© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs224980. doi:10.1242/jcs.224980

RESEARCH ARTICLE Src-mediated phosphorylation of βPix-b regulates dendritic spine morphogenesis Mi-seon Shin1, Sang-ho Song1,*, Jung Eun Shin1,‡, Seung-Hye Lee1,§, Sung-Oh Huh2 and Dongeun Park1,¶

ABSTRACT differentiate into mature spines with various morphologies such as PAK-interacting guanine nucleotide exchange factor (βPix; also stubby, thin, mushroom-shaped and cup-shaped protrusions (Harris, known as Arhgef7) has been implicated in many actin-based 1999; Hering and Sheng, 2001; Peters and Kaiserman-Abramof, cellular processes, including spine morphogenesis in neurons. 1970). The morphology of dendritic spines is largely sustained by However, the molecular mechanisms by which βPix controls spine activity-dependent rearrangement of actin cytoskeleton (Calabrese morphology remain elusive. Previously, we have reported the et al., 2006; Cingolani and Goda, 2008; Fortin et al., 2012; Lai and expression of several alternative spliced βPix isoforms in the brain. Ip, 2013; Matsuzaki et al., 2004; Matus, 2000; Saneyoshi and Here, we report a novel finding that the b isoform of βPix (βPix-b) Hayashi, 2012). Hence, regulatory proteins of the dynamic actin mediates the regulation of spine and synapse formation. We found cytoskeleton play essential roles in spine morphogenesis (Calabrese that βPix-b, which is mainly expressed in neurons, enhances spine et al., 2006; Fortin et al., 2012; Lin and Webb, 2009; Tolias et al., and synapse formation through preferential localization at spines. In 2011). The key regulators for the organization of actin cytoskeleton neurons, glutamate treatment efficiently stimulates Rac1 GEF activity in spines are Rho family GTPases including RhoA, Rac1 and of βPix-b. The glutamate stimulation also promotes Src-mediated Cdc42 (Newey et al., 2005). Among Rho GTPases, Rac1 is well phosphorylation of βPix-b in both an AMPA receptor- and NMDA established for its role in spine morphogenesis in both animals receptor-dependent manner. Tyrosine 598 (Y598) of βPix-b is and cultured neurons (Govek et al., 2005). The activity of identified as the major Src-mediated phosphorylation site. Finally, Rho GTPases is tightly modulated through GDP/GTP cycling Y598 phosphorylation of βPix-b enhances its Rac1 GEF activity that is between inactive GDP-bound and active GTP-bound forms. Rho critical for spine and synapse formation. In conclusion, we provide a GTPases are activated by guanine nucleotide exchange factors novel mechanism by which βPix-b regulates activity-dependent (GEFs), which catalyze the exchange of GDP for GTP on spinogenesis and synaptogenesis via Src-mediated phosphorylation. Rho GTPases (Schmidt and Hall, 2002), and inactivated by GTPase-activating proteins (GAPs), which facilitate hydrolysis KEY WORDS: Spine, Synapse, βPix-b, Rac1 GEF, Src family kinases, of GTP to GDP (Bernards and Settleman, 2004). For example, Phosphorylation several Rac1 GEFs including Tiam1, kalirin and GEFT (also known as Arhgef25) have been reported to be localized in dendritic INTRODUCTION spines and to promote their development (Bryan et al., 2004; Dendritic spines are small actin-rich protrusions on the shaft of Penzes et al., 2001; Tolias et al., 2005; Xie et al., 2007; Zhang and neuronal dendrites, which establish postsynaptic sites of most Macara, 2006). excitatory synapses for receiving synaptic input and transmitting βPix (also known as Arhgef7) is a Rac1 GEF involved in information induced by synaptic activity (Koch and Zador, 1993; synaptogenesis (Saneyoshi et al., 2008; Zhang et al., 2003, 2005). Korobova and Svitkina, 2010; Shepherd, 1996; Yuste and βPix is a multidomain protein consisting of an Src homology 3 Majewska, 2001). Because spines play a vital role in building (SH3) domain, a Dbl homology (DH)-Pleckstrin homology (PH) synaptic connectivity in neurons, spine development and Rac/Cdc42 GEF domain, a GIT1-binding domain (GBD) and a maintenance are critical to normal cognitive function and underlie proline-rich region (Rosenberger and Kutsche, 2006). βPix interacts neural processes such as learning and memory. Abnormal spine with Rac1 or PAK proteins, downstream effector kinases of Rac1, formation and morphology are associated with numerous through its SH3 domain and has GEF activity through its DH neurological disorders (Calabrese et al., 2006; Lai and Ip, 2013; domain (Bagrodia et al., 1998; ten Klooster et al., 2006). In neurons, Penzes et al., 2011; van Spronsen and Hoogenraad, 2010). Spines βPix has been shown to be localized to spines and involved in the are highly dynamic structures: immature filopodia-like protrusions promotion of spine formation in a CaMKI-dependent mechanism (Saneyoshi et al., 2008). Previously, we reported several spliced variants of βPix including βPix-a, -b and -d, showing different 1School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea. 2Department of Pharmacology, College of Medicine, Institute of Natural expression patterns (Kim et al., 2000; Kim and Park, 2001; Oh et al., Medicine, Hallym University, Chuncheon 24252, Republic of Korea. 1997). Of these, βPix-a is expressed in all tissues, whereas βPix-b *Present address: Lee Kong Chian School of Medicine, Nanyang Technological β University and Institute of Molecular and Cell Biology, Singapore 138673, and -d, the Pix isoforms containing the unique insert region, are Singapore. ‡Present address: KU Advanced Graduate Program for Life Science, mainly expressed in the brain, suggesting that βPix-b or -d may play Korea University, Seoul 02841, Republic of Korea. §Present address: Department of an important role in the regulation of neuron-specific processes. Neuroscience, Genentech, Inc., South San Francisco, CA 94080, USA. Interestingly, overexpressing the ubiquitous βPix-a isoform has ¶Author for correspondence ([email protected]) been reported to increase immature filopodia-like protrusions while inhibiting the formation of mature dendritic spines in cultured M.-s.S., 0000-0002-3377-6038; J.E.S., 0000-0002-8502-7979; S.-H.L., 0000- 0003-2328-9938; D.P., 0000-0003-2010-4598 hippocampal neurons (Zhang et al., 2003). These findings suggest the specific role of neuronal βPix isoforms in spine maturation, yet

Received 7 September 2018; Accepted 24 December 2018 their potential function remains largely unexplored. Journal of Cell Science

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Here, we investigated the roles of βPix-b in spine and synapse domain structures were identical to those of βPix-a, except for the formation and uncover the molecular mechanisms underlying the presence of the neuronal isoform-specific insert region (Fig. 1). To regulation of its activity. We demonstrate that βPix-b is highly examine the roles of βPix-b in spine development, 7 days in vitro expressed in neurons, and overexpression of βPix-b enhances spine (DIV) rat hippocampal neurons were transfected with GFP, GFP- development and synapse formation. In addition, glutamate βPix-a or GFP-βPix-b constructs. Their effects on spines and stimulation specifically increases the GEF activity of βPix-b synapses were then analyzed at 19 DIV. Synapses were identified by among βPix isoforms. We also found that only βPix-b displays immunostaining for synaptophysin (presynaptic marker) (Fig. 2A). high levels of tyrosine phosphorylation upon glutamate stimulation First, we examined the fluorescence intensity profiles of βPix-a and its GEF activity correlates with its phosphorylation level. and -b to compare their localization in spines. The preferential Tyrosine phosphorylation of βPix-b upon glutamate stimulation is localization to spines was assessed using the spine localization mediated by Src family kinases. We identified that tyrosine 598 index described in the Materials and Methods. The index indicates (Y598) of βPix-b is the major Src-mediated phosphorylation site, the ratio of fluorescence intensity in the spine to that in the shaft; and demonstrate that Y598 phosphorylation of βPix-b plays an when the value is greater than 1, it indicates that the fluorescence essential role in regulating its GEF activity, which is critical for signal localizes more in the spine than in the shaft. In the neurons spine development and synapse formation in neurons. These overexpressing βPix-a, the spine localization index was increased findings suggest a novel function of βPix-b in neurons and provide by 57.5% when compared with control (GFP only), supporting new understanding of the molecular mechanisms underlying localization of βPix-a in spines. Notably, the localization index in the activity-dependent spine morphogenesis and synaptic plasticity. neurons overexpressing GFP-βPix-b was significantly higher (160% increase compared with control) than that of GFP-βPix-a, indicating RESULTS that βPix-b more preferentially localizes in spine structures than Expression patterns of βPix isoforms in tissues βPix-a (Fig. 2B). This finding is consistent with the notion that βPix- Previous reports have demonstrated the presence of βPix splice b is the βPix isoform that plays a primary role in the regulation of variants in the brain (Bagrodia et al., 1998; Kim et al., 2000; Kim spine development. Indeed, neurons overexpressing GFP-βPix-b and Park, 2001; Koh et al., 2001). The modular structure of βPix strongly increased spine density without altering filopodia density isoforms varies; βPix-b and -d isoforms, but not the ubiquitous compared with that in GFP control (Fig. 2C). In contrast, GFP-βPix- βPix-a, contain a common insert region (Fig. 1A). In addition, the a overexpression resulted in decreased dendritic spine density but βPix-d isoform also lacks the C-terminal coiled-coil domain that is increased filopodia density, consistent with Zhang et al. (2003). In known to mediate dimerization of βPix (Kim and Park, 2001). addition, GFP-βPix-a overexpression reduced synapse formation, However, the differential molecular function of these isoforms in whereas GFP-βPix-b overexpression enhanced synapse formation the brain remains unclear. To investigate the role of the βPix (Fig. 2D). Taken together, these results indicate that, unlike βPix-a, isoforms, we first examined their expression levels in rat tissues. βPix-b overexpression enhances the formation of spines and Western blot analysis of rat tissue samples with anti-βPix antibody synapses, suggesting differential roles of βPix-a and βPix-b in recognizing its SH3 domain or the insert domain showed various spine morphogenesis and synapse formation. expression patterns of the βPix isoforms. βPix-b and -d were mainly Next, we asked how spine localization of βPix-b is regulated. expressed in the brain. In contrast, βPix-a was expressed in all Previous studies demonstrated that βPix-a can homo- or heterodimerize tissues investigated (Fig. 1B). To further characterize whether via its C-terminal region including coiled-coil domain and the PDZ expression of βPix-b and -d is specific to neurons, immunoblotting binding motif that is important for the intracellular localization of for the βPix isoforms in cultured rat hippocampal neurons, cortical GEF function in both non-neuronal cells and neurons (Kim et al., 2001; neurons and glial cells was performed. βPix-b and -d were detected Koh et al., 2001; Zhang et al., 2003). Because βPix-balsohasthesame in hippocampal and cortical neurons, but not in glial cells, whereas C-terminal region, we examined whether the C-terminal region of βPix-a was expressed in both neurons and glial cells (Fig. 1C). βPix-b affects its localization in neurons using a mutant, βPix-bΔCC Immunohistochemistry of rat brain sections with anti-βPix insert (a βPix-b construct devoid of coiled-coil domain and PDZ binding antibody also showed that βPix-b and -d, the insert region-containing motif) (Fig. S1). Neurons expressing GFP-βPix-bΔCC showed βPix isoforms, were specifically expressed in neurons of the impaired spine localization of the mutant protein and disturbed spine cerebral cortex and hippocampus (Fig. 1D, arrows). To examine development and synapse formation. These data show that βPix-b the expression levels of βPix isoforms during brain development, localizes to spine through its C-terminal region that is critical for the embryonic (E18) and adult rat brain extracts were analyzed by development of spine and synapse. immunoblotting with anti-βPix SH3 antibody. Interestingly, βPix-a was abundant in E18 embryonic brains, but its expression was βPix-b has higher GEF activity than βPix-a drastically reduced in adult brains. In contrast, βPix-b and -d In addition to the differences in spine localization between βPix-a expression were consistently high at both stages (Fig. 1E). These data and -b, their differential GEF activity for Rac1 may contribute to the suggest that βPix-b and -d may play important roles in neuronal distinct phenotypes of βPix-a and -b in spine formation. Llano et al. development and neuronal maintenance in adult brains. (2015) showed that βPix-b-induced Rac1 activation is drastically inhibited in neurons expressing βPix-b construct devoid of DH βPix-b enhances spine development and synapse formation domain (βPix-bDHm), indicating that the DH domain of βPix-b has through its localization at spines GEF activity for Rac1. Here, we tested whether there is a difference In a previous study, it was shown that expression of βPix-a enhanced in GEF activity between βPix-a and -b. To compare Rac1-GTP the formation of dendritic filopodia in cultured neurons. However, levels activated by βPix-a and -b, Rac1 activation assay using these filopodia failed to develop into mature spines and synapses glutathione S-transferase (GST)-PBD (p21-binding domain of PAK (Zhang et al., 2003). Here, we examined whether βPix-b is involved that binds to GTP-bound Rac1) was performed in Cos7 cells in the formation of mature spines and synapses. Here, we focused on (Fig. 3A). Expression of GFP-βPix-b caused a significant increase the b isoform because its expression was enriched in neurons and its in active Rac1 (Rac1-GTP) levels (2.68-fold increase compared Journal of Cell Science

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Fig. 1. Expression patterns of βPix isoforms. (A) Schematic representation of βPix isoforms. CC, coiled-coil domain; DH, Dbl homology domain; GBD, GIT1-binding domain; INS, insert region; PH, Pleckstrin homology domain; PXXP, proline-rich region; SH3, Src homology 3 domain. (B) Expression of βPixisoformsisshown in adult rat tissues. Lysates were analyzed by immunoblotting with anti-SH3 or anti-insert antibodies. Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; Mu, muscle; Sp, spleen; Th, thymus. (C) βPix-b is specifically expressed in neurons. 18 DIV rat hippocampal neurons (Hp), cortical neurons (Ct) and 21 DIV glial cells (Gl) were analyzed by immunoblotting with anti-SH3 or anti-insert antibodies. (D) Immunohistochemistry of adult rat brain with anti-insert antibody in the cerebral cortex and hippocampal CA1 region is shown. Positive staining was observed as dark-brown precipitates of diaminobenzidine (arrows). Scale bar: 100 µm. (E) βPix expression is developmentally regulated. Brain extracts of E18 embryos and adult rats were analyzed by immunoblotting with anti-SH3 antibody. with control), whereas expression of GFP-βPix-a did not. Similar Next, we further examined the GEF activity of βPix-b in results were also observed in Neuro2a cells (Fig. 3B), indicating that glutamate-stimulated neurons. Glutamate is known to activate

βPix-b has higher GEF activity for Rac1 than βPix-a. glutamate receptors and alter the status of phosphorylation in Journal of Cell Science

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Fig. 2. βPix-b, a major neuronal βPix isoform, enhances dendritic spine development and synapse formation. (A,B) βPix-b preferentially localizes to dendritic spines. Rat hippocampal neurons were transfected with GFP (control), GFP-βPix-a or GFP-βPix-b at 7 DIV, and Dsred (volume marker) was co- expressed to show the overall morphology of the neurons. Transfected neurons were fixed and immunostained for synapses with anti-synaptophysin antibodies. Arrowheads indicate spines. Scale bars: 5 µm. The bar graph illustrates the spine:shaft fluorescence intensity ratio of primary, secondary and tertiary dendrites randomly selected (246–405 dendrite segments per groups from 59–88 neurons). (C,D) βPix-b enhances spine and synapse formation. Quantification of spine density and synapse formation in neurons transfected with the indicated constructs. C illustrates the number of dendritic spines and filopodia per 10 µm. D illustrates the synapse:spine percentage in neurons. Dendritic spines were randomly selected from 246–405 dendrite segments (662–1317 spines per groups). *P<0.05, ***P<0.00001 for spines and synapses; #P<0.05, ###P<0.00001 for filopodia; ns, not significant; ANOVA followed by Bonferroni-corrected t-test. Error bars indicate s.e.m. numerous proteins that is essential for normal brain function. Matsuzaki et al., 2004; Matus, 2000). Tiam1 and kalirin-7, Above all, neuronal activity induced by glutamate has been Rac1 GEFs, induce actin remodeling in dendritic spines in reported to affect the morphology and plasticity of dendritic response to glutamate receptor stimulation (Tolias et al., 2005; spines through regulators of actin cytoskeleton (Lai and Ip, 2013; Xie et al., 2007). In active GEF assay using GST-Rac1G15A Journal of Cell Science

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Fig. 3. See next page for legend.

(a nucleotide-free form of Rac1 that binds to activated Rac1 level was further increased (by 127%) by glutamate stimulation. GEF proteins) (Fig. 3C), βPix-a or -d isoforms with active GEF Together, these results show that βPix-b is a more efficient function were not detected even after glutamate treatment. GEF protein than ubiquitous βPix-a and the GEF activity of In contrast, βPix-b with active GEF function was present βPix-b can be regulated effectively in spines by glutamate even in the basal state (without stimulation), and the GEF activity stimulation. Journal of Cell Science

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Fig. 3. βPix-b has higher Rac1 GEF activity than other βPix isoforms. tyrosine phosphorylation of βPix-b is mediated by an Src- (A,B) βPix-b has higher GEF activity for Rac1 than βPix-a. (A) Cos7 cells were dependent mechanism. β β transfected with construct expressing GFP (control), GFP- Pix-a or GFP- Pix-b. Next, we searched for glutamate receptors involved in the activation After 17–20 h, cell lysates were subjected to GST-PBD (p21-binding domain) pulldown assay (PD; Rac1 activation assay) and immunoblotted (IB) with anti- of Src family kinases. Previous studies demonstrated the activation Rac1 antibodies. Ratio of active Rac1 to input Rac1 levels was normalized to of Src family kinases through α-amino-3-hydroxy-5-methyl-4- GFP expression level. *P<0.05; ANOVA followed by Bonferroni-corrected isoxazolepropionic acid (AMPA) receptor activation (Hayashi et al., t-test. Error bars indicate s.e.m. (B) Lysates from Neuro2a cells expressing 1999; Socodato et al., 2012; Wu et al., 2004) and AMPA receptor- Flag (control), Flag-βPix-a or Flag-βPix-b were subjected to GST-PBD mediated synaptic stimulation involved in the maintenance pulldown assay and immunoblotted with anti-Rac1 antibodies. Ratio of active of dendritic spines (Mateos et al., 2007; McKinney et al., 1999; Rac1 to input Rac1 level was normalized to Flag expression level. (C) Glutamate stimulation increases the GEF activity of βPix-b, but not that of Takahashi et al., 2009). As mentioned, NMDA receptor-dependent βPix-a and -d. Left: 19 DIV rat cortical neurons were treated with 50 µM neuronal activity also plays an important role in the enhancement of glutamate/5 µM glycine for 5 min. Cell lysates were subjected to GST- spine and synapse formation through phosphorylation of βPix-a for Rac1G15A pulldown assay (active GEF assay) and immunoblotted with anti- Rac1 activation in dendritic spines (Saneyoshi et al., 2008). However, insert (βPix) and anti-SH3 (βPix) antibodies. Right: quantification of active the major βPix isoform(s) actually responsible for activity-dependent βPix-b levels normalized to input βPix-b. *P<0.05; Student’s t-test. Error bars Rac1 activation in neurons has yet to be clarified in the signaling indicate s.e.m. CB, Coomassie Blue staining. pathway from AMPA or NMDA receptors to actin reorganization. Our findings indicated that βPix-b appeared to be more effective at Glutamate stimulation triggers Src-mediated tyrosine activating Rac1 compared with βPix-a (Fig. 3). To investigate which phosphorylation of βPix-b in an AMPA receptor- and NMDA glutamate receptor is involved in the regulation of βPix-b, we receptor-mediated manner employed the specific antagonists for each glutamate receptor and first Next, we set out to investigate the mechanism by which the GEF examined their effects on βPix-b phosphorylation. Rat cortical activity of βPix-a and -b is differentially regulated upon glutamate neurons at 19 DIV were pretreated with CNQX, an AMPA receptor stimulation. In previous studies, several phosphorylated residues antagonist, or D-AP5, an NMDA receptor antagonist, before of βPix-a were uncovered (Mayhew et al., 2007), and the glutamate application and immunoprecipitation experiments were phosphorylation of βPix-a is known to be essential for regulation of performed. Following pretreatment with either CNQX or D-AP5, the its GEF activity in neurons and non-neuronal cells (Feng et al., 2006, glutamate-induced phosphorylation of βPix-b tyrosine residues was 2010; Saneyoshi et al., 2008; Shin et al., 2002, 2004). Moreover, reduced to the control level (Fig. 4C), showing that both AMPA neuronal activity induces phosphorylation of βPix-a at serine 516 by a receptor- and NMDA receptor-dependent neuronal activity induces Ca2+-dependent mechanism through N-methyl-D-aspartate (NMDA) Src-mediated tyrosine phosphorylation of βPix-b. Since both receptor stimulation, which is critical for regulating spine formation inhibitors completely blocked the tyrosine phosphorylation of βPix- (Saneyoshi et al., 2008). We hypothesized that glutamate stimulation b, we additionally investigated whether the inhibitors may affect triggers specific phosphorylation of βPix-b to activate its GEF baseline phosphorylation without glutamate stimulation. However, activity. To test this possibility, we examined phosphorylation levels the baseline phosphorylation of CNQX or D-AP5 treatment in of βPix-b in glutamate-stimulated neurons. Immunoprecipitation of unstimulated neurons was not significantly different from that of the βPix isoforms in 19 DIV glutamate-stimulated cortical neurons and control (Fig. S2). This result suggests that AMPA- and NMDA- immunoblotting with anti-4G10 (phosphotyrosine) antibody showed receptor pathways may interact to synergistically induce the tyrosine that tyrosine phosphorylation of βPix-b was elevated by glutamate phosphorylation of βPix-b upon glutamate stimulation. Together, stimulation, by 108% compared with control (Fig. 4A). In contrast, these data suggest that βPix-b is specifically phosphorylated by Src there was no significant increase in phosphotyrosine levels of βPix-a family kinases in an AMPA receptor- and NMDA receptor-dependent and -d, correlating with the GEF activity assay results in manner, and the phosphorylation levels correlate with the GEF Fig. 3C. These data suggest that the glutamate-dependent increase activity shown in Fig. 3. in both tyrosine phosphorylation and GEF activity is probably specific for βPix-b. Tyrosine 598 (Y598) residue in the insert region of βPix-b is We next investigated which tyrosine kinase is responsible for the the major phosphorylation site by Src phosphorylation of βPix-b in neurons. Among several candidates, Among the βPix isoforms, glutamate stimulation exclusively Src family kinases are known as key kinases for regulation of elevates phosphorylation levels of the βPix-b isoform. Hence, we various signaling cascades in neurons and have recently been hypothesized that there is a βPix-b-specific residue(s) for which revealed as important regulators of dendritic spine formation phosphorylation is relevant to the regulation of its GEF function. (Morita et al., 2006; Repetto et al., 2014; Webb et al., 2007). Because the insert region is unique structural differences between Moreover, Src was previously reported as an upstream kinase for βPix-b and βPix-a (Fig. 5A), we investigated the putative tyrosine phosphorylation of βPix-a (Feng et al., 2006, 2010). In non- phosphorylation sites in the insert region. The amino acid sequence of neuronal cells, βPix-a was phosphorylated at tyrosine 442 in an Src- βPix-b was analyzed by the NetPhos program (http://www.cbs.dtu.dk/ dependent manner. Hence, we examined whether Src family kinases services/NetPhos/) to assess the probability of phosphorylation. The phosphorylate βPix-b in glutamate-stimulated neurons. Glutamate analysis revealed that tyrosine 598 (Y598) residue in the insert region with PP2, a specific Src-family kinase inhibitor, or PP3, the inactive has the highest probability (probability=0.971, Fig. 5B) among analog of PP2, was applied to 19 DIV cortical neurons and tyrosine residues in βPix-b. To examine whether Y598 is the authentic phosphorylation levels of βPix-b were examined with anti-4G10 phosphorylation site, we generated a βPix-b mutant, Flag-βPix- antibody (Fig. 4B). Immunoprecipitation in glutamate-stimulated bY598F, by introducing a phenylalanine substitute for Y598 to block rat cortical neurons at 19 DIV showed that phosphorylated tyrosine phosphorylation and compared tyrosine phosphorylation between levels of βPix-b were increased by glutamate treatment either with wild-type Flag-βPix-b and Flag-βPix-bY598F. Immunoblotting with or without PP3 pretreatment. However, PP2 pretreatment blocked anti-4G10 antibody in Neuro2a cells revealed that wild-type βPix-b tyrosine phosphorylation, indicating that glutamate-induced was strongly phosphorylated at tyrosine, whereas the βPix-bY598F Journal of Cell Science

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Fig. 4. See next page for legend. mutant was not noticeably phosphorylated (Fig. 5C). Interestingly, development (Kuo et al., 1997). To investigate whether Y598 is Y598 phosphorylation seems to be spontaneous in Neuro2a cells phosphorylated by Src, constitutively active Src was co-expressed grown in serum-containing media. It is known that Src is basally active with Flag-βPix-b or Flag-βPix-bY598F in Cos7 cells and their in Neuro2a cells and that neurons have high Src activity during tyrosine phosphorylation was examined (Fig. 5D). Unlike Neuro2a Journal of Cell Science

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Fig. 4. Glutamate stimulation increases Src-mediated tyrosine that Src-mediated Y598 phosphorylation of βPix-b can be phosphorylation of βPix-b in an AMPA receptor- and NMDA receptor- modulated by glutamate-induced neuronal activity. β β dependent manner. (A) Among the Pix isoforms, Pix-b is preferentially From these data, we also found that although βPix-d has Y598 phosphorylated upon glutamate stimulation. Left: 19 DIV rat cortical neurons β were stimulated with 50 µM glutamate/5 µM glycine for 5 min. Cell lysates were residue in the insert region that is identical to Pix-b, its Y598 incubated with anti-SH3 antibody for immunoprecipitation (IP) and phosphorylation was not enhanced upon glutamate stimulation immunoblotted (IB) with anti-4G10 (phosphotyrosine) and anti-SH3 (βPix) (Fig. 6A), consistent with the results shown in Fig. 4. Our data antibodies. Right: quantification of βPix-b phosphotyrosine level normalized to suggest that Y598 phosphorylation is a specific modification on immunoprecipitated βPix-b. (B) Src family kinases mediate tyrosine βPix-b among the βPix isoforms in neurons. phosphorylation of βPix-b. Left: 50 µM glutamate/5 µM glycine treatment was administered to 19 DIV rat cortical neurons for 5 min. 10 µM PP2 or 10 µM PP3 Y598 phosphorylation of βPix-b enhances its GEF activity for pretreatment was administered for 15 min before adding glutamate. Cell lysates were incubated with anti-SH3 antibody for immunoprecipitation and Rac1 that is critical for spine development and synapse immunoblotted with anti-4G10 and anti-SH3 antibodies. Right: quantification of formation phosphotyrosine levels of βPix-b normalized to immunoprecipitated βPix-b. Activated Rac1 GTPase regulates actin cytoskeleton that is essential (C) AMPA receptor and NMDA receptor mediate phosphorylation of βPix-b. for development, maintenance and plasticity of dendritic spines in Left: 100 µM D-AP5 or 30 µM CNQX pretreatment was administered to 19 DIV neurons (Tashiro and Yuste, 2004). Hence, we investigated whether rat cortical neurons for 15 min before adding glutamate. Right: quantification of Y598 phosphorylation of βPix-b affects the regulation of its GEF phosphotyrosine levels of βPix-b normalized to immunoprecipitated βPix-b. *P<0.05; ns, not significant; Student’s t-test. Error bars indicate s.e.m. function for Rac1 activation. First, active GEF assay with GST- Rac1G15A in Cos7 cells showed that GEF activity of wild-type GFP-βPix-b was markedly elevated when constitutively active Src cells, Cos7 cells did not exhibit spontaneous Y598 phosphorylation. was co-expressed (2.7±0.36 vs 7.3±2.63; mean±s.e.m.) (Fig. 7A). Expression of active Src strongly induced tyrosine phosphorylation of However, GEF activity of GFP-βPix-bY598F was not significantly βPix-b. However, this phosphorylation was drastically reduced in changed by co-expression of active Src (not significant; 1.37±0.63). βPix-bY598F, indicating that Y598 of βPix-b is the major Src- Rac1 activation assay with GST-PBD also showed similar results. mediated phosphorylation site. Rac1-GTP levels in cells expressing GFP-βPix-bY598F with active To further examine the phosphorylation of βPix-b, we generated Src were modestly increased compared with GFP control (3.42 an anti-pY598 antibody specific for Y598 phosphorylation. This ±1.48), whereas expression of GFP-βPix-b with active Src antibody specifically recognized serum- and Src-induced drastically increased Rac1 activation (15.1±3.13) (Fig. 7B). phosphorylation of βPix-b in Neuro2a cells and did not recognize Interestingly, βPix-bY598F exhibited some interesting differences Flag-βPix-bY589F (Fig. 5E), showing its specificity for pY598 of in two different assays: active GEF assay and Rac1 activation assay. βPix-b. Using anti-pY598 antibody, we investigated the localization As mentioned above, the active GEF level of βPix-bY598F was of the endogenous βPix-b phosphorylated at Y598 (Fig. S3). similar to that of control, while its Rac1-GTP level remained higher Hippocampal neurons at 25 DIV were immunostained with anti- than that of control (3.42±1.48). We suppose that the discrepancy is pY598 antibody, and the pattern of immunostaining was compared due to active Src co-expression affecting broad Src-dependent with those of total βPix isoforms and various synaptic markers. pathways inducing Rac1 signaling in cells. pY598 immunostaining in dendrites overlapped with the total βPix To confirm the effect of Y598 phosphorylation on βPix-b- immunostaining by anti-βPix SH3 antibody only at the spine-like mediated Rac1 activation in neurons, we performed a fluorescence structures (Fig. S3, top row). In addition, the anti-pY598 resonance energy transfer (FRET)-based assay in cultured immunostaining colocalized well with the postsynaptic marker, hippocampal neurons using a Rac1 activation biosensor, Raichu- PSD95, and the AMPA receptor subunit, GluR2, and with a Rac1 construct (Itoh et al., 2002). We transfected Flag-βPix-b or somewhat lesser degree with the presynaptic marker, synaptophysin Flag-βPix-bY598F mutant with Raichu-Rac1 probe to 7 DIV (Syn; also known as Syp) (Fig. S3, bottom three rows), showing that hippocampal neurons from neuron-specific βPix isoform knockout Y598 phosphorylation of βPix-b occurs spontaneously in neurons (KO) mice and analyzed the FRET signals at 19 DIV. These KO and specifically localizes to spines. mice lack expression of neuron-specific βPix isoforms, βPix-b and -d. We found that βPix-b overexpression increased Rac1 activation Glutamate induces Y598 phosphorylation of βPix-b in dendrites and protrusions by 49% (Fig. 7C), consistent with the in an Src-dependent manner effect of βPix-b expression in a previous FRET study (Llano et al., Our data demonstrate that βPix-b is phosphorylated by Src family 2015). Importantly, this effect was fully abolished by the Y598F kinases upon glutamate stimulation (Fig. 4B) and Y598 of βPix-b is mutation. These results suggest that Y598 phosphorylation the major phosphorylated residue (Fig. 5). Therefore, we examined regulates activity of βPix-b as a Rac1 GEF in both Cos7 cells and whether Y598 phosphorylation can be modulated by glutamate- neurons. mediated synaptic activity. Glutamate treatment was administered to Finally, we tested whether Y598 phosphorylation of βPix-b is 19 DIV rat cortical neurons, and Y598 phosphorylation of βPix-b critical for regulation of spine morphogenesis and synapse was examined with anti-pY598 and anti-4G10 antibody. Glutamate plasticity. Hippocampal neurons from neuron-specific βPix treatment increased Y598 phosphorylation of βPix-b, whereas PP2 isoform KO mice were cultured and transfected at 7 DIV with pretreatment blocked the phosphorylation (Fig. 6A), suggesting that GFP, GFP-βPix-b, GFP-βPix-bY598E or GFP-βPix-bY598F glutamate induces Y598 phosphorylation of βPix-b in an Src- constructs, as indicated in Fig. 8A. βPix-bY598E, a phospho- dependent manner. This was also confirmed by immunostaining in mimicking mutant, was characterized in Neuro2a cells in which the 25 DIV hippocampal neurons using anti-pY598 antibody (Fig. 6B). Y598 phosphorylation occurs spontaneously due to the Src being Glutamate-induced pY598 βPix-b that colocalized with GluR2 basally active. We found that βPix-bY598E was recognized by (Fig. 6B, top row) disappeared with PP2 pretreatment (Fig. 6B, anti-pY598 antibody and exhibited higher Rac1 GEF activity than middle row), and reappeared with glutamate treatment after washing βPix-bY598F. Notably, no further enhancement of the GEF activity out PP2 (Fig. 6B, bottom row). Taken together, these results suggest was observed by βPix-bY598E expression compared with the Journal of Cell Science

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Fig. 5. Tyrosine 598 (Y598) of βPix-b is the major site for Src-mediated phosphorylation. (A,B) Predicted tyrosine phosphorylation sites in the insert region of βPix-b. (A) Schematic diagram of βPix-a and βPix-b domain structures. (B) Potential tyrosine phosphorylation sites in the βPix-b sequence were predicted by NetPhos program. Residues showing score >0.5 were defined as predicted phosphorylation sites. Scores closer to 1 indicate higher probability of phosphorylation. The sequence containing the Y598 in the insert region of βPix-b exhibits the highest probability. (C,D) Y598 of βPix-b is the major phosphorylation site by Src. (C) Lysates from Neuro2a cells expressing Flag- βPix-b or Flag-βPix-bY598F were immunoprecipitated (IP) with anti-Flag (M2) antibody. The immunoprecipitates were subjected to immunoblotting (IB) with anti-4G10 and anti-Flag (M2) antibodies. (D) Cos7 cells expressing Flag (control), Flag-βPix-b or Flag-βPix- bY598F were co-expressed with or without active Src. Cell lysates were immunoprecipitated and subjected to immunoblotting as in C. (E) Anti-pY598 antibody specifically recognizes Y598 phosphorylation of βPix-b. Neuro2a cells expressing Flag (control), Flag-βPix-b or Flag-βPix-bY598F were co-expressed with or without active Src and incubated in the presence or absence of serum. Cell lysates were immunoprecipitated and subjected to immunoblotting with anti-pY598 (Y598-phosphorylated βPix-b) and anti-Flag (M2) antibodies.

expression of wild-type βPix-b (Fig. S4). Synapses were stained all localized preferentially at dendritic spines without noticeable using anti-synaptophysin antibody and analyzed at 19 DIV. differences (Fig. 8B), indicating that Y598 phosphorylation of Expression of wild-type GFP-βPix-b and GFP-βPix-bY598E βPix-b does not affect the localization of βPix-b. In addition, we strongly increased the density of dendritic spine, whereas neurons observed that GFP-βPix-bY598E did not significantly enhance expressing GFP-βPix-bY598F, non-phosphorylatable mutant, did spine density and synapse formation when compared with wild- not significantly change the spine density (Fig. 8A,C). Filopodia type βPix-b (not significant). This is consistent with the results density was not significantly changed by any of the βPix-b shown in Fig. S4, suggesting that wild-type βPix-b may already be constructs. In addition, GFP-βPix-b and GFP-βPix-bY598E fully activated in neuronal cells and neurons in an active increased the formation of synapses, but GFP-βPix-bY598F did Src-dependent manner. Collectively, these results demonstrate that not (Fig. 8D), suggesting that phosphorylation of βPix-b at Y598 Src-mediated Y598 phosphorylation of βPix-b regulates its GEF regulates spine development and synapse formation. Interestingly, function for Rac1 activation that is critical for spine development wild-type GFP-βPix-b, GFP-βPix-bY598E and GFP-βPix-bY598F and synapse formation. Journal of Cell Science

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Fig. 6. Glutamate induces Src-mediated Y598 phosphorylation of βPix-b at dendritic spines. (A) Glutamate stimulation induces Src-dependent Y598 phosphorylation of βPix-b. 50 µM glutamate treatment was administered to 19 DIV cortical neurons for 10 min. 10 µM PP2 pretreatment was administered for 15 min before adding glutamate, where indicated. Cell lysates were immunoprecipitated with anti-insert antibodies and subjected to immunoblotting (IB) with anti- 4G10 and anti-pY598 antibodies. (B) Glutamate induces Y598 phosphorylation of βPix-b at dendritic spines. 25 DIV hippocampal neurons were treated with 50 µM glutamate for 10 min with or without PP2 pretreatment, fixed and immunostained with anti-pY598 and anti-GluR2 antibodies. The top row shows control neurons without PP2 pretreatment; the middle row shows neurons pretreated with 10 µM PP2 for 15 min before glutamate addition; the bottom row shows neurons treated with 50 µM glutamate (Glu) for 10 min after washing out the pretreated 10 µM PP2. Scale bar: 10 µm.

DISCUSSION by Y598 phosphorylation upon neuronal activation. We previously Here, we demonstrate that Src-mediated Y598 phosphorylation of reported that several βPix isoforms are present in the brain. Among βPix-b regulates spine and synapse development. This conclusion is those, βPix-a is ubiquitously expressed, whereas βPix-b and -d are based on our findings that (1) βPix-b is mainly expressed in neurons; highly enriched in brain tissues, suggesting that βPix-b and -d are (2) βPix-b overexpression enhances spine development and synapse involved in neuronal processes (Kim et al., 2000; Kim and Park, formation with its preferential localization at spines; (3) βPix-b 2001; Oh et al., 1997). In our present data, we confirmed that βPix-b exhibits higher basal GEF activity than ubiquitous βPix-a and its GEF and -d are mainly expressed in neurons (Fig. 1). βPix functions have activity is further elevated upon glutamate-stimulation in neurons; been implicated in spine and synapse formation (Saneyoshi et al., (4) glutamate induces Src-mediated tyrosine phosphorylation of 2008). However, a previous study showed that βPix-a overexpression βPix-b; (5) tyrosine 598 (Y598) in the insert region is the major in cultured neurons inhibited the development of mature spine and phosphorylation site in βPix-b upon glutamate stimulation; and synapse, but enhanced the formation of dendritic filopodia (Zhang (6) inhibiting Y598 phosphorylation (βPix-bY598F) drastically et al., 2003), suggesting that βPix-a overexpression induces an decreases the GEF activity of βPix-b for Rac1 signaling and further antimorphic phenotype of βPix, possibly due to mislocalization of disrupts spine development and synapse formation. Our data provide βPix-a in neurons. Because the molecular structure of βPix-b is evidence that βPix-b acts as a positive regulator to enhance identical to that of βPix-a, except for the presence of the insert region spinogenesis and synaptogenesis through its GEF activity regulated in βPix-b, we primarily compared the roles of these two isoforms in Journal of Cell Science

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Fig. 7. See next page for legend. Journal of Cell Science

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Fig. 7. Y598 phosphorylation enhances Rac1 GEF activity of βPix-b. antagonists for the AMPA receptor and NMDA receptor, (A,B) Src-mediated Y598 phosphorylation enhances Rac1 GEF activity of respectively. These data indicate that Src activation induces the β β β Pix-b. (A) Left: GFP (control), GFP- Pix-b or GFP- Pix-bY598F were co- phosphorylation of βPix-b by both AMPA receptor- and NMDA expressed with or without active Src in Cos7 cells. After 17–20 h, cell lysates were subjected to active GEF assay (PD) and immunoblotted (IB) with anti- receptor-mediated pathways. SH3 antibodies. Right: ratio of active βPix-b or βPix-bY598F to input actin Because βPix-b has the insert region, which is deficient in βPix-a, (loading control) levels were normalized to GFP expression level. (B) Left: cell we hypothesized that an unknown tyrosine phosphorylation site(s) lysates were prepared as in A. After 17–20 h, cell lysates were subjected to in the insert region of βPix-b is critical for regulation of its GEF Rac1 activation assay (PD) and immunoblotted with anti-Rac1 antibodies. activity. We found that Y598 in the insert region of βPix-b is the Right: ratio of active Rac1 to input Rac1 levels were normalized to GFP major phosphorylation site by Src (Figs 5 and 6; Fig. S3). Among expression level. (C) Y598 phosphorylation of βPix-b induces Rac1 activation in neurons. Dissociated hippocampal neurons from P0 neuron-specific βPix several prospective tyrosine phosphorylation sites, Y598 in the isoform KO mice were transfected with Raichu-Rac1 (Rac1 activation insert region showed the highest probability for phosphorylation. To biosensor), together with Flag (control), Flag-βPix-b or Flag-βPix-bY598F at confirm the site of phosphorylation, βPix-bY598F mutant for 7 DIV and imaged at 19 DIV. Left: representative FRET images of Rac1 blocking Y598 phosphorylation and anti-pY598 antibody for activation are shown in the intensity-modulated display mode. In the intensity- targeting Y598 phosphorylation were generated. We observed that modulated mode, eight colors from red to blue were used to represent the the Y598F mutation blocks tyrosine phosphorylation of βPix-b even FRET:CFP ratio. Scale bar: 10 µm. Right: quantification of Raichu-Rac1 FRET when Src is constitutively activated, indicating that Y598 of βPix-b signal in dendrites (13–17 neurons per condition). The values were normalized to that of cells expressing Raichu-Rac1 only. *P<0.05, **P<0.001; ns, not in the insert region is the major phosphorylation site (Fig. 5). In significant; ANOVA followed by Bonferroni-corrected t-test. Error bars indicate neurons, we also confirmed that Y598 phosphorylation can be s.e.m. CB, Coomassie Blue staining. modulated by glutamate-induced Src activation (Fig. 6). The anti- pY598 antibody reveals that Y598-phosphorylated βPix-b is highly spine development in this study. Interestingly, overexpression of enriched in spines and that Y598 phosphorylation of βPix-b occurs βPix-b, but not βPix-a, enhances spine development and synapse spontaneously in neurons (Fig. S3). Our other results also support formation in cultured neurons (Fig. 2). Our data support that the that Y598-phosphorylated βPix-b localizes in spines. First, βPix-b is functional difference between βPix-a and -b is due to their preferentially localized in spines (Fig. 2). Second, βPix-b is localization (Fig. 2) and GEF activity (Fig. 3). First, we found that phosphorylated by Src family kinases in a stimulation-dependent overexpressed βPix-b preferentially localizes in the spines and, at the manner (Figs 4–6). Third, wild-type βPix-b, the phospho- same time, spine density and synapse formation are enhanced. In the mimicking mutant βPix-bY598E and the phospho-blocking case of βPix-a overexpression, however, spine density and synapse mutant βPix-bY598F do not display different spine localization formation are inhibited, consistent with Zhang et al. (2003), although indices (Fig. 8B), indicating that Y598 phosphorylation of βPix-b its localization still slightly prefers spines to shafts. Our data show that takes place in spines, but the phosphorylation itself does not affect overexpression of βPix-b elevates its localization at spine heads and the localization of βPix-b. Furthermore, we found that βPix-b is the results in promotion of spine and synapse formation, suggesting that only Y598-phosphorylated βPix isoform detected in neurons βPix-b is important for the regulation of molecular effectors in spine (Fig. 6A). The βPix-d isoform, containing the identical insert development. region, does not show a detectable Y598 phosphorylation upon Next, our results show that βPix-b exhibits a higher GEF activity glutamate stimulation. This result is consistent with the finding that than βPix-a toward Rac1, which is known as a downstream target of only βPix-b is tyrosine phosphorylated upon glutamate stimulation βPix (Klooster et al., 2006) (Fig. 3), suggesting that βPix-b is a more (Fig. 4), suggesting that the Y598 phosphorylation is a specific efficient GEF protein than βPix-a. In addition, our GEF activity modification for βPix-b. Why is Y598 of βPix-d not phosphorylated assay of βPix in glutamate-stimulated neurons shows that only βPix- by Src in glutamate-stimulated neurons? We suppose that lack of b responds to the stimulation with enhanced GEF activity. Taken C-terminal domain in βPix-d interrupts its localization at spines, together, these results indicate that differences in localization and resulting in inefficient Y598 phosphorylation. βPix-d has a unique GEF activity between βPix-a and -b lead to their distinct roles in the 11-amino-acid stretch instead of the C-terminal domain found in regulation of spine morphology and synapse plasticity in neurons. other βPix isoforms that is necessary for proper intracellular What could be a possible mechanism regulating the GEF activity localization, as shown in Fig. S1. Indeed, βPix-d expression shows of βPix-b? Previous studies reported that βPix-a has numerous diffused localization in both spines and dendrites (data not shown), phosphorylated residues that also exist in βPix-b, and its similar to the localization of βPix-bΔCC, which is structurally phosphorylation is known as a critical modification to activate GEF identical to βPix-d except for the 11 amino acids unique to βPix-d function (Feng et al., 2006, 2010; Mayhew et al., 2007; Saneyoshi (Fig. S1). Unlike βPix-b, βPix-d probably cannot properly localize et al., 2008; Shin et al., 2002; Shin et al., 2004). We predicted that the to spines due to the lack of C-terminal domain, leading to spatial phosphorylation of βPix-b is correlated with its GEF activity. To test sequestration of βPix-d from Src that localized at the membrane this hypothesis, we examined the phosphorylation level of βPix-b in of spines. glutamate-stimulated neurons (Fig. 4). As predicted, our data suggest Finally, we asked what the role of Y598 phosphorylation of that tyrosine phosphorylation of βPix-b regulates its GEF function. βPix-b in neuronal development was (Figs 7 and 8). We observed To understand the upstream mechanisms for the phosphorylation that introducing a non-phosphorylatable mutation to βPix-b Y598 of βPix-b, we investigated which kinases cause phosphorylation of residue (Y598F) abrogates the GEF activity of βPix-b for Rac1 in βPix-b (Fig. 4). A glutamate stimulation-dependent increase in the Rac1 activation assays in Cos 7 cells and in neurons. Furthermore, tyrosine phosphorylation levels of βPix-b was inhibited when PP2 βPix-bY598F is functionally inactive and its overexpression fails to was pretreated, showing that Src family kinases mediate the recover spine and synapse formation in hippocampal neurons from phosphorylation of βPix-b. Further, we investigated which neuron-specific βPix isoform KO mice, unlike the expression of glutamate receptor is involved in the activation of Src (Fig. 4). wild-type βPix-b. These observations demonstrate that Y598 Our results show that the increase in the tyrosine phosphorylation of phosphorylation of βPix-b regulates GEF function for Rac1

βPix-b was abrogated by both CNQX and D-AP5, which are activation, and this regulation is critical for spine development Journal of Cell Science

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Fig. 8. See next page for legend. Journal of Cell Science

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Fig. 8. Y598 phosphorylation of βPix-b is critical for spine development Yes (also known as Yes1) are also ubiquitously expressed, and Lyn and synapse formation. (A,B) Y598 phosphorylation does not affect the and Lck are expressed in brain (Thomas and Brugge, 1997). Thus, localization of βPix-b to spines. Hippocampal neurons cultured from P0 β these Src family kinases can potentially be involved in Y598 neuron-specific Pix isoform KO mice were transfected at 7 DIV with Dsred and β one of the following constructs: GFP (control), GFP-βPix-b, GFP-βPix-bY598E phosphorylation of Pix-b. In addition, it is also possible that the Src or GFP-βPix-bY598F. Synapses were labeled by anti-synaptophysin family kinases might indirectly regulate the phosphorylation of immunostaining. Arrowheads indicate spines. Scale bars: 5 µm. The bar βPix-b by activating other kinases. graphs illustrate the spine:shaft fluorescence intensity ratio in randomly The functional roles and the regulatory mechanisms of βPix-d are selected dendrites (112–140 dendrite segments per groups from 31–36 also to be discovered in future studies. Like βPix-b, βPix-d is highly β neurons). (C,D) Y598 phosphorylation of Pix-b regulates spine and synapse expressed in neurons and contains the insert domain (Fig. 1). formation. Quantification of spine density and synapse formation in neurons However, βPix-d has a unique stretch of 11 amino acids replacing transfected with the indicated constructs. C illustrates the number of dendritic β spines per 10 µm. D illustrates the synapse:spine percentage in neurons. the C-terminal region of other Pix isoforms and is not Dendritic spines were randomly selected from 112–140 dendrite segments phosphorylated by Src, suggesting that βPix-d is regulated via (317–452 spines per groups). **P<0.001, ***P<0.00001; ns, not significant; different mechanisms from those regulating βPix-b. ANOVA followed by Bonferroni-corrected t-test. Error bars indicate s.e.m. In conclusion, we identify βPix-b as a major neuronal βPix (E) A prospective model for a novel role of βPix-b in activity-dependent isoform primarily regulating the formation of dendritic spines and β spinogenesis and synaptogenesis. Pix-b preferentially localizes at the spine synapses through its GEF activity for Rac1. The regulatory pathway and promotes spine and synapse formation. Once glutamate-dependent β neuronal activity activates Src family kinases in an AMPA receptor- and NMDA requires Src-mediated Y598 phosphorylation of Pix-b that is receptor-mediated manner, Y598 of βPix-b is specifically phosphorylated. necessary for its GEF activity. We show that modulation of the GEF Y598 phosphorylation of βPix-b increases its GEF activity for Rac1 and activity by Y598 phosphorylation is critical for regulating spine enhances spine development and synapse formation. development and synapse formation. These findings provide novel understanding of the molecular mechanisms underlying activity- dependent spine morphogenesis and synapse formation. and synapse formation. Interestingly, we also show that there are no differences in localization between wild-type βPix-b and its MATERIALS AND METHODS mutants, the phospho-mimicking βPix-bY598E and the phospho- Antibodies and reagents blocking βPix-bY598F, indicating that Y598 phosphorylation of β β Rabbit polyclonal antibodies for the insert and the SH3 domains of Pix Pix-b regulates its GEF activity, but not its localization in spines. were prepared as described previously (Kim et al., 2000; Oh et al., 1997). Based on these data, we suggest that βPix-b expression and its Antibodies specifically recognizing phosphorylated Y598 of βPix-b were Y598 phosphorylation in dendritic spines regulate the proper generated by Labfrontier (Republic of Korea) by immunizing rabbits with morphology and development of spines in both basal state and phosphorylated peptides EDSE(pY598)DSIW (where pY is phosphorylated stimulated condition. A prospective model of this regulation is Tyr) purchased from BioSynthesis (USA). Specific phosphotyrosine schematically described in Fig. 8E. Although our study reveals the antibodies were purified from the rabbit serum with immobilized peptides, important role of βPix-b in spinogenesis, a few questions remain first with non-phosphorylated peptides for negative selection and then unanswered. with phosphorylated peptides for positive selection. The following We have not yet identified the reason why βPix-b is more antibodies were purchased: monoclonal anti-phosphotyrosine 4G10 (#05- β 321) from Upstate; monoclonal anti-Rac1 (#610650) from BD Transduction localized at spine heads than ubiquitous Pix-a (Fig. 2). We suppose Laboratories; monoclonal anti-Cdc42 (#C70020) from Transduction that there are probably binding partners specifically interacting with Laboratories; polyclonal anti-phospho-Src(Tyr416) (#2101) from Cell the βPix-b isoform for spine localization. Recent studies reported Signaling Technology; monoclonal anti-Flag M2 (#F7425), monoclonal that KCC2, a neuron-specific KCl co-transporter that localizes at the anti-β-tubulin (#T4026) and monoclonal anti-actin (#A4700) from Sigma- dendritic spines, plays a critical role in the regulation of spine actin Aldrich; polyclonal anti-synaptophysin (#MAB5258), polyclonal anti-PSD- dynamics via interaction with βPix-b (Llano et al., 2015). It will be 95 (#MAB1596) and polyclonal anti-GluR2 (#AB1506) from Chemicon. interesting to examine whether KCC2 specifically interacts with Reagents purchased were PP2 (#529573), a specific Src family kinase βPix-b among βPix isoforms and whether KCC2 recruits βPix-b to inhibitor, and PP3 (#529574), a negative control for PP2, from Calbiochem; proper localization at spines. and CNQX (#1045), AMPA receptor antagonist, and D-AP5 (#0106), In neurons, Rac1 and Cdc42 are important for promoting the NMDA receptor antagonist, from Tocris. formation, development and maintenance of spines and synapses DNA constructs (Newey et al., 2005; Tolias et al., 2011). Because βPix is known as The coding region of βPix-b was subcloned into pFlag-CMV2 (Eastman the GEF protein for Cdc42 as well as Rac1 (Bagrodia et al., 1998; Kodak Co.) and pEGFP-N1 (Clontech) by PCR. To generate βPix-b mutant β Manser et al., 1998), we also examined whether Pix-b has the constructs, site-directed mutagenesis was performed using a QuikChange potential to promote Cdc42 activity. Cos7 cells expressing GFP- site-directed mutagenesis kit (Stratagene), following the manufacturer’s βPix-b showed higher Cdc42 activity than those expressing GFP- instruction. The following mutagenic primers were used: pEGFPN1-βPix- βPix-a (2.76-fold increase compared with control) (Fig. S5). This b(Y598E), 5′-TCGGAAGACTCTGAGGAAGACAGTATATGGACA-3′ result is similar to the results of the Rac1 activation assay shown in and 5′-TGTCCATATACTGTCTTCCTCAGAGTCTTCCGA-3′;pEGFP- Fig. 3A, suggesting that Cdc42 activity can also be regulated by N1-βPix-b(Y598F), 5′-TCGGAAGACTCTGAGTTTGACAGTATATGG- ′ ′ ′ βPix-b. Whether Y598 phosphorylation of βPix-b modulates spine ACA-3 and 5 -TGTCCATATACTGTCAAACTCAGAGTCTTCCGA-3 ; β Δ ′ ′ development through the Cdc42-activating ability is to be pEGFPN1- Pix-b( CC), 5 -GACAAGCTTCGATGACTGATAACAAC-3 and 5′-CGCGTCGACGTACTCTATCACTGTCTG-3′. These primers were investigated in future studies. also used for mutagenesis of pFlagCMV2-βPix-b(Y598F). The mutants Next, because PP2 is a broad-spectrum inhibitor of Src family were verified by automated DNA sequencing. We used a GST-PBD construct kinases, we cannot exclude the possible involvement of other Src described previously (Park et al., 2012). GST-Rac1G15A construct was a kind family kinases in βPix-b phosphorylation. Among the Src family gift from J. G. Hanley (University of Bristol, Bristol, UK). Raichu-Rac1 probe kinases, Src is expressed ubiquitously, but shows a high expression for FRET was kindly provided by M. Matsuda (Kyoto University, Kyoto, level especially in neurons (Thomas and Brugge, 1997). Fyn and Japan). Journal of Cell Science

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Mice 30 min before treatment with 50 μM glutamate and 5 μM glycine for 5 min. Neuron-specific βPix isoform KO mice were generated by replacing exon 19 The bead-bound proteins were denatured in Laemmli sample buffer, with a neomycin-resistance cassette flanked by LoxP sites to specifically delete separated by SDS-PAGE and immunoblotted using anti-SH3 (βPix), anti- the insert domain-coding region. In order to allow expression of the ubiquitous Rac1 and anti-Cdc42 antibodies. The GST fusion proteins were visualized βPix-a isoform, the mouse line described above was crossed to Sox2-Cre mice by Coomassie Blue staining. to excise the neomycin-resistance cassette in the germ line (Hayashi et al., 2002; Kos, 2004). In these mice, expression of βPix-b and -d containing the Immunoprecipitation insert region was removed, but βPix-a remained expressed in brain (data not Protein G sepharose beads (GE Healthcare) and appropriate antibodies were shown). Their genotypes were confirmed using PCR techniques. All micewere incubated for 1 h at 4°C. Transiently transfected cells were washed with maintained and used according to the guidelines of the Seoul National PBS and lysed in immunoprecipitation buffer (50 mM HEPES, pH7.4, 1% University Institutional Animal Care and Use Committees. NP-40, 300 mM NaCl, 15 mM NaF, 1 mM sodium vanadate, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 1 µg/ml pepstatin). Lysates were clarified Cell line culture and transfection by centrifugation at 22,500 g for 30 min. Protein concentrations were Cos7 cells were cultured in Dulbecco’s modified Eagle medium supplemented determined using Bradford reagent. Clarified lysates were added to samples with 10% fetal bovine serum (c-FBS, Welgene) and 1% penicillin- and incubated for 1 h at 4°C. The immunoprecipitates were washed five streptomycin (Gibco) at 37°C with 5% CO2. Neuro2a cells were cultured in times with immunoprecipitation buffer to remove unbound proteins. The minimum essential medium (MEM) supplemented with 10% c-FBS at 37°C same protocol was performed with cultured rat neurons. Rat cortical neurons with 5% CO2. Cos7 cells and Neuro2a cells were transfected using at 19 DIV were incubated in Tyrode solution (119 mM NaCl, 2.5 mM KCl, Lipofectamine Plus (Invitrogen) according to the manufacturer’sinstructions. 2 mM CaCl2, 2 mM MgCl2 and 30 mM glucose in 25 mM HEPES, pH 7.4) for 30 min before stimulation with 50 µM glutamate and 5 µM glycine for Primary neuronal culture and transfection 5 or 10 min. The following reagents were administered as pretreatment Dissociated hippocampal (for staining experiments) and cortical (for for 15 min before adding glutamate: 10 µM PP2, 10 µM PP3, 30 µM CNQX biochemical experiments) neurons were prepared from embryonic day 18 or 100 µM D-AP5. Proteins bound to antibodies were loaded in Laemmli (E18) Sprague-Dawley rat embryos and postnatal day 0 (P0) pups of neuron- sample buffer, separated by SDS-PAGE and immunoblotted. specific βPix isoform KO mice of either sex as described previously (Beaudoin et al., 2012; Park et al., 2012). We used protocols described in Immunoblotting Park et al. (2012) and Beaudoin et al. (2012) for neuron cultures, with minor For examination of βPix isoform expression levels, adult rat brain and organ modifications. In brief, dissociated hippocampal and cortex tissues were tissues were homogenized in 8 ml homogenization buffer (50 mM Tris- treated with papain (20 µg/ml, Worthington) and DNase (10 U/µl, Sigma- HCl, pH 8.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2 mM sodium Aldrich) for 25 min at 37°C. The tissues were dissociated by trituration with vanadate, 15 mM NaF, 1 µg/ml leupeptin and 1 µg/ml pepstatin) with a a glass Pasteur pipette and then plated in 60-mm or 100-mm dishes coated Polytron homogenizer and incubated for 1 h at 4°C after addition of 1% with poly-D-lysine (10 ng/ml, Sigma-Aldrich). Cultures were grown in Triton X-100. The homogenates were centrifuged at 22,500 g for 30 min at Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and 4°C and the supernatants used for immunoblotting. Lysates from cultured 0.5 mM L-glutamine (Welgene). The cultures were maintained by cell lines or primary neurons were prepared by washing with PBS and lysing discarding and replacing half of the original medium with fresh medium in SDS sample buffer (62.5 mM Tris-HCl, pH 6.7, 10% glycerol and 2% every 4–7 days. Hippocampal neurons were transiently transfected at 7 DIV SDS) or in assay buffer. Protein concentrations were determined with BCA with the calcium phosphate method using a CalPhos Transfection Kit reagent (Pierce) or Bradford reagent (Bio-Rad) by using bovine serum (Calbiochem). Glial cells were cultured from 18-day embryos as described albumin solutions as standards. Samples were loaded in 1× Laemmli sample previously (Goslin et al., 1998). Dissociated cells were plated in 60-mm or buffer (5× sample buffer: 60 mM Tris-HCl, pH 6.8, 30% glycerol, 2% SDS, 100-mm diameter dishes coated with poly-D-lysine (1 mg/ml). Cultures 14.4 mM β-mercaptoethanol and 0.2% Bromophenol Blue) and equal were grown in MEM (Invitrogen) supplemented with 5% FBS (Invitrogen), amounts of protein were resolved by SDS-PAGE and transferred to a PVDF penicillin-streptomycin, 0.4% glucose and 0.5 mM L-glutamine. or nitrocellulose membrane. Blots were blocked with 5% bovine serum albumin (BSA) in 1× TBS-T (0.1% Tween 20 in 1× TBS-T) for 30 min. The Active GEF assay, Rac1 activation assay and Cdc42 activation blots were incubated with primary antibodies for 1 h and washed three times assay for 10 min with TBS-T. Then, the blots were incubated with horseradish Active GEF assay and Rac1 activation assay were performed with GST peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch fusion proteins expressed in Escherichia coli (BL21) cells and purified by Laboratories) for 45 min, washed three times with TBS-T and analyzed by glutathione sepharose beads (GE Healthcare). The fusion proteins bound to enhanced chemiluminescence. the beads were then eluted by glutathione elution buffer (20 mM glutathione, 100 mM Tris-HCl, pH 8.0, 120 mM NaCl) and quantified Immunohistochemistry using Bradford quantification method (Bio-Rad). Activity of GEF proteins We performed fixation and sectioning according to the procedures of was assessed by measuring the amount of GEF protein bound to Schaeren-Wiemers and Gerfin-Moser (1993). Frozen sections of adult rat GST-Rac1G15A (nucleotide-free Rac1 GTPase) proteins, which form a brains on slides were sectioned into 40-μm slices, fixed with 4% high-affinity complex with active Rac1 GEFs, as described previously paraformaldehyde for 15 min and washed with PBS. All procedures were (Blanco-Suárez et al., 2014). Active Rac1 levels were measured using GST- performed at room temperature (RT). Specimens were incubated with 0.3% PBD (p21-binding domain of PAK) proteins, which bind to GTP-bound H2O2/methanol solution for quenching of endogenous peroxidase activity Rac1 (Shin et al., 2004). Briefly, glutathione sepharose beads and 5 μg GST for 20 min and washed three times with PBS. Non-specific signals were fusion proteins were incubated in assay buffer (50 mM HEPES, pH7.4, 1% blocked by incubation with 1.5% horse serum/0.3% Triton X-100 in PBS. NP-40, 150 mM NaCl, 15 mM NaF, 1 mM sodium vanadate, 1 µg/ml Then, specimens were incubated with primary antibodies, biotinylated leupeptin, 1 µg/ml aprotinin and 1 µg/ml pepstatin) for 1 h at 4°C. Cells secondary antibodies and Ultra-Sensitive ABC Peroxidase reagent (Pierce). transfected with the indicated constructs for 17–20 h were washed with PBS Signals were visualized by diaminobenzidine staining (Pierce). and lysed with assay buffer for 20 min at 4°C. Cell lysates were centrifuged at 22,500 g for 30 min at 4°C, and then the supernatant was added to the pre- Immunocytochemistry and microscope image acquisition incubated mixture of GST fusion proteins and beads and incubated for 1 h at Hippocampal neurons seeded on 18-mm coverslips were fixed at 19 or 25 4°C. The beads were washed three times with assay buffer. These assays DIV in PBS containing 3.7% paraformaldehyde for 10 min at RT. Where were also performed with rat neuronal cultures. Rat cortical neurons at 19 indicated, cells were treated with 10 µM PP2 for 15 min and then 50 µM DIV were incubated in Tyrode solution (119 mM NaCl, 2.5 mM KCl, 2 mM glutamate for 10 min in Tyrode solution for 30 min before fixation. Fixed

CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.4, and 30 mM glucose) for neurons were permeabilized with 0.5% Triton X-100 in PBS for 10 min and Journal of Cell Science

15 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs224980. doi:10.1242/jcs.224980 incubated in blocking solution (3% BSA, 0.5% gelatin, 0.1% Triton X-100 in Investigation: M.S., S.S., J.E.S., S.-H.L., S.-O.H.; Resources: M.S., S.S.; Data PBS) for 30 min. Blocked coverslips were then incubated with primary curation: M.S., S.S.; Writing - original draft: M.S., S.S., D.P.; Writing - review & antibodies diluted in blocking solution for 1 h at RT. After washing with PBS editing: J.E.S., S.-H.L.; Visualization: M.S., S.S.; Supervision: D.P.; Project containing 0.1% Triton X-100, the neurons were incubated with secondary administration: D.P.; Funding acquisition: D.P. antibodies for 45 min at RT. Coverslips were mounted with Vectashield Funding mounting medium (Vector Laboratories). Fluorescent images were acquired This work was supported by the Basic Science Research Program through the with a Zeiss Axiovert fluorescence microscope equipped with a 100×, 1.4 National Research Foundation of Korea [NRF-2016R1D1A1B03934362 and NA plan-apochromat objective lens and a Zeiss Axiocam HRm CCD NRF-2017R1A2B4006259]. camera. Quantification of various parameters of spine morphology was performed with ImageJ (NIH) software. Supplementary information Supplementary information available online at Analysis of neuronal morphology http://jcs.biologists.org/lookup/doi/10.1242/jcs.224980.supplemental For quantification of spine:shaft fluorescence intensity ratio, spine density and synapse formation percentage (%), neuron images were processed Reference equally by Adobe Photoshop. All quantifications were repeated, with at least Bagrodia, S., Taylor, S. J., Jordon, K. A., Van Aelst, L. and Cerione, R. A. (1998). – – A novel regulator of p21-activated kinases. J. Biol. Chem. 273, 23633-23636. three independent experiments, and 317 1317 spines per group in 116 405 Beaudoin, G. M. J., III, Lee, S.-H., Singh, D., Yuan, Y., Ng, Y.-G., Reichardt, L. F. primary, secondary and tertiary dendrites were randomly selected from and Arikkath, J. (2012). Culturing pyramidal neurons from the early postnatal 31–88 neurons. The fluorescence intensity ratio of spine:shaft, defined as mouse hippocampus and cortex. Nat. Protoc. 7, 1741-1754. ‘spine localization index’, was measured to assess spine accumulation of Bernards, A. and Settleman, J. (2004). GAP control: regulating the regulators of indicated proteins as previously described (Okuno et al., 2012): small GTPases. Trends Cell Biol. 14, 377-385. ́ Spine localization index=(GFP-spine/GFP-shaft)/(Dsred-spine/Dsred- Blanco-Suarez, E., Fiuza, M., Liu, X., Chakkarapani, E. and Hanley, J. G. (2014). Differential Tiam1/Rac1 activation in hippocampal and cortical neurons mediates shaft). differential spine shrinkage in response to oxygen/glucose deprivation. J. Cereb. Briefly, the images were analyzed by line-scan analyses of ImageJ Blood Flow Metab. 34, 1898-1906. software to obtain intensity plots. The intensity values were taken at the peak Bryan, B., Kumar, V., Stafford, L. J., Cai, Y., Wu, G. and Liu, M. (2004). GEFT, a of the green fluorescence intensity plot for GFP value and at the peak of the Rho family guanine nucleotide exchange factor, regulates neurite outgrowth and red fluorescence intensity plot for Dsred value. dendritic spine formation. J. Biol. Chem. 279, 45824-45832. Protrusions were classified into spines and filopodia as previously Calabrese, B., Wilson, M. S. and Halpain, S. (2006). Development and regulation described (Llano et al., 2015; Park et al., 2012; Zhang and Macara, 2006). of dendritic spine synapses. Physiology (Bethesda) 21, 38-47. Cingolani, L. A. and Goda, Y. (2008). Actin in action: the interplay between the actin Filopodia were defined as protrusions without a distinguishable head or with cytoskeleton and synaptic efficacy. Nat. Rev. Neurosci. 9, 344-356. a length of >3.5 µm, whereas spines were defined as protrusions with a head, Feige, J. N., Sage, D., Wahli, W., Desvergne, B. and Gelman, L. (2005). PixFRET, as determined by head/neck ratio >1.2. Spines were measured manually an ImageJ plug-in for FRET calculation that can accommodate variations in using ImageJ software. Synapses were defined as spines that colocalized with spectral bleed-throughs. Microsc. Res. Tech. 68, 51-58. a presynaptic marker, synaptophysin, or a postsynaptic marker, PSD-95. For Feng, Q., Baird, D., Peng, X., Wang, J., Ly, T., Guan, J.-L. and Cerione, R. A. the number of synapses, the synaptophysin-positive spines were counted. (2006). Cool-1 functions as an essential regulatory node for EGF receptor- and Src-mediated cell growth. Nat. Cell Biol. 8, 945-956. Feng, Q., Baird, D., Yoo, S., Antonyak, M. and Cerione, R. A. (2010). FRET Phosphorylation of the cool-1/beta-Pix protein serves as a regulatory signal for Cultured hippocampal neurons from neuron-specific βPix isoform KO mice the migration and invasive activity of Src-transformed cells. J. Biol. Chem. 285, were transfected with Raichu-Rac1 probe (Itoh et al., 2002) and Flag-tagged 18806-18816. βPix constructs at 7 DIV. At 19 DIV, neurons were fixed by adding 3.7% Fortin, D. A., Srivastava, T. and Soderling, T. R. (2012). Structural modulation of dendritic spines during synaptic plasticity. Neuroscientist 18, 326-341. paraformaldehyde for 10 min at RT and washed three times in PBS. Goslin, K., Asmussen, H. and Banker, G. (1998). Rat hippocampal neurons in low- Coverslips were mounted and then imaged using a Zeiss LSM 700 confocal density culture. In Culturing Nerve Cells (ed. G. Banker and K. Goslin), pp. laser-scanning microscope equipped with a 40×, 1.20 NA C-Apochromat 339-370. MA: MIT University Press: Cambridge. objective. Images were obtained with the following settings: FRET, Govek, E.-E., Newey, S. E. and Van Aelst, L. (2005). The role of the Rho GTPases excitation (Ex) 405 nm/emission (Em) 500–600 nm; CFP, Ex 405 nm/Em in neuronal development. Genes Dev. 19, 1-49. 420–490 nm; YFP, Ex 488 nm/Em 500–600 nm. After acquisition of a Harris, K. M. (1999). Structure, development, and plasticity of dendritic spines. Curr. Opin. Neurobiol. 9, 343-348. FRET image, intensity of the FRET signal was calculated using the ImageJ Hayashi, T., Umemori, H., Mishina, M. and Yamamoto, T. (1999). The AMPA plug-in PixFRET (NIH) (Feige et al., 2005). receptor interacts with and signals through the protein tyrosine kinase Lyn. Nature 397, 72-76. Statistics Hayashi, S., Lewis, P., Pevny, L. and McMahon, A. P. (2002). Efficient gene All statistical analyses were performed from at least three independent modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Mech. Dev. 119 Suppl. 1, S97-s101. experiments by Student’s t-test or one-way analysis of variance (ANOVA) t- Hering, H. and Sheng, M. (2001). Dendritic spines: structure, dynamics and followed by Bonferroni-corrected test using Origin 8.5 software regulation. Nat. Rev. Neurosci. 2, 880-888. (OriginLab Co., Northampton, MA). Actual P-values are indicated in the Itoh, R. E., Kurokawa, K., Ohba, Y., Yoshizaki, H., Mochizuki, N. and Matsuda, figure legends. All histograms representing western blots and morphometric M. (2002). Activation of rac and cdc42 video imaged by fluorescent resonance data in neurons are represented as mean±s.e.m. energy transfer-based single-molecule probes in the membrane of living cells. Mol. Cell. Biol. 22, 6582-6591. Kim, T. and Park, D. (2001). Molecular cloning and characterization of a novel Acknowledgements mouse betaPix isoform. Mol. Cells 11, 89-94. We thank Dr Heiner Westphal and Alex Grinberg, who were former members of Kim, S., Kim, T., Lee, D., Park, S. H., Kim, H. and Park, D. (2000). Molecular Mammalian Genes and Development, Eunice Kennedy Shriver National Institute of cloning of neuronally expressed mouse betaPix isoforms. Biochem. Biophys. Res. Child Health and Human Development, National Institutes of Health, Bethesda, Commun. 272, 721-725. USA, for advice and technical assistance for generating βPix KO mice. Kim, S., Lee, S. H. and Park, D. (2001). Leucine zipper-mediated homodimerization of the p21-activated kinase-interacting factor, beta Pix. Implication for a role in Competing interests cytoskeletal reorganization. J. Biol. Chem. 276, 10581-10584. Koch, C. and Zador, A. (1993). The function of dendritic spines: devices subserving The authors declare no competing or financial interests. biochemical rather than electrical compartmentalization. J. Neurosci. 13, 413-422. Koh, C. G., Manser, E., Zhao, Z. S., Ng, C. P. and Lim, L. (2001). Beta1PIX, the Author contributions PAK-interacting exchange factor, requires localization via a coiled-coil region to Conceptualization: M.S., S.S., D.P.; Methodology: M.S., S.S., D.P.; Validation: M.S., promote microvillus-like structures and membrane ruffles. J. Cell Sci. 114,

S.S., J.E.S., S.-H.L., S.-O.H.; Formal analysis: M.S., S.S., J.E.S., D.P.; 4239-4251. Journal of Cell Science

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