup>, which
 were placed on a Millicell cell culture insert with a pore size of 0.4 μm
 and a diameter of 30 mm on the 6-well plate containing 1.2 ml of culture
 medium. The culture medium was composed of 95.5 ml Opti-MEM (Gibco), 50 ml
 of HBSS (Gibco), 50 ml of Hof Horseshoe serum (Gibco), and 2 ml of
 D-glucose (50%), and the pH was set to 7.2. After plating, the hippocampal
 slices were kept at 37°C in 5% CO2. At DIV3, anti-mitotic (0.5 mM uridine,
 0.5 mM ARA-C, 0.5 mM 5-Fluoro-deoxy-uridine) was added to inhibit the
 glial growth. At DIV4, the media was replaced with fresh media. Later, the
 culture media were refreshed twice a week. At DIV10, slices were treated
 either with 180 ng of BDNF for 24 hours or 30 μM Bicuculine for 1 hour,
 diluted in culture media. After treatments, the slices were put back into
 refreshed culture media and incubated for 4 h, 1 day, and 2 days from the
 start of the treatment. Slices were then snap-frozen and stored at -80°C
 until protein extraction.
 <strong>Characterization of ABBA
 antibody</strong> In western blotting and
 immunostaining, we used an earlier characterized rabbit ABBA antibody.
 This was originally generated in the Lappalainen laboratory against the
 central region of the mouse ABBA protein that is not conserved in other
 I-BAR-domain proteins, such as the loss homologue MIM or Irsp53. This
 study demonstrated with Western blot analysis that the antibody detected
 only a single band of expected mobility (around 100 kDa) from brain
 lysates and in NIH3T3 cells expressing a GFP-fusion protein of ABBA.
 Furthermore, the antibody did not recognize other I-BAR-domain proteins,
 IRSp53 and MIM, on a western blot, suggesting that the antibody is
 specific to ABBA. The antibody was later reproduced in Rivera´s
 laboratory, and in their recent study, this antibody was used to detect
 decreased ABBA expression in shRNA ABBA-treated C6 cells, indicating that
 it is indeed ABBA that the antibody recognizes. Uncropped western blots
 shown in Figure 1 are provided as Supplementary Figure 1.  
 <strong>Immunostaining/immunohistochemistry</strong> Both paraffin sections (5 µm, Fig. 1) and cryosections (50 µm, Fig. 2) were used for stainings. Briefly, paraffinized brain sections went through deparaffinization and rehydration. After antigen retrieval (1h boiling in 0.01 M citrate buffer, pH 6.0), blocking was done by using 2% BSA, 5% milk, 5% sucrose, and 1% Triton X-100 in PBS. Cryosections were first permeabilized with 0.1% Triton X-100 in PBS and then blocked for 1.5 hours in a blocking buffer containing 0.3% Triton X-100 and 5% goat serum in PBS. The sections were then incubated in blocking solution with primary antibodies: rabbit anti-ABBA (1:500 [5], guinea pig polyclonal antiserum against parvalbumin (1:500, synaptic system, 195004), chicken polyclonal anti-GFAP (1:4000, Abcam, Ab4674), and chicken monoclonal recombinant IgY anti-Iba1 (1:1000, synaptic system, 234009) overnight at 4°C. The next day, sections were incubated in secondary antibodies: anti-rabbit Alexa-488, anti-rabbit Alexa-647 (1:1000, ThermoFisher, A21206, A31573), anti-guinea pig Alexa-568 (1:1000, ThermoFisher, A11075), and anti-chicken Alexa-647 (1:1000, ThermoFisher, A-21449) for 2h and mounted on glass coverslips using Immu-mount (Thermo Scientific,999041). The stained fixed brain slices were scanned using a 3DHISTECH Panoramic 250 FLASH III digital slide scanner using a 20X/0.8 NA objective at the FIMM digital microscopy and molecular pathology unit. For better resolution and 3D images, the brain slices were further imaged through a Zeiss LSM880 inverted confocal microscope using 20X/0.80 NA dry or 63X 1.4 NA oil immersion objectives. <strong>Western blotting</strong> The hippocampus, cortex, and cerebellum of wild-type mice were homogenized and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with 10% protease inhibitor, 10% phosphatase inhibitor cocktail (Roche), and 1% SDS, using a bead mill homogenizer with 1.4 mm ceramic beads for 15-20 seconds. Organotypic brain slices were similarly processed with sonication. Protein concentration was measured using the BCA Protein Assay (ThermoFisher Scientific, 23227). For analysis, 20 µg of protein was separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked for 5 minutes in Bio-Rad EveryBlot blocking buffer (Bio-Rad, 12010020) and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: Rabbit anti-ABBA (1:1000 [5], rabbit anti-srGap3 (1:1000, Novus, NBP1-88831), and mouse anti-Gas7 (1:1000, Santa Cruz, SC-365385). Membranes were washed three times with TBS-T and incubated with StarBright secondary antibodies (anti-rabbit, 1:2500, Invitrogen, 1200Y161; anti-mouse, Invitrogen, 12005866) diluted in blocking buffer and 0.02% SDS for 1 hour at room temperature. Membranes were again washed three times with TBS-T and imaged using the ChemiDocTM MP Imaging System (Bio-Rad). The protein levels were quantified using Image Lab software (Bio-Rad) and normalized against total protein per lane. Precision Plus Protein Unstained (Bio-Rad, 161- 363) and All Blue Standards (Bio-Rad, 1610373EDU) were used as molecular weight references. <strong>Hippocampal cultures and transfections</strong> The plasmids pmCherry-C1 and pEGFP-C1 were purchased from Clontech Laboratories, Inc. GFP-ABBA and mCherry-ABBA were gifts from Juha Saarikangas. The RFP-LifeAct construct was a gift from Roland Wedlich-Söldner (University of Münster, Münster, Germany). GFP-LifeAct was a gift from Emmanuel Lemichez (an Institute Pasteur, Université Paris Cité, France). The murine N-WASP-mCherry construct was provided by Maria Vartiainen (Institute of Biotechnology, University of Helsinki). mCherry-actin plasmid was a gift from Martin Bähler (Westfälische Wilhelms-University, Münster, Germany). The Scar W and WA constructs [13] were a gift from Laura Machesky (Beatson Institute for Cancer Research, Glasgow, United Kingdom).  mCherry-Akt-PH was provided by Vesa Olkkonen (Minerva Foundation Institute for Medical Research). GFP-Tubby was given by Lawrence Shapiro (Columbia University, USA). GFP-Rac1 N17 and GFP-Rac1 V12 were gifts from Johan Peränen (University of Helsinki). The hippocampal culture preparation was done by the previously proposed method. The dissection and dissociation of cells were carried out by the Neuroscience Center core facility (HiLIFE, University of Helsinki). Briefly, hippocampi were dissected from embryonic day 16–17 Wistar rat fetuses. Cells were dissociated in 0.05% papain and triturated in Ca2+ and Mg2+-free HBSS medium with 1mM sodium pyruvate and 10mM HEPES (pH 7.2). The obtained cells were then plated on 13mm glass coverslips (VWR) (100000 cells) or 13 mm high-precision glass coverslips for SIM (Marienfeld) (15000 cells) coated with poly-L-Lysine (0.01 mg/ml, Sigma Aldrich) in a 24-well plate. The culturing of the cells was done in a Neurobasal medium (Invitrogen) supplemented with L-glutamine (Invitrogen), B-27 (Invitrogen), and primocin (InvivoGen) in humidified incubators at 37°C and 5% carbon dioxide (CO2) with media refreshing twice a week. The transfection was performed at DIV14 by using Lipofectamine 2000 (Invitrogen) with 0.5 μg plasmid/well in 24-well plates, as described earlier [15]. Normally, the transfected cultures were fixed after 24 hours of the transfection. For testing effects of F-actin depolymerization or PI3-kinase activity, cells were treated with 5 mM Latrunculin B (Sigma-Aldrich) or 100 mM PI3-kinase inhibitor LY294002 (Sigma-Aldrich) before fixation. <strong>Immunostaining</strong> Hippocampal cultures were fixed with pre-warmed (37°C) 4% paraformaldehyde (PFA) for 13 minutes at room temperature (RT), and after this, they were permeabilized using 0.2% TritonX-100 in PBS. Blocking was done for 30 minutes using 3% normal donkey serum and 0.5% bovine serum albumin (BSA) in PBS. The fixed cells were incubated with primary antibodies in a blocking solution for 1 hour at RT, followed by incubation with secondary antibodies for 1 hour. The coverslips with cells were mounted on glass slides by using Shandon Immu-Mount (Thermo Fisher Scientific,9990402) or Prolong Gold (ThermoFisher, P10144). The primary antibodies used were rabbit anti-ABBA (1:400 [5], mouse anti-myc (9E10) (1:200, ThermoFisher, MA1-980), rabbit monoclonal anti-GAD65/67 (1:400, Abcam, AB 183999), guinea pig polyclonal antiserum against parvalbumin (1:500, Synaptic Systems, 195004), and chicken polyclonal anti-GFAP (1:4000, Abcam, Ab4674). The Secondary antibodies were anti-rabbit Alexa-488, anti-rabbit Alexa-647 (1:1000, ThermoFisher, A21206, A21446), anti-guinea pig Alexa-568 (1:1000, ThermoFisher, A11075), and anti-chicken Alexa-647 (1:1000, ThermoFisher, A-21449). F-actin was visualized using Alexa 488-conjugated phalloidin, Alexa 568-conjugated phalloidin, or Alexa 633-conjugated phalloidin (1:200, ThermoFisher, A12379, A12380, and A22284). <strong>Imaging</strong> Confocal images were obtained using either a Zeiss LSM780 or LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature, and the live cell imaging was performed in a chamber where the temperature was maintained at 37°C and the CO2 levels were 5%.  The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Cells or fields of view were selected randomly for imaging. The laser power and gain settings were adjusted to maximize the signal-to-noise ratio. The Fiji software was used to process the image files obtained from the imaging. The used imaging system was the DeltaVision OMX SR system (GE Healthcare Life Sciences) with a 60X 1.42 NA PlanApo N oil immersion objective. AcquireSR software was used for acquisition, and SoftWoRx for image reconstruction and alignment. <strong>Dendritic spine morphology and density analysis</strong> Neuron Studio was used for the analysis of dendritic spine morphology and density. Neuron Studio is a software package designed for three-dimensional detection of dendritic spines from fluorescence microscopy images. It also allows the classification of spines into different types based on their morphology. Images were re-numbered and ordered randomly before analysis, so that the analysis was done blinded to the experimental condition. The Fiji software was used to convert Zeiss files with the z-stacks of 20–30 optical sections to the TIFF files. The images had a voxel size of 0.066 μm x 0.066 μm x 0.2 μm, and the EGFP or the mCherry channels were used for analyzing the dendritic spine morphology and density. After modeling the dendrite surface, protrusions with a minimum volume of 5 voxels (0.020 μm<sup>3</sup>), a length between 0.1 μm and 5 μm, and a maximal width of 3 μm were retained as spines. Following the default settings of the program and the empirical classification rule defined by spines with a minimum head diameter of 0.35 μm and a minimum head vs. neck ratio of 1.1, were classified as mushroom spines. Non-mushroom spines with a minimum volume of 10 voxels (0.040 μm<sup>3</sup>) were classified as stubby spines. All other spines were considered thin. The spines detected and modeled by the software were carefully verified and corrected manually. The wrongly labeled structures were removed or corrected, and the spines that were not detected by the software were added and classified. Measurements obtained by Neuron Studio were transferred to a spreadsheet application (MS Excel) for further analysis. <strong>Co-localization Analysis</strong> The co-localization analysis between GFP and mCherry-tagged constructs was done on the maximum intensity projections obtained from 3D Z-stacks. After making a maximum intensity projection, the GIMP software was used to outline the region of interest of the image for co-localization analysis. GIMP is an open-source drawing and annotation software (https://www.gimp.org/). The co-localization analysis was carried out with an EzColocalization Plugin in the Fiji software by following the procedure created, used, and reported in previous research. Pearson’s correlation coefficient (PCC) value was determined for each image to measure the co-occurrence of two-channel signals. The range of PCC values is from 1 to -1, indicating a strong co-localization and a strong anti-co-localization, respectively. PCC=0 reflects no correlation between the two signals. <strong>Analyzing the membrane vs diffuse protein ratio for GFP-Tubby</strong> The quantification of fluorescence intensity was done using single confocal focal planes in the middle (in the z direction) of the measured dendrites. We first draw lines along the sides presenting the plasma membrane, and in the middle of a dendrite, presenting diffuse protein using Fiji software. Measurements were averaged to get one average value for the membrane and one for diffuse protein, and the ratio was calculated using these average values. <strong>Statistical analyses</strong> Statistics were made in GraphPad Prism (GraphPad Software Inc.). We used either the two-sample t-test or two-way ANOVA for parametric data and the Kruskal-Wallis test or the Mann-Whitney U test for non-parametric data. The tests used are specified for each analysis in the Figure legends. <strong>Figures</strong> Microscope images were processed in Fiji, Imaris (Oxford Instruments), and Photoshop (Adobe). Graphs were done in Prism (GraphPad Software Inc.). The final layout of figures was done in Inkscape.
description: # Data from: Expression, subcellular localization, and mechanistic
 analysis of intellectual disability syndrome protein ABBA Dataset DOI:
 [10.5061/dryad.ttdz08m8g](10.5061/dryad.ttdz08m8g) ## Description of the
 data and file structure ### Files and variables ``` Title of Dataset ```
 Expression, subcellular localization, and mechanistic analysis of
 Intellectual disability syndrome protein ABBA Dataset
 DOI: [10.5061/dryad.ttdz08m8g](applewebdata://53C9C7CE-7FDF-4E59-8945-B1955748EB01/10.5061/dryad.ttdz08m8g) ``` Brief summary of dataset contents, contextualized in experimental procedures and results: ``` Data used for article Jabeen A, Khanal P, Toissalo E, Lahti L, Minkeviciene R, Kramm A, Rivera C, Hotulainen P. Expression, Subcellular Localization, and Mechanistic Analysis of Intellectual Disability Syndrome Protein ABBA. Mol Neurobiol. 2025 Dec 8;63(1):271. doi: 10.1007/s12035-025-05475-3. PMID: 41359239. Link to article: [https://link.springer.com/article/10.1007/s12035-025-05475-3](https://link.springer.com/article/10.1007/s12035-025-05475-3) Data is organized by article figures so that all data used for main results presented in Figures 1-7 are put to same .zip folder named Figure_1.zip, Figure_2.zip etc. Opened .zip folder contains data for A,B,C, etc. panels of figures and a subfolder name Figure 1AB indicates for which figure panel A, B, C, etc. the folder has data. Additionally, there is a zipped folder for Supplementary figure 3. It is easiest to understand the structure of the data with article. Excel files are saved also as .csv files. If Excel files have more than one sheet, there are more than one .csv-file, one for each Excel sheet. Excels / .csv files have analyses done in article, they are not necessary to analyze figures, only showing numbers behind averages shown in article. ``` Description of the data and file structure ``` ``` Map for main and subfolders ``` Figure_1.zip Unzip to get Folder Figure 1 &gt; Folder Figure 1A,B Subfolder Figure 1AB includes Figure 1A (Western blot images of Figure 1A), Figure 1B (Western blot images for Figure 1B), “Figure 1AB Western blots.xlsx” Excel file (Analysis of Figure 1 Western blots) and .csv files (1A and 1B) of Excel sheets. The protein levels were quantified using Image Lab software (Bio-Rad) and normalized against total protein per lane. --- Figure_2.zip Unzip to get Folder Figure 2 &gt; Folder Figure 2C Subfolder Figure 2C includes 6 folders with 6 different experiments (called First – Sixth batch) containg Western blot images for each experiment, “All organotypic western results FINAL, dryad.xlsx” Excel file (Analysis of Figure 2C Western blots), and .csv file of Excel file. The protein levels were quantified using Image Lab software (Bio-Rad) and normalized against total protein per lane. --- Figure_3.zip Unzip to get Folder Figure 3 &gt; Folders Figure 3B, Figure 3C, Figure 3D Figure 3B: SIM super-resolution images. Only 1 is shown in the article. File format: 110424.PHL488-ABBA-568-1_A_SIR_ALX.dv Super-resolution SIM image. Stack. Opens with FIJI. Imaging date: 110424 11th April 2024 Phalloidin-Alexa488, ABBA antibody – Alexa 568-secondary antibody The used imaging system was the DeltaVision OMX SR system (GE Healthcare Life Sciences) with a 60x 1.42 NA PlanApo N oil immersion objective. AcquireSR software was used for acquisition, and SoftWoRx for image reconstruction and alignment. ABBA-568+phl488 sim imaging.pptx shows maximum projections in power point. Figure 3C Maximum projection of a time lapse video shown in Figure 3C Cultured neurons transfected with GFP-ABBA and RFP-Lifeact. File format: MAX_v051023ABBA-GFPlifeactRFPC04Z25cycles30.44slicesoriginal.tif Opens with FIJI. Imaging date: 05th October 2023 The frames are imaged every 60 seconds. The video has 30 frames. Figure 3D Maximum projection of a time lapse video shown in Figure 3D Cultured neurons transfected with GFP-ABBA and RFP-Lifeact. File format: MAX_281123.ABBA-GFP+lifeactin-RFP.C02.Z2.5.interval0.25+cycle20.33sliceTYPICAL.tif Opens with FIJI. Imaging date: 28th November 2023 The frames are imaged every 60 seconds. The video has 20 frames. --- Figure_4.zip Unzip to get Folder Figure 4 &gt; Folders Figure 4A, Figure 4B, Figure 4C, 4D and 4E F \---- Figure 4A: Image Folder: mCherry-ABBAwithGFP File format: 110523.mCherry-ABBA+GFP.C01.Z2.czi Primary hippocampal neurons expressing mCherry-ABBA and GFP Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Image Folder: mCherry-ABBAwithGFPTubby File format: 17.7.23.GFP-Tubby+mCherry-ABBA.c01.z2.czi Primary hippocampal neurons expressing mCherry-ABBA and GFP-Tubby Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. In addition Power point summaries of maximum projections ABBA-mCherry+GFPfor4A.pptx mCherry-ABBA+GFP-TUBBY.pptx Figure 4B: Image Folder: GFPABBAmCherry File format: 121022-GFP-ABBA+mCherry-C01.Z2.DIV15.czi Primary hippocampal neurons expressing GFP-ABBA and mCherry Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Image Folder: GFPABBAwithmCherryAktPH File format: 121022GFP-ABBA+mCherry-Akt-PH c01Z2.czi Primary hippocampal neurons expressing GFP-ABBA and mCherry-Akt-PH In addition Power point summaries of maximum projections GFPABBAmCherrycontrolsforAkt.pptx GFP-ABBA+mCherry-Akt-PH.pptx Figure 4C: Excel file for colocalization results: ABBA colocalisation analysis Tubby and Akt.xlsx and .csv file of Excel file. Figure 4E F Image Folder: DMSOtreatedcontrols File format: 120623.ABBA-GFP+mcherry+DMSO.10 min.C02.Z2.czi Primary hippocampal neurons expressing GFP-ABBA and mCherry, treated with DMSO (=control) Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Image Folder: PI3Ktreatedcells File format: 120623.ABBA-GFP+mCherry+PI3KI 10 mim.C05.Z2.czi Primary hippocampal neurons expressing GFP-ABBA and mCherry, treated with PI3K inhibitor 100 µM LY294002 for 10 minutes. Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. In addition Power point summary of maximum projections ABBA-GFP PI3K images to analysis.pptx. --- Figure_5.zip Unzip to get Folder Figure 5 &gt; Folders Figure 5A, 5B, 5C, 5D, 5E, 5FG Figure 5A: Image Folder GFPABBAmCherry File format: 100821_15DIV_24h_ABBA GFP_mCherry_C01_Z2.czi Primary hippocampal neurons expressing GFP-ABBA and mCherry Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Image Folder GFPABBA_mCherryNWASP File format: 100821_15DIV_24h_GFP-ABBA_Nwasp mCherry_C01_Z2.czi Primary hippocampal neurons expressing GFP-ABBA and NWASP-mCherry Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Image Folder GFPABBA_mCherryactin File format: 121022_15DIV_24h_GFP-ABBA_Actin mCherry_C06.Z2.czi Primary hippocampal neurons expressing GFP-ABBA and mCherry-actin Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. In addition Power point files present summary of maximum projections ABBA GFP + mCherry_15DIV.pptx GFP-ABBA + NWASP mCherry.pptx ABBA GFP+Actin mCherry_15DIV..pptx Figure 5B In contrast to other folders, this folder has each Experiment in separate folder. Therefore 13 folders. Folders have images for mCherry-ABBA and GFP / GFP-Rac1 N17 / GFP-Rac1 V12 Some folders have subfolders for cells marked C01, C02, C03…etc. File format: 011024.ABBA-mCherry+GFP.Z0.7.C01.czi Z0.7 means Software Zoom 0.7 which gives overview of a cell 011024.ABBA-mCherry+GFP.Z2.C01.czi Z2 means Software Zoom 2 which gives higher magnification image of a dendrite used for analyses All maximum projections are summed in ABBARAC1images.pptx Power point file. Figure 5C has Excel file for colocalization analysis results ColocalizationABBA_NWASP_actin.xlsx and same data saved as .csv file. Figure 5D has Excel file for colocalization analysis results ABBA_Rac1_colocalization analysis.xlsx and same data saved as .csv file. Figure 5FG has Video folders Before and After 5 μM Latrunculin B treatment GFP-ABBA and RFP-LifeAct transfected hippocampal DIV15 neuron. File format: MAX_281123.ABBA-GFP+lifeactin-RFP.C01.Z2.5.interval0.25+cycle20.68slice.afterlatb.tif Maximum projection of confocal video. Opens with Fiji. Confocal images were obtained using LSM880 inverted confocal microscope. The live cell imaging was performed in a chamber where the temperature was maintained at 37°C and CO2 levels were 5%.  The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The frames are imaged every 60 seconds. Videos are summed up to Power Point file: ABBA-GFP+Lifeact-RFP-videos.pptx Figure_6.zip Unzip to get Folder Figure 6  Folders Figure 6AB, 6CD Figure 6AB: Image Folder mCherry+GFP-lifeact File format: 031023.mCherry+Lifeact-GFP.C04.Z2.czi Primary hippocampal neurons expressing GFP-LifeAct and mCherry Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Image Folder mCherry-ABBA+GFP-lifeact File format: 2_151123.ABBA-mCherry+lifeact-RFP.C01.Z2.czi Primary hippocampal neurons expressing GFP-LifeAct and mCherry-ABBA Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. In addition Power point files present summary of maximum projections mCherry-ABBA+GFP-lifeact.pptx mCherry+GFP-lifeact.pptx Figure 6CD contains Image Folders for DMSO-treated control cells and LatB- treated cells (LatA is uncorrect labeling in folders) at different time points 5, 10 and 15 minutes. File Format: 100821_15DIV_ABBA GFP_5uM DMSO_5min_phalloidin 633_C01_Z0.6.czi Primary hippocampal neurons expressing GFP-ABBA and stained with Phalloidin-Alexa633, treated with LatB 5 µM for 5-15 minutes. Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 0.6 with software. Opens with Fiji. Power Point file GFP-ABBA+Phalloidin_LatB treatment.pptx summarizes maximum projection images. Excel File GFP-ABBA+phl Dmso, latb treatments.xlsx summarizes analysis. Same data saved as .csv file. --- Figure_7.zip Unzip to get Folder Figure 7 &gt; Folders Figure 7A, 7C, 7E, 7F Figure 7A Image Folders mCherry_GFP and mCherryABBA_GFP contain images used for spine density analysis. Primary hippocampal neurons expressing GFP-mCherry or GFP-mCherry-ABBA, repectively.  Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Power Point files summarize all maximum projections: GFP+mCherry.pptx ABBA mCherry+GFP_15DIV.pptx Figure 7C Image Folders GFP_GAD6567 and GFPABBA_GAD6567 contain images used for spine density analysis of inhibitory neurons. GAD65/67-positive neurons expressing GFP or GFP-ABBA, repectively. Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Power Point file summarize all maximum projections: ABBA with GAD.pptx Figure 7E Image Folders GFPABBA_W and GFPABBA_WA contain images used for spine density analysis. Primary hippocampal neurons expressing GFP-ABBA and W (control) or WA (sequesters Arp2/3), repectively.  Confocal images were obtained using a Zeiss LSM880 inverted confocal microscope. The fixed samples were imaged at room temperature. The 63X 1.4 NA oil immersion objective was used for Z-stack imaging of each neuron. The step size was set to 0.2 μm. Zoom 2 with software. Opens with Fiji. Power Point files summarize all maximum projections: ABBA-GFP+W-myc647.pptx ABBA-GFP+WA-myc647.pptx Figure 7F is empty here but images are same as in Figure 5B Folder. In addition Excel file SpineDensitySummaryall.xlsx summarizes all Spine density results and .csv files for 7AB, 7CD, 7EF and 7GH data. --- Supplementaryfigure_3.zip Unzip to get Folder SupplementaryFigure_3 Subfolders 070725_PI3KI and 260625_PI3KI have images imaged at two different days. However, cells are from same experiment. Folders have subfolders for GFP_Tubby_DMSO and GFP_Tubby_PI3KI having Zeiss Airyscan confocal stacks for different cells. Folders have an overview taken at zoom 0.7 (Objective 63x) called C01.Z0.7.czi and zoom-in figure, which is taken at Zoom 2 and is called C01.Z2.czi. Running number 1, 2, 3 in C01 indicates cell number. Additionally zipped folder contains Power point file which has maximum projections of all analyzed cells to help to get overview of the experiment. This presentation is called GFP_tubby.pptx.  Analysis of the membrane vs diffuse protein ratio for GFP-Tubby can be found from Tubby analysis.xlsx Excel file, also saved as .csv file. The quantification of fluorescence intensity was done using single confocal focal planes in the middle (in the z direction) of the measured dendrites. We first draw lines along the sides presenting the plasma membrane, and in the middle of a dendrite, presenting diffuse protein using Fiji software. Measurements were averaged to get one average value for the membrane and one for diffuse protein, and the ratio was calculated using these average values. ``` Code/software ``` Image files open with Fiji (just drag and drop), Excel files with Excel and Powerpoint files with Powerpoint.