RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

Cross-correlated fluctuation analysis reveals phosphorylation-regulated paxillin-FAK complexes in nascent adhesions.

We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions

Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.

BackgroundA complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.ResultsWe introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.ConclusionsThese observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes

Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.

BackgroundA complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.ResultsWe introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.ConclusionsThese observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes

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

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