Loss of α-actinin-2 prevents synapse formation.

<p><b>A)</b> Actively firing pre-synaptic boutons do not synapse with spines on neurons lacking α-actinin-2. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, treated with FM4-64 for 5 min on DIV 19 and observed live. Arrows mark FM4-64 juxtaposition to spines or the dendrite. The fraction of spines juxtaposed to FM4-64 is reduced in neurons lacking α-actinin-2 (see results). For each condition, 14 neurons from 2 separate cultures were analyzed. <b>B)</b> Loss of α-actinin-2 prevents synapse formation with excitatory axon boutons. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 21, and immunostained for VGLUT1. Arrows mark VGLUT1 juxtaposition to spines or the dendrite. The fraction of spines juxtaposed to VGLUT1 is reduced in neurons lacking α-actinin-2 (see results). For each condition 23–26 neurons from 2 separate cultures were analyzed.</p

Knockdown of α-actinin-2 prevents spine maturation in response to NMDA receptor activation.

<p><b>A)</b> When α-actinin-2 is knocked down, spines do not shorten or assume a “mushroom” morphology in response to glycine. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2. Neurons were chronically treated with AP-5, an NMDA receptor antagonist, to inhibit spine maturation. At DIV 19–21, neurons were acutely stimulated by the addition of 200 µM glycine and AP-5 withdrawal, while control neurons were continuously treated with AP-5. <b>B</b>–<b>C)</b> Quantification of spine morphology in response to α-actinin-2 inhibition and glycine stimulation. Fraction of spines with a large head, spine tip width >0.4 µm, increases in response to glycine stimulation but is prevented by α-actinin-2 knockdown, B. In contrast to stimulated controls, inhibition of α-actinin-2 does not increase the fraction of mushroom-shaped spines and decrease filopodia-like spines, C. For each condition, 556–1721 spines of 16–24 neurons from 2 separate cultures were analyzed. <b>D</b>–<b>E)</b> α-Actinin-2 knockdown prevents enrichment of actin filaments in spines. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 21, and stained for rhodamine-phalloidin. Arrowheads mark actin enrichment in spines of control neurons and arrows point to the lack of actin in spines of neurons with α-actinin-2 knocked down, D. The fluorescent intensity of rhodamine-phalloidin is reduced in spines with α-actinin-2 knocked down, E. For each condition, 87–97 spines from 5 neurons were analyzed. Error bars represent SEM. p-values were derived using the paired t-test. Scale = 5 µm.</p

α-actinin-2 localizes to post-synaptic sites in dendritic spines on hippocampal neurons.

<p><b>A)</b> An anti-α-actinin antibody (ab68167) recognizes α-actinin-2 and not α-actinin-1 or α-actinin-4. CHO-K1 cells were transfected with human α-actinin-1-GFP, α-actinin-2-GFP, or α-actinin-4-GFP. Cells were lysed and immunoblotted for α-actinin-2 and GFP. <b>B</b>) α-Actinin-2 is enriched in hippocampal neurons but not in glia cells or COS-7 cells, which lacks α-actinin-2. Cells were lysed and immunoblotted for α-actinin-2. Actin is the loading control. <b>C)</b> α-Actinin-2 localizes to dendritic spines. Hippocampal neurons were transfected at DIV 17 with GFP (green), and fixed, and immunostained for endogenous α-actinin-2 (magenta) at DIV 21. <b>D)</b> α-Actinin-2 co-localizes with post-synaptic markers, but not with a pre-synaptic marker. Hippocampal neurons were fixed at DIV 16 or 21 and immunostained for endogenous α-actinin-2 (green) and either endogenous synaptophysin, PSD-95, or the NR1 subunit of the NMDA receptor (magenta). <b>E</b>–<b>G)</b> The siRNA is specific for α-actinin-2. Hippocampal neurons were co-transfected at DIV 17 with GFP and either a control empty vector (pSUPER), or a vector containing siRNA against α-actinin-2 (pSUPER-α-actinin-2), or the α-actinin-2 siRNA-containing vector plus a α-actinin-2 vector conferring resistance to RNAi (pSUPER-α-actinin-2+ α-actinin-2-SS). The cells were fixed at DIV 21 and immunostained for endogenous α-actinin-2. Arrows point to the neurons co-expressing GFP and its immunostaining for α-actinin-2. For each condition (55 control cells and 46 α-actinin-2 knockdown cells), the integrated density of the cell body and dendrites were measured from the transfected neuron and adjacent untransfected neuron of the same image and the percent change was plotted, F. Error bars represent SEM. p-values were derived using the paired t-test. <b>G)</b> CHO-K1 cells were co-transfected with GFP, pSUPER or pSUPER-α-actinin-2, plus either α-actinin-2-Flag or α-actinin-2-SS-Flag. Transfection efficiency was close to 100% as >95% of the cells in each condition exhibited GFP fluorescence (data not shown). Cells were lysed 72 hours after transfection and immunoblotted for α-actinin-2 and α-actinin-4. Tubulin is the loading control.</p

Loss of α-actinin-2 prevents assembly of the post-synaptic density.

<p><b>A–C)</b> α-Actinin-2 knockdown inhibits PSD assembly in spines. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 21, and immunostained for PSD-95. Arrows mark localization of PSD-95 in spines or in the dendrite shaft, A. The fraction of spines with localized PSD-95 is reduced in neurons with α-actinin-2 knocked down, B. PSD-95 localizes to the dendrite shaft with increased frequency in neurons lacking α-actinin-2, C. For each condition, 37-45 neurons from 5 separate cultures were analyzed. <b>D</b>–<b>F)</b> Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER, pSUPER-α-actinin-2, or pSUPER-α-actinin-2 plus α-actinin-2-SS, D. The fraction of spines with PSD-95 is rescued in neurons expressing exogenous α-actinin-2-SS, E. The area of PSD-95 is significantly reduced in spines lacking α-actinin-2 and rescued in neurons expressing exogenous α-actinin-2-SS, F. For each condition, PSD-95 area was measured from 543–991 spines of 23–27 neurons from 3 separate cultures. <b>G</b>–<b>I)</b> α-Actinin-2 knockdown prevents the recruitment of the NMDA receptor to the spine. Hippocampal neurons were co-transfected at DIV 6 with SEP-NR1 and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 22, and immunostained for GFP and rhodamine-phalloidin. Arrows mark SEP-NR1 localization in either spines or in the dendrite, G. The fraction of spines co-localized with SEP-NR1 is reduced in neurons with α-actinin-2 knocked down, H. For each condition, 12–19 neurons from 3 separate cultures were analyzed. <b>I)</b> Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER-α-actinin-2 or pSUPER-α-actinin-2 plus α-actinin-2-SS, fixed on DIV 21, and immunostained for NMDAR1. Error bars represent SEM. p-values were derived using the paired t-test.</p

α-Actinin-2 contributes to development of dendritic arbors and spine morphology in early cultures.

<p><b>A–C)</b> Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2 and fixed on DIV 22. Note the reduced size and number of dendrite arbors on neurons lacking α-actinin-2. While there is no difference in the number of primary dendrites, the number of secondary and tertiary dendrites on neurons lacking α-actinin-2 is reduced, B. The length of primary and secondary dendrites is smaller in neurons with α-actinin-2 knocked down, C. The branching complexity is reduced in neurons lacking α-actinin-2, D. Dendrites from 17 control neurons and 21 α-actinin-2 knockdown neurons from 2 different cultures were analyzed. <b>E)</b> Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2. Neurons were fixed on DIV 21 and scored for <b>(E</b>–<b>I)</b> changes in spine density, length, head width, and morphology. Two examples of control neurons and α-actinin-2 knockdown neurons are shown. Arrows point to irregularly shaped protrusions containing numerous filopodia, which is observed in several α-actinin-2 knockdowns. Inhibition of α-actinin-2 increases the number of spines per µm length of the dendrite (spine density), F. Spine density was quantified from 73 control neurons and 65 α-actinin-2 knockdown neurons from more than 3 cultures. The fraction of spine head widths >0.4 µm is significantly reduced in neurons with α-actinin-2 knocked down, G. Spine length is shifted to the right (longer) in neurons lacking α-actinin-2, H. α-Actinin-2 knockdown creates an increase in the fraction of filopodia-like spines (long protrusions without a spine head) and a concomitant decrease in the fraction of mushroom-shaped spines, I. For quantification of spine width, length, and morphology, 1875–2245 spines from 29 control neurons and 35 α-actinin-2 knockdown neurons from more than 3 cultures were analyzed. Error bars represent standard error of the mean (SEM). p-values were derived using the paired t-test (B, C, D, H, I) and Mann-Whitney test (F, G).</p

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

Knockdown of α-actinin-2 increases spine number and inhibits spine maturation.

<p><b>A)</b> Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER, pSUPER-α-actinin-2, or pSUPER-α-actinin-2 plus α-actinin-2-SS. Neurons were fixed on DIV 21 and scored for <b>(B</b>–<b>D)</b> changes in spine density, head width, and morphology. Inhibition of α-actinin-2 at DIV 17 increases spine density, B. For each condition, spine density was analyzed on 35–42 dendrites from 3 separate cultures. The fraction of spine head widths >0.4 µm is significantly reduced in neurons with α-actinin-2 knocked down at DIV 17, C. α-Actinin-2 knockdown creates an increase in the fraction of filopodia-like spines and a decrease in the fraction of mushroom-shaped spines, thin spines (long protrusions with small head at tip), and stubby spines, D. For quantification of spine head width and morphology, 1444–2081 spines from 51 control neurons, 40 α-actinin-2 knockdown neurons, and 35 rescue neurons of 3 separate cultures were analyzed. <b>E)</b> Hippocampal neurons were transfected at DIV 6 with either GFP alone or GFP + α-actinin-2-SS. Neurons were fixed on DIV 22 and scored for <b>(F</b>–<b>H)</b> changes in spine length, head width, and morphology. Overexpression of α-actinin-2-SS increases spine length (F) and reduces spine head width (G). α-Actinin-2-SS overexpression creates an increase in the fraction of filopodia-like spines and a decrease in the fraction of stubby spines, H. For quantification of spine length, head width, and morphology, 311–610 spines from 15 control neurons and 21 α-actinin-2-SS overexpression neurons of 3 separate cultures were analyzed. Error bars represent SEM. p-values were derived using the paired t-test. Scale = 5 µm.</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

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