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The Novel Synaptogenic Protein Farp1 Links Postsynaptic Cytoskeletal Dynamics and Transsynaptic Organization

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The Novel Synaptogenic Protein Farp1 Links Postsynaptic Cytoskeletal Dynamics and Transsynaptic Organization Published December 3, 2012 JCB: Article The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization Lucas Cheadle and Thomas Biederer Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 ynaptic adhesion organizes synapses, yet the sig- (FERM) domain binds SynCAM 1, assembling a synaptic naling pathways that drive and integrate synapse complex. Farp1 increases synapse number and modulates S development remain incompletely understood. We spine morphology, and SynCAM 1 requires Farp1 for pro- screened for regulators of these processes by proteomically moting spines. In turn, SynCAM 1 loss reduces the ability of Downloaded from analyzing synaptic membranes lacking the synaptogenic Farp1 to elevate spine density. Mechanistically, Farp1 acti- adhesion molecule SynCAM 1. This identified FERM, Rho/ vates the GTPase Rac1 in spines downstream of SynCAM ArhGEF, and Pleckstrin domain protein 1 (Farp1) as strongly 1 clustering, and promotes F-actin assembly. Farp1 further- reduced in SynCAM 1 knockout mice. Farp1 regulates den- more triggers a retrograde signal regulating active zone dritic filopodial dynamics in immature neurons, indicating composition via SynCAM 1. These results reveal a postsyn- roles in synapse formation. Later in development, Farp1 aptic signaling pathway that engages transsynaptic inter- jcb.rupress.org is postsynaptic and its 4.1 protein/ezrin/radixin/moesin actions to coordinate synapse development. on December 10, 2012 Introduction Synapse formation in the brain involves concerted steps. reorganization contributes to the formation and structural Axons and dendrites of developing neurons interact through plasticity of spines (Bonhoeffer and Yuste, 2002). Regulators exploratory filopodia (Ziv and Smith, 1996; Fiala et al., 1998), of postsynaptic actin include members of the Rho GTPase and contact triggers cytoskeletal rearrangements, resulting in family—RhoA, Rac1, and Cdc42—that have distinct func- shorter and wider filopodia as stable synapses form (Hotulainen tions in modulating spine turnover and morphology (Tashiro and Hoogenraad, 2010). Adhesion molecules guide these stages, et al., 2000; Tada and Sheng, 2006). Cell surface interactions THE JOURNAL OF CELL BIOLOGY assembling into transsynaptic complexes to regulate synapse can activate theses GTPases, notably via Ephrin-B receptors number and morphology (Missler et al., 2012). These param- that bind guanine nucleotide exchange factors (GEFs) and, eters are critical for neuronal connectivity (Kasai et al., 2003; additionally, promote kinase signaling (Penzes et al., 2003; Chklovskii et al., 2004; Yuste, 2011). Moeller et al., 2006; Tolias et al., 2007). The actin cytoskeleton is prominent in dendritic spines, The understanding of synapse organization will benefit the postsynaptic specializations of mature excitatory synapses, from additional insight into the signaling pathways underly- and shapes these protrusions, anchors receptors, and partici- ing dendritic contact exploration and spine development. To pates in signaling (Okamoto et al., 2004; Frost et al., 2010). identify novel regulators of synapse formation, we focused on Spine actin is highly dynamic (Fischer et al., 1998), and its synaptic cell adhesion molecule 1 (SynCAM 1)-mediated syn- aptogenesis. SynCAM 1 (also known as Cadm1 and nectin-like 2 protein) is well-suited to study synaptic signaling because it Correspondence to Thomas Biederer: [email protected] Abbreviations used in this paper: DH, Dbl oncogene homology; div, days in vitro; Farp1, FERM, Rho/ArhGEF, and Pleckstrin domain protein 1; FERM, 4.1 © 2012 Cheadle and Biederer This article is distributed under the terms of an Attribution– protein/ezrin/radixin/moesin; GEF, guanine nucleotide exchange factor; GPI, Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- glycosylphosphatidylinositol; KO, knockout; Lat-A, Latrunculin A; P, postnatal lication date (see http://www.rupress.org/terms). After six months it is available under a day; PH, pleckstrin homology; PSD, postsynaptic density; SynCAM, synaptic cell Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, adhesion molecule; WT, wild type. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Supplemental Material can be found at: http://jcb.rupress.org/content/suppl/2012/11/29/jcb.201205041.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 199 No. 6 985–1001 www.jcb.org/cgi/doi/10.1083/jcb.201205041 JCB 985 Published December 3, 2012 first promotes excitatory synapse numbers and then acts in the KO mice by 79 ± 22% (P = 0.02; Fig. 1 B), validating its pro- mature brain to maintain this increase (Biederer et al., 2002; teomic identification. Fogel et al., 2007; Robbins et al., 2010). Further, it has an intra- Farp1 promotes dendrite outgrowth in motor neurons of the cellular motif predicted to interact with 4.1 protein/ezrin/ chick spinal cord (Zhuang et al., 2009). However, the expression, radixin/moesin (FERM) domains present in cytoskeletal regula- interactions, and functions of Farp1 protein in the mammalian tors (Biederer, 2006). central nervous system were unknown. Immunoblotting showed In an unbiased proteomic analysis of synaptic membranes that Farp1 is most prominent in the brain (Fig. 1 C) and is ex- from SynCAM 1 knockout (KO) mice, we have now identi- pressed across brain regions (Fig. 1 D). At postnatal day 13 (P13), fied FERM, Rho/ArhGEF, and Pleckstrin domain protein 1 when synapse formation in rat hippocampus is rapid (Fiala et al., (Farp1) as a novel synapse-organizing molecule that binds 1998), Farp1 was found in synaptosomes and enriched in synap- via its FERM domain to the cytosolic tail of SynCAM 1. Func- tic plasma membranes, cofractionating with SynCAM 1 (Fig. 1 E, tional studies revealed that Farp1 promotes the structural lanes 4 and 6). Farp1 was present in Triton X-100–resistant frac- dynamics of dendritic filopodia and their stability early in tions from synaptosomes, which is indicative of a cytoskeletal as- development. In mature neurons, Farp1 is enriched at post- sociation, and a small fraction of Farp1 cofractionated with synaptic sites and regulates the number of spines in dissociated postsynaptic densities (Fig. 1 F). neurons and organotypic slice culture. Notably, SynCAM 1 Our antibody detected Farp1 when it was overexpressed requires Farp1 to promote synapse formation, and the syn- in neurons (Fig. S1), but was not suitable for staining endog- aptogenic activity of Farp1 is reduced in absence of SynCAM 1. enous protein. To localize Farp1, we generated a full-length Downloaded from Biochemical assays and live imaging of an optical probe construct tagged at its amino terminus with GFP. In dissoci- demonstrate that Farp1 specifically binds the GTPase Rac1 ated cultures of rat hippocampal neurons at 15 d in vitro (div), and activates it in postsynaptic protrusions. In turn, Farp1 GFP-Farp1 was present in dendrites but not axons (Fig. S2). increases F-actin polymerization in spine heads. Moreover, GFP-Farp1 was enriched at 12 div in most dendritic protrusions SynCAM 1 and postsynaptic Farp1 signal retrogradely across positive for the excitatory postsynaptic protein Shank (Fig. 1 G). the synaptic cleft to modulate the composition of presyn- GFP-Farp1–containing protrusions were additionally marked aptic active zones. These results identify a novel signaling by the F-actin probe UtrCH-Cherry (utrophin calponin homol- jcb.rupress.org pathway that coordinates synaptic adhesion and pre- and post- ogy domain fused to Cherry; Burkel et al., 2007), which we synaptic organization. used as marker for spine heads, as they contain dense actin networks (Matus et al., 1982). These results show that Farp1 is a novel postsynaptic protein that is strongly reduced at syn- aptic membranes lacking SynCAM 1. Results on December 10, 2012 Proteomic identification of Farp1 SynCAM 1–Farp1 form a synaptic complex We performed a proteomic screen to compare the composition through a FERM domain interaction of synaptic membranes from forebrains of KO mice lacking In hippocampal neurons, GFP-Farp1 was enriched in spine SynCAM 1 (Robbins et al., 2010) versus wild-type (WT) litter- heads relative to the volume marker Cherry, and colocalized mates. This approach followed the rationale that intracellular with endogenous SynCAM 1 (Fig. 2, A and B). In support of synaptogenic signaling partners of SynCAM 1 may be recruited an interaction between SynCAM 1 and Farp1 at synapses and to or stabilized at synaptic membranes by this adhesion mol- consistent with the rationale of our proteomic screen, Farp1 ecule, resulting in lower levels of such partners at synapses coimmunoprecipitated with SynCAM 1 from synaptosomes lacking SynCAM 1. Isobaric tagging for relative and abso- (Fig. 2 C). The high degree of sequence similarity of the cyto- lute quantitation (iTRAQ) mass spectrometry identified 24 solic FERM domain–binding motifs of SynCAMs 1–4 (Biederer, proteins that increased above a 1.3-fold cutoff in SynCAM 1 2006) raised the possibility that Farp1 may bind more than one KO synaptic plasma membranes compared with WT. These family member. However, affinity chromatography of forebrain hits included neurexin 1, neuroligin 2, and EphA4, synapse- extracts on GST fusions of different SynCAM cytosolic tails organizing proteins that may be increased to compensate for showed that Farp1 only binds to SynCAM
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