Regulators of spine maturation are distinct from regulators of spine precursor formation.

<p><b>A)</b> Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. <b>B)</b> Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 <i>Ophn-1</i> shRNA #1, 5 <i>Ophn-1</i> shRNA #2, 17 β<i>-pix</i> shRNA #1, 7 β<i>-pix</i> shRNA #2, 15 <i>Frabin</i> shRNA #1, 8 <i>Frabin</i> shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β<i>-pix</i> shRNA #1 which was determined by Mann-Whitney Rank Sum Test). <b>C)</b> <i>Arhgap23</i> shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 <i>Arhgap23</i> shRNA #1, and 12 <i>Arhgap23</i> shRNA #2 neurons; p = 0.02 for Control vs <i>Arhgap23</i> shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs <i>Arhgap23</i> shRNA #2 (Mann-Whitney Rank Sum Test). <b>D)</b> Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 <i>Ophn-1</i> shRNA #1, 130 <i>Ophn-1</i> shRNA #2, 729 β<i>-pix</i> shRNA #1, 449 β<i>-pix</i> shRNA #2, 556 <i>Frabin</i> shRNA #1, 688 <i>Frabin</i> shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). <b>E)</b> <i>Arhgap23</i> and <i>Vav2</i> shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 <i>Arhgap23</i> shRNA #1, 1182 <i>Arhgap23</i> shRNA #2, 962 <i>Vav2</i> shRNA #1, and 551 <i>Vav2</i> shRNA #2 spines; p < 0.001 for Control vs <i>Arhgap23</i> shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs <i>Arhgap23</i> shRNA #2 (Mann-Whitney Rank Sum Test), p = 0.006 for Control vs <i>Vav2</i> shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs <i>Vav2</i> shRNA #2 (Mann-Whitney Rank Sum Test).</p

Synaptic Regulators of RhoGTPase Signaling.

<p>Synaptic Regulators of RhoGTPase Signaling.</p

ARHGAP23 is a novel Rac GAP that regulates adhesion maturation.

<p><b>A)</b> Representative images of ARHGAP23-GFP or GFP control CHO.K1 cells plated on fibronectin and stained for the adhesion marker, paxillin, and actin filaments (rhodamine phalloidin). <b>B)</b> Quantification of ARHGAP23 puncta size (n = 44 cells) in ARHGAP23 GFP-expressing CHO.K1 cells and paxillin puncta size in either ARHGAP23 GFP-expressing CHO.K1 cells (n = 35 cells) or control CHO.K1 cells (n = 12 cells); p = 0.015 for GAP23 vs paxillin puncta size in GAP23 GFP-expressing CHO.K1 cells (Mann-Whitney Rank Sum Test), p = 0.012 for paxillin puncta size in GAP23 GFP-expressing vs control CHO.K1 cells (Mann-Whitney Rank Sum Test). <b>C)</b> Representative images of CHO.K1 cells transfected with GFP and either control empty pSUPER vector or ARHGAP23 shRNA and plated on fibronectin. Cells were stained for the adhesion marker, paxillin, and actin filaments (rhodamine phalloidin). <b>D)</b> Quantification of adhesion size in control (n = 24 cells) or <i>Arhgap23</i> shRNA (n = 25 cells) CHO.K1 cells; p = 0.006 (Mann-Whitney Rank Sum Test). <b>E)</b> Ratiometric FRET images of control or <i>Arhgap23</i> shRNA CHO.K1 cells co-transfected with the WT Raichu Rac FRET probe or constitutively active control, Raichu Rac V12, and plated on fibronectin. The top panel shows the intensity of the CFP donor of the FRET probe in each cell. <b>F)</b> Quantification of FRET intensity in control or <i>Arhgap23</i> shRNA cells expressing Raichu Rac probes. n = 31 control WT Raichu Rac, 24 control Raichu Rac V12, 11 <i>Arhgap23</i> shRNA #1 WT Raichu Rac cells, 7 <i>Arhgap23</i> shRNA #1 Raichu Rac V12 cells, 18 <i>Arhgap23</i> shRNA #2 WT Raichu Rac cells, 13 <i>Arhgap23</i> shRNA #2 Raichu Rac V12 CHO.K1 cells; p < 0.001 for WT Raichu Rac vs Raichu Rac V12 in control CHO.K1 cells (t-test), but WT Raichu Rac is not statistically different from Raichu Rac V12 when CHO.K1 cells are transfected with either <i>Arhgap23</i> shRNA sequence (t-test).</p

Rac drives spine precursor formation, while myosin-II and Cdc42 activity regulate spine length.

<p><b>A)</b> Rac1 photoactivation increases spine precursor formation. DIV14-21 primary rat hippocampal neurons expressing either photoactivatable Rac1 or as a positive control, constitutively activated Rac1 (‘Lit’ PA-Rac), were kept in dark (black bars) or exposed to room lighting for 10min (white bars). The resulting spine density is expressed as percent control unactivated PA-Rac-expressing neurons. n = 24 PA-Rac neurons kept in dark, 23 PA-Rac light-exposed neurons, 8 ‘lit’ PA-Rac neurons kept in dark, and 9 ‘lit’ PA-Rac light-exposed neurons; p = 0.047 PA-Rac dark vs light-acivated (t-test). <b>B)</b> Acute Rac1 photoactivation does not affect spine length, unlike constitutive Rac1 activity (‘Lit’ control). <b>C)</b> Representative images of neurons expressing either photoactivable Rac1 (PA-Rac1, top panel) or the constitutively active ‘lit’ Rac1 control (bottom panel) that were either kept in the dark (left panel) or exposed to room lighting for 10 min (light-activated, right panel). <b>D)</b> DIV-9/10 primary rat hippocampal neurons transfected with WT Raichu Cdc42 were treated with 50μM Blebbistatin for 1 hour or left untreated. FRET was calculated as the ratio of FRET signal to CFP donor signal. Blebbistatin treatment increases Cdc42 activity by ~7%. n = 50 spine precursors each for untreated and Blebbistatin-treated, p = 0.016 (t-test).</p

Regulators of spine precursor formation and elongation.

<p><b>A)</b> Representative Images of GFP-expressing DIV-9 neurons transfected with the indicated shRNA targeting sequence for 72 hours. <b>B)</b> β<i>-pix</i> shRNA targeting sequences significantly decrease spine precursor density in DIV-9/10 neurons. Spine density is expressed as the percentage of the average control spine density. n = 24 control neurons, 15 β-PIX shRNA #1 neurons, and 7 β-PIX shRNA #2 neurons; p = 0.001 for Control vs β<i>-pix</i> shRNA #1 (t-test) and p = 0.007 for Control vs β<i>-pix</i> shRNA #2 (t-test). <b>C)</b> Cumulative distribution plot of spine length in DIV-9/10 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. shRNAs against either the RhoA-GAP, <i>Oligophrenin-1</i>, or Cdc42-GEF, <i>Frabin</i>, significantly decrease spine length, whereas shRNAs against β<i>-pix</i> significantly increase spine length. n = 1114 control, 680 <i>Oligophrenin-1</i> shRNA #1, 115 <i>Oligophrenin-1</i> shRNA #2, 613 <i>Frabin</i> shRNA #1, 393 <i>Frabin</i> shRNA #2, 465 β<i>-pix</i> shRNA #1, and 166 β<i>-pix</i> shRNA #2 spines; p = 0.005 for Control vs β<i>-pix</i> shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs β<i>-pix</i> shRNA #2 (Mann-Whitney Rank Sum Test), p = 0.001 for Control vs <i>Frabin</i> shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.01 for Control vs <i>Frabin</i> shRNA #2 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs <i>Oligophrenin-1</i> shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs <i>Oligophrenin-1</i> shRNA #2 (Mann-Whitney Rank Sum Test).</p

Expression of RhoGTPase regulators during synapse development.

<p><b>A)</b> Primary rat hippocampal neurons expressing GFP were stained for the dendritic marker, MAP-2 (MAP-2). At ~1 week in culture (days in vitro, DIV-10), filopodia-like spine precursors extend from the dendritic shaft. However, after 2 weeks, spines begin to mature into a polarized mushroom-shape, characterized by a bulbous spine head atop a thin spine neck. <b>B)</b> Real-Time RT-PCR of RhoGTPase regulators from RNA harvested from primary rat hippocampal neurons grown for either 1 or 2 weeks in culture (DIV-7, grey bars; DIV-14, black bars). RNA expression levels are expressed as a percentage of actin RNA. (n ≥ 5 neuron cultures for each time point; p<0.001 for <i>Arhgdia</i> expression at DIV-7 vs DIV-14 (Mann-Whitney Rank Sum Test) and p = 0.033 for <i>Arhgdig</i> expression at DIV-7 vs DIV-14 (t-test); RNA expression levels of all other regulators was not statistically significant between DIV-7 and DIV-14.</p

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development - Fig 6

<p><b>A & B) RhoGDIα and</b> γ <b>maintain spine maturation.</b> Quantification of Spine Length in GFP-expressing DIV16-27 neurons transfected with the indicated shRNA targeting sequence<b>s</b> or control neurons. Spine Length is normalized to the average control spine length for each neuronal culture. n = 1232 control spines, 1757 <i>Arhgdi-</i>α shRNA spines, 1305 <i>Arhgdi-</i>β shRNA spines, and 1526 <i>Arhgdi-</i>γ shRNA spines; p < 0.001 for control neurons vs either <i>Arhgdi-</i>α shRNA or <i>Arhgdi-</i>γ shRNA neurons, but is not statistically different from <i>Arhgdi-</i>β shRNA neurons (Mann-Whitney Rank Sum Test).</p