Research Articles: Cellular/Molecular Abl2:cortactin interactions regulate dendritic spine stability via control of a stable filamentous actin pool https://doi.org/10.1523/JNEUROSCI.2472-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.2472-20.2021 Received: 21 September 2020 Revised: 15 January 2021 Accepted: 16 February 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 the authors 1 Abl2:cortactin interactions regulate dendritic spine stability via control of a stable 2 filamentous actin pool. 3 4 Abbreviated title: Abl2 and cortactin regulate spine actin stability. a,d* a,b,c,* 5 Juliana E. Shawa, Michaela B. C. Kilanderd, Yu-Chih Lin and Anthony J. Koleske a b c 6 Departments of Molecular Biophysics and Biochemistry, Neuroscience, Interdepartmental 7 Neuroscience Program, Yale University, New Haven, CT 06520, U.S.A. 8 dProgram in Neuroscience, Hussman Institute for Autism, Baltimore, MD 21201, U.S.A. * 9 Co-corresponding authors: [email protected] and [email protected] 10 Number of Pages: 41 11 Number of Figures: 7 12 Number of Tables: 1 13 Number of multimedia: 0 14 Number 3D models: 0 15 Number of words for abstract: 220 words 16 Number of words for introduction: 652 words 17 Number of words for discussion: 1,655 words 18 19 Conflict of interest statement: The authors declare no competing financial interests. 20 21 Acknowledgements: We thank Aaron Levy, Josie Bircher, Melissa Carrizales and Amanda 22 Jeng for helpful comments on this manuscript, as well as other members of the Koleske lab for 23 their critical discussion and suggestions. This work was supported by NIH grants NS089662 24 (A.J.K.), NS105640 (A.J.K. and Michael J. Higley), MH115939 (A.J.K.), F31 MH116571 (J.E.S.), 25 and J.E.S. was supported on NIH training grant T32GM007223 (Susan J. Baserga). 26 1 27 Abstract 28 Dendritic spines act as the receptive contacts at most excitatory synapses. Spines are enriched 29 in a network of actin filaments comprised of two kinetically distinct pools. The majority of spine 30 actin is highly dynamic and regulates spine size, structural plasticity, and postsynaptic density 31 organization. The remainder of the spine actin network is more stable, but the function of this 32 minor actin population is not well understood, as tools to study it have not been available. 33 Previous work has shown that disruption of the Abl2/Arg nonreceptor tyrosine kinase in mice 34 compromises spine stability and size. Here, using cultured hippocampal neurons pooled from 35 both sexes of mice, we provide evidence that binding to cortactin tethers Abl2 in spines, where 36 Abl2 and cortactin maintain the small pool of stable actin required for dendritic spine stability. 37 Using fluorescence recovery after photobleaching of GFP-actin, we find that disruption of 38 Abl2:cortactin interactions eliminates stable actin filaments in dendritic spines, significantly 39 reducing spine density. A subset of spines remaining after Abl2 depletion retain their stable 40 actin pool and undergo activity-dependent spine enlargement, associated with increased 41 cortactin and GluN2B levels. Finally, tonic increases in synaptic activity rescue spine loss upon 42 Abl2 depletion by promoting cortactin enrichment in vulnerable spines. Together, our findings 43 strongly suggest Abl2:cortactin interactions promote spine stability by maintaining pools of 44 stable actin filaments in spines. 45 46 Significance Statement 47 Dendritic spines contain two kinetically distinct pools of actin. The more abundant, highly 48 dynamic pool regulates spine shape, size, and plasticity. The function of the smaller, stable 49 actin network is not well understood, as tools to study it have not been available. We 50 demonstrate here that Abl2 and its substrate and interaction partner, cortactin, are essential to 51 maintain the stable pool in spines. Depletion of the stable actin pool via disruption of Abl2 or 52 cortactin, or interactions between the proteins, significantly reduces spine stability. We also 2 53 provide evidence that tonic increases in synaptic activity promote spine stability via enrichment 54 of cortactin in spines, suggesting synaptic activity acts on the stable actin pool to stabilize 55 dendritic spines. 3 56 Introduction 57 Dendritic spines serve as receptive post-synaptic compartments on excitatory neurons 58 and defects in their formation, density and shape are hallmarks of many brain disorders (Fiala et 59 al., 2002; Penzes et al., 2011). Spine shape and stability are principally supported by a 60 filamentous actin network, which also organizes scaffolding proteins and neurotransmitter 61 receptors at the post- synaptic density (Fischer et al., 1998; Alvarez and Sabatini, 2007; 62 Schubert and Dotti, 2007; Lin and Webb, 2009; Hotulainen and Hoogenraad, 2010). Spine actin 63 filaments assemble into higher-order structures that undergo dynamic rearrangements and 64 provide the forces to drive changes in spine shape and size (Matus, 2000; Luo, 2002; Sekino et 65 al., 2007; Chazeau and Giannone, 2016). Indeed, molecules that regulate spine plasticity and 66 stability, including cell surface receptors and kinases, often converge on signaling pathways that 67 control polymerization, stability, and contractility of actin networks. Therefore, the cytoskeletal 68 machinery is a key module to regulate spine plasticity and stability. 69 Fluorescence recovery after photobleaching (FRAP) experiments of GFP-actin have 70 determined that most (80-85%) spine actin filaments are dynamic, undergoing ongoing 71 polymerization and turnover within 10s of seconds, while the remainder of actin is more stable. 72 These distinct kinetic pools of actin likely play unique roles in the spine. Dynamic filaments 73 mediate a net retrograde flow of actin from the spine periphery toward the spine core and are 74 perfectly positioned to promote changes in spine size and shape (Honkura et al., 2008; Frost et 75 al., 2010) and also regulate neurotransmitter receptor localization or gating properties 76 (Rosenmund and Westbrook, 1993; Borgdorff and Choquet, 2002; Kerr and Blanpied, 2012). 77 Photoactivation of mEOS3.2-actin reveals that the small, stable pool of actin is enriched at the 78 spine base and turns over much more slowly (t ~17 minutes)(Honkura et al., 2008). The relative 79 proportion of stable actin increases during synapse maturation (Koskinen et al., 2014) and may 80 serve as a base for polymerization of new actin during spine enlargement (Mikhaylova et al., 4 81 2018). The functions of the stable actin pool have been largely understudied, primarily because 82 we lack an in-depth understanding of the molecules and mechanisms that regulate it. 83 The Abl2/Arg nonreceptor tyrosine kinase interacts with its substrate and binding partner 84 cortactin to regulate actin-based structures in many cellular contexts (MacGrath and Koleske, 85 2012a; Schnoor et al., 2018). Both proteins localize to dendritic spines where they promote 86 spine stability (Hering and Sheng, 2003; Iki et al., 2005; Sfakianos et al., 2007; Lin et al., 2013; 87 MacGillavry et al., 2016; Mikhaylova et al., 2018). Abl2 binds actin filaments cooperatively 88 (Wang et al., 2001) and increases the binding stoichiometry of cortactin for actin filaments 89 (MacGrath and Koleske, 2012b). These interactions significantly impact actin filament stability – 90 at saturated binding, Abl2 and cortactin each stabilize actin filaments (Courtemanche et al., 91 2015; Scherer et al., 2018), but mixtures of Abl2 and cortactin at concentrations far below 92 saturated binding also stabilize actin filaments (MacGrath and Koleske, 2012b; Courtemanche 93 et al., 2015). While Abl2 and cortactin synergize to stabilize actin filaments in vitro, it is unclear 94 whether or how they contribute to actin stability in neurons or how this mechanism might impact 95 dendritic spine shape or stability. 96 Here, we show that cortactin anchors Abl2 in dendritic spines via an 97 SH2:phosphotyrosine interface, and mutational disruption of this interface dislodges Abl2 from 98 spines. Depletion of either protein eliminates the stable actin pool in spines, as measured by 99 FRAP of GFP-actin. Disruption of this stable pool correlates with a significant loss of dendritic 100 spines. We find that a subset of spines remaining after Abl2 depletion exhibit increased spine 101 head widths, which is tightly associated with increased cortactin and GluN2B levels, and 102 retention of the stable actin pool. Finally, tonic increases in activity can rescue spine loss upon 103 Abl2 depletion by promoting cortactin enrichment in vulnerable spines that are otherwise lost in 104 basal conditions. Together, our data indicate that Abl2 and cortactin synergize to maintain 105 dendritic spine stable actin, which is critical for spine stability. 5 106 Materials and Methods: 107 Animals 108 All animal procedures were compliant with federal regulations and approved by the Animal Care 109 and Use Committees at Yale University, University of Maryland at Baltimore and Hussman 110 Institute for Autism. Balb/c mice were purchased from Charles River Laboratories. Animals were 111 housed and cared for in a Yale-sponsored OPRR approved animal facility. Neonatal mice of 112 both sexes were sacrificed for neuronal culture preparation. 113 114 Cell culture and transfections 115 HEK293 cells were plated in 6-well plates and maintained in high glucose DMEM (Invitrogen) 116 growth media supplemented with 1% pen/strep (Gibco), 2 mM L-glutamine (Gibco), and 10% 117 fetal bovine serum (Sigma-Aldrich). Primary neuronal cultures were prepared from postnatal day 118 1 mouse hippocampus as previously described (Lin et al., 2013). Neurons were maintained in 119 serum free media containing 1% pen/strep, 2 mM L-glutamine, and 2% B27-supplement (Gibco) 120 in Neurobasal media (NB-SFM) (Invitrogen).