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• W4309300949 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rat neuron culture and mouse brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date and defined novel classes of putative interactors, creating a framework for understanding gephyrin’s nonsynaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner. Editor's evaluation This article describes and validates new tools to study gephyrin biology in the brain, a critical regulator of synaptic inhibition and metabolism. The experiments are compelling, carefully controlled, and lead to a fundamental advance in neuroscience. This article will be of interest to a broad range of neuroscientists including those in synaptic, cellular, and circuit areas. https://doi.org/10.7554/eLife.80895.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Biological research has relied for decades on the accuracy and precision of specific antibodies to morphologically describe protein localization and dynamics, or to biochemically describe protein interaction partners, using techniques such as immunolabeling, immunoprecipitation, and immunoassays, among others. While antibody-based tools have been invaluable, for a given protein we often lack a variety of binders that perform excellently across applications. Antibodies that detect fixed proteins in tissue (which are typically partially denatured) may not bind with the same affinity or specificity to the same protein in a lysate (which may retain a more native confirmation). The heterogeneous quality of some commercial antibodies presents an additional challenge as the often ambiguous or unknown antibody sequence, provenance, and specificity of poly- and monoclonal antibodies alike lead to false information and ultimately a high additional cost to research (Bradbury and Plückthun, 2015; Smith, 2015). This problem is especially relevant for the study of synaptic proteins, be they receptors or scaffolds, as these proteins are often used as markers to define the presence, plasticity, and regulation of synapses as a strong correlate for synaptic function. For example, ionotropic glutamate receptor subunits and the scaffolding molecule PSD-95 are frequently used to define the excitatory postsynapse, while GABAA receptors (GABAARs) and the scaffolding protein gephyrin define the inhibitory postsynapse (Micheva et al., 2010). Gephyrin is a highly conserved signaling scaffold that oligomerizes into multimers and binds to cognate inhibitory synaptic proteins to functionally tether GABAARs at postsynaptic sites in apposition to presynaptic GABA release sites (Tyagarajan and Fritschy, 2014). Gephyrin is composed of three major domains: the N-terminal G domain and C-terminal E domain facilitate self-oligomerization of gephyrin underneath inhibitory postsynaptic sites, and they are linked together by the C domain, which is a substrate for diverse posttranslational modifications (Sander et al., 2013; Tyagarajan and Fritschy, 2014). Gephyrin mediates its scaffolding role by coordinating the retention of inhibitory synaptic molecules (Figure 1A), including GABAA and glycine receptors (GABAARs, GlyRs), collybistin, and neuroligin 2 through interactions at locations within the E domain or E/C domain interface (Choii and Ko, 2015; Tyagarajan and Fritschy, 2014), with additional protein interactors binding to the G and C domains. These protein interactions can act synergistically to enhance postsynaptic density assembly and alter gephyrin lattice compaction (Groeneweg et al., 2018). Therefore, via homo- and heterophilic protein–protein interactions, gephyrin can control inhibitory postsynaptic function. Figure 1 with 2 supplements see all Download asset Open asset In vitro selection and generation of anti-gephyrin DARPins. (A) Diagram of gephyrin function at the inhibitory postsynapse via its scaffolding role. (B) Gephyrin domain structure and location of key phosphoserine residues S268 and S270, the commonly used antibody clone for detection of gephyrin (Ab7a) is phospho-S270-specific. (C) The antibody Ab7a does not detect gephyrin clusters colocalized with the γ2 GABAA receptor subunit (GABRG2) in a phospho-null mouse model where S268 and S270 are mutated to alanines. (D) DARPins are an order of magnitude smaller than conventional antibodies and achieve target binding specificity by varying the sequence of ankyrin repeats (A.R.) with variable residues (magenta). (E) DARPin library design, with residues in magenta randomized in the original design and additional residues randomized in the caps (green). An N3C structure is shown with the N-terminal cap as a green ribbon and the C-terminal cap as a cyan ribbon with green side chains. (F) Schematic of anti-gephyrin DARPin selection and screening. (G) Structure of DARPin-FLAG clones used for initial validation experiments contain an N-terminal His8 tag and C-terminal FLAG tag for purification and detection, respectively. (H) Coomassie-stained gel of the nonbinding control (E3_5) and eight anti-gephyrin DARPin binders. Figure 1—source data 1 Raw image and annotated uncropped Coomassie gel from Figure 1H. https://cdn.elifesciences.org/articles/80895/elife-80895-fig1-data1-v3.pdf Download elife-80895-fig1-data1-v3.pdf Gephyrin’s scaffolding role is dynamically regulated by its post-translational modifications (PTMs). Gephyrin phosphorylation at several defined serine residues controls gephyrin oligomerization and compaction, thereby affecting GABAergic transmission (Battaglia et al., 2018; Ghosh et al., 2016; Petrini and Barberis, 2014; Zacchi et al., 2014). Two of these phosphosites, serines S268 and S270, are targeted by the kinases ERK1/2 and GSK3ß or cyclin-dependent kinases (CDKs), respectively, to downregulate gephyrin clustering (Figure 1B), thereby controlling postsynaptic strength (Tyagarajan et al., 2013). These phosphorylation events directly regulate gephyrin conformation via packing density changes to alter GABAA receptor dwell time (Battaglia et al., 2018), by altering gephyrin interacting partners (Zhou et al., 2021), or some combination of the two (Specht, 2019). Unfortunately, the most widely used anti-gephyrin antibody for identifying inhibitory postsynaptic sites, monoclonal antibody clone Ab7a, is sensitive to phosphorylation at serine 270 (Kalbouneh et al., 2014; Kuhse et al., 2012; Zhou et al., 2021), thus complicating interpretation of inhibitory postsynaptic presence, size, or dynamics. In addition to PTMs, gephyrin is regulated by alternative splicing by a suite of exonic splice cassette insertions (annotation outlined in Fritschy et al., 2008). While the principal (P1) isoform of gephyrin in neurons facilitates its synaptic scaffolding role, gephyrin is also a metabolic enzyme that participates in molybdenum cofactor (MOCO) biosynthesis (Nawrotzki et al., 2012; Schwarz and Mendel, 2006; Tyagarajan and Fritschy, 2014). MOCO synthesis can be mediated in non-neuronal cells by an isoform that includes the C3 splice cassette (Licatalosi et al., 2008; Meier et al., 2000; Smolinsky et al., 2008), suggesting that gephyrin harbors both isoform- and cell-type-specific functions. Gephyrin has been reported to complex with a wide variety of proteins as determined by both targeted and unbiased interaction studies (Fuhrmann et al., 2002; Sabatini et al., 1999; Uezu et al., 2016). These screens have implicated gephyrin in nonsynaptic processes, including regulation of mTOR signaling (Sabatini et al., 1999; Wuchter et al., 2012), and motor protein complexes (Fuhrmann et al., 2002). Furthermore, these interactomes have identified novel proteins such as InSyn1, with implications for understanding the heterogeneity of inhibitory synapse organization (Uezu et al., 2019). Still, the overlap in coverage of gephyrin’s interactome in each study has been variable with respect to identification of canonical inhibitory synaptic proteins due to limitations of each screening technique. Taken together, there is a need to generate and characterize molecular tools that can (1) interrogate gephyrin in different applications, (2) be functionally validated for the experiment in question, and (3) be diverse enough in their mode of interaction to not limit the different protein functional states that can be probed. Designed Ankyrin Repeat Proteins (DARPins) represent an attractive alternative tool compared to conventional antibodies as they are highly stable and specific synthetic protein binders that can be generated via high-throughput in vitro selection and screening (Binz et al., 2004; Kohl et al., 2003). Since they possess a defined genetic sequence, they can be adapted into diverse fusion constructs, and their structural stability facilitates their engineering to achieve differential binding (Harmansa and Affolter, 2018; Plückthun, 2015). DARPins are composed of a variable number (typically 2–3) ankyrin repeats containing randomized residues, flanked by N- and C-terminal capping repeats with a hydrophilic surface that shield the hydrophobic core. Each repeat forms a structural unit, which consists of a β-turn followed by two antiparallel α-helices and a loop reaching the turn of the next repeat. The randomized residues on adjacent repeats within the β-turn turns and on the surface of the α-helices form a variable and contiguous concave surface that mediates specific interactions with target proteins. Using a DARPin library with high diversity (~1012 unique DARPins), DARPins can be selected using ribosome display and then screened for particular binding characteristics (Dreier and Plückthun, 2012; Douthwaite and Jackson, 2012). Using this approach, DARPins have been shown to selectively bind to different conformations of proteins, including those brought about by phosphorylation (Kummer et al., 2012; Plückthun, 2015). Despite being used extensively as both experimental tools for structural biology as well as therapeutics (Plückthun, 2015; Tamaskovic et al., 2012), DARPins have not yet been applied to neuroscience research in the current literature. In order to generate a new toolset of anti-gephyrin binders, we screened a DARPin library for binding to different gephyrin phosphorylation mutants and characterized the resulting DARPins in both morphological and biochemical applications. We validated the use of anti-gephyrin DARPins to understand how different binders can reveal novel aspects of gephyrin and inhibitory synapse biology highlighting heterogeneity of inhibitory postsynapse morphology and composition. Results Generation and selection of anti-gephyrin DARPins Gephyrin clusters GABAA receptors and other inhibitory molecules such as neuroligin 2 and collybistin at postsynaptic sites (Figure 1A), where its clustering role is modified by phosphorylation, importantly at serines S268 and S270 (Figure 1B). This phosphorylation of gephyrin links upstream signaling (e.g., neurotrophic factors, activity) to downstream gephyrin regulation of inhibitory synaptic function (Groeneweg et al., 2018; Tyagarajan and Fritschy, 2014). The commonly used commercial antibody clone for morphological detection of synaptic gephyrin (clone Ab7a) has been employed extensively for almost four decades in the literature to identify inhibitory synapses (Pfeiffer et al., 1984). Though, rather than binding gephyrin regardless of its modified state, this antibody was recently demonstrated to specifically recognize gephyrin phosphorylated at serine S270 (Kuhse et al., 2012). This antibody’s specificity for phospho-gephyrin complicates interpretation of synaptic gephyrin cluster identification when using clone Ab7a and prevents accurate detection of postsynaptic gephyrin clusters when gephyrin S270 phosphorylation is low or blocked. This is illustrated by the lack of binding of Ab7a to gephyrin in brain tissue derived from a phospho-S268A/S270A phospho-mutant mouse line, in which serines S268 and S270 are mutated to alanines (Figure 1C). Therefore, to generate protein binders that can more robustly identify gephyrin independently of its phosphorylation status, we looked beyond antibody-based binders to (DARPins). DARPins are small (~12–15 kDa) compared to conventional antibodies (Figure 1D), and their binding to specific target proteins is mediated by several randomized residues contained within assemblies of 2–3 variable ankyrin repeats (AR) flanked by capping repeats (Binz et al., 2004). This basic DARPin structure creates a rigid concave shape with enhanced thermostability (Figure 1E). In addition, DARPins do not contain cysteines, allowing for functional cytoplasmic recombinant expression in Escherichia coli as well as cytoplasmic expression and functional studies in mammalian cells. We performed a ribosome display selection, followed by screening of individual clones against recombinant gephyrin (P1 principal isoform) containing either S268A/S270A or S268E/S270E mutations (Figure 1F), which mimic the respective dephosphorylated and phosphorylated state, thus representing functionally distinct gephyrin conformations (Battaglia et al., 2018; Tyagarajan et al., 2013). This allowed us to define sensitivity toward the modified state and widen the spectrum of DARPins obtained from the selection. Single DARPin clones were expressed in E. coli containing an N-terminal MRGS(H)8 (His8) tag and C-terminal FLAG tag (Figure 1G). Initial screening was performed with 376 DARPin clones using a high-throughput HTRF assay with crude extracts derived from 96-well expression plates. Of the initial hits, 32 were sequenced and 25 unique DARPins identified. These DARPins were further screened using an ELISA-based assay for relative binding to the phospho-null or phospho-mimetic gephyrin isoforms, or the absence of target as control (Figure 1—figure supplement 1). From this screen, eight DARPins were chosen for expression/purification and further analysis due to their high signal-to-background characteristics, as well as for equal binding to both phospho-mutant forms of gephyrin (Figure 1H, Figure 1—figure supplement 1). These eight DARPins showed diversity in the variable residues in the target protein interaction surface, highlighting the broad spectrum of binders that were obtained with this technology, and suggesting that they likely interact with gephyrin using different binding orientation or epitopes and independent of phosphorylation (Figure 1—figure supplement 2). Characterization of anti-gephyrin DARPins as morphological tools The antibody clone Ab7a has been used extensively to both define the location, size, and dynamics of postsynaptic gephyrin puncta (Bausen et al., 2010; Kalbouneh et al., 2014; Niwa et al., 2019). However, this antibody reacts preferentially with gephyrin phosphorylated at S270, and sometimes also labels nonspecific structures such as the nucleus (Figure 2A). Alternative anti-gephyrin antibodies exist such as clone 3B11, which can be used for immunoprecipitation of gephyrin and detection on immunoblots, but leads to high background when used to label synapses (Figure 2—figure supplement 1B). To determine whether anti-gephyrin DARPins function as antibody-like tools in synaptic staining (in addition to binding recombinant gephyrin in vitro), we compared FLAG-tagged anti-gephyrin DARPins against antibody clone Ab7a for staining in primary rat hippocampal neuron culture at 15 days in vitro (DIV) (Figure 2—figure supplement 1A–C). While the unselected control DARPin clone E3_5-FLAG (Binz et al., 2003) did not present with detectable signal (Figure 2A), DARPin-FLAG clones 27B3, 27D3, 27F3, and 27G2 labeled gephyrin puncta with high specificity (Figure 2A, Figure 2—figure supplement 1C). Clone 27D5-FLAG produced no detectable signal, and clones 27B5, 27H2, and 27G4 labeled gephyrin puncta but produced considerable background comparable to another commercial anti-gephyrin antibody (clone 3B11) (Figure 2—figure supplement 1A, B). Moreover, clones 27B3, 27D3, 27F3, and 27G2 colocalized with presynaptic vesicular GABA transporter (VGAT)-containing axon terminals (Figure 2B). We compared the fraction of detected gephyrin puncta colocalized with VGAT, as well as the size of detected gephyrin clusters, using both the antibody Ab7a and selected DARPin-FLAG clones that displayed low background, namely, 27B3, 27D3, 27F3, and 27G2 (Figure 2C and D). We found no differences between DARPin-FLAG 27B3 or 27G2 and Ab7a colocalization with VGAT, indicating equal functionality in morphological applications. DARPin-FLAG 27D3 and 27F3 labeled puncta of a smaller size, which could relate either to their affinity for synaptic gephyrin or heterogeneity in epitope accessibility as different postsynaptic gephyrin puncta may differ in their isoform or post-translationally modified state. Figure 2 with 1 supplement see all Download asset Open asset Anti-gephyrin DARPins specifically label gephyrin at inhibitory postsynaptic sites. Native gephyrin in fixed hippocampal neuron cultures (DIV15) probed using DARPin-FLAG clones, subsequently detected with anti-FLAG antibodies, and compared to staining with commercial anti-gephyrin antibody clone Ab7a. (A) Representative images of DARPin-FLAG clones 27B3, 27D3, 27F3, and 27G2 gephyrin puncta colocalized to Ab7a signal compared to the control DARPin E3_5. (B) Higher-magnification images of dendrite segments showing detected DARPin-FLAG signal colocalized with presynaptic VGAT. (C) Colocalization analysis indicating the fraction of gephyrin puncta that colocalize with VGAT along a proximal dendrite segment (>30 neurons/group pooled across three experiments). (D) Average puncta size identified by antibody Ab7a or DARPin-FLAG clones averaged by cell (pooled across neurons, >1100 synapses/group pooled across three experiment). Statistics: (C, D) one-way ANOVA, Tukey’s post-hoc test comparing all groups ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05. Figure 2—source data 1 Data and statistical analysis to generate the violin plot in Figure 2C and D. https://cdn.elifesciences.org/articles/80895/elife-80895-fig2-data1-v3.xlsx Download elife-80895-fig2-data1-v3.xlsx Anti-gephyrin DARPin-hFc fusion construct identifies phosphorylated and nonphosphorylated gephyrin clusters in mouse brain tissue Identification of inhibitory synapses often involves the co-labeling of both pre- and postsynaptic structures using multiple antibodies raised in different species. In order to label gephyrin clusters in the brain, we replaced the His8 and FLAG epitope tags from DARPin-FLAG clones 27B3, 27F3, 27G2, and the control clone E3_5 with an N-terminal human serum albumin (HSA) leader sequence and C-terminal human Fc (hFc) tag for mammalian recombinant production and purification and detection (Figure 3—figure supplement 1). The addition of the hFc tag allows for use in tandem with essentially all primary antibodies targeting synaptic markers raised in commonly used species such as rat, mouse, rabbit, goat, and guinea pig. Furthermore, it makes the construct bivalent. Consistently, DARPin-hFc 27G2 specifically labeled gephyrin puncta apposed to presynaptic VGAT terminals in both hippocampal neuron culture and mouse brain tissue (Figure 3—figure supplement 2). The specificity of this labeling could be confirmed by incubating DARPin-hFc 27G2 with a molar excess of recombinant gephyrin as a competitor, which led to the loss of immunofluorescent signal (Figure 3—figure supplement 3). A significant fraction of synaptic gephyrin clusters are phosphorylated at serine 270, and therefore lead to an uncertain interpretation when their size and dynamics are assessed using the phospho-specific antibody Ab7a (Kalbouneh et al., 2014; Specht, 2019; Zhou et al., 2021). As predicted, DARPins-hFc 27G2 can label gephyrin puncta in both wildtype and phospho-S268A/S270A mutant mouse tissue while the commercial pS270-specific antibody Ab7a does not (Figure 3A). Figure 3 with 5 supplements see all Download asset Open asset Phospho S270-insensitive DARPin-hFc 27G2 multiplexed with antibody Ab7a can assess synapse-specific gephyrin S270 phosphorylation. (A) Representative images of DARPin-hFc 27G2 (but not antibody Ab7a) labeling gephyrin puncta in both wildype (WT) and phospho-mutant gephyrin S268A/S270A mutant mouse brain tissue (somatosensory cortex layer 2/3). (B) Representative images from hippocampal neuron culture showing the relative Ab7a signal (indicating S270 phosphorylation) varies by synapse and between neurons. (C) Representative image showing DARPin-hFc 27G2 binding at synaptic puncta in primary hippocampal neuron culture is preserved after inhibition of CDKs following 24 hr treatment with 5 µM aminopurvalanol (PurvA) while Ab7a staining is severely reduced. (D) The relative fluorescence intensity at individual synapses (pooled from 30 neurons per group) showing a pronounced decrease in the average Ab7a/DARPin-hFc 27G2 intensity ratio. Quantification of Ab7a/DARPin-hFc 27G2 fluorescence signal averaged across cells pooled from three independent experiments, n = 30 cells/group. (E) Representative images of hippocampal neuron culture used for quantification of relative Ab7a/DARPin-hFc labeling of clusters on the soma, proximal dendrites, or the axon-initial segment (A.I.S.) (AnkG). (F) Ab7a/DARPin intensity ratio of individual synapses pooled from 45 cells over three independent experiments showing a decrease in A.I.S. cluster Ab7a staining. Lower: quantification indicates significantly reduced A.I.S. Ab7a labeling of clusters compared to dendritic or somatic compartments. Statistics: (D) one-way ANOVA; (F) repeated-measures one-way ANOVA. All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Mean and SD are presented. Figure 3—source data 1 Values and statistical results used to generate Figure 3D and F. https://cdn.elifesciences.org/articles/80895/elife-80895-fig3-data1-v3.xlsx Download elife-80895-fig3-data1-v3.xlsx The relative amount of Ab7a to anti-gephyrin DARPin signal could be used as a proxy to estimate relative gephyrin S270 phosphorylation at synapses. Indeed, we found that the Ab7a signal varied considerably both between adjacent synapses within a neuron and between neurons (Figure 3B, Figure 3—figure supplement 4). We confirmed the phosphosensitivity of this analysis method by inhibiting CDKs (upstream of gephyrin S270 phosphorylation) using 5 µM aminopurvalanol A applied for 24 hr. This treatment reduced Ab7a but not DARPin-hFc 27G2 signal as indicated by the decrease in the ratio between these two intensities seen both for individual synapses and when averaged by neuron (Figure 3C and D). We therefore examined the Ab7a/DARPin-hFc 27G2 intensity ratio between the somatic, dendritic, and axon-initial segment (A.I.S.) compartments in primary hippocampal neuron culture (Figure 3E and F), finding a significant reduction in Ab7a signal within the A.I.S. as defined by AnkyrinG immunolabeling (AnkG). Our results demonstrate that gephyrin phospho-S270 status varies between two neighboring clusters within a dendrite segment and also for the first time we can label gephyrin within the A.I.S. To test whether application of anti-gephyrin binders may affect the quantification of gephyrin clusters, we transfected hippocampal neuron cultures with EGFP-gephyrin plasmid and quantified cluster size along the principal dendrites of neurons using fluorescent signal from EGFP. This analysis demonstrated that compared to the untreated condition, application of either DARPin-hFc clones or antibody Ab7a does not influence the median size of EGFP gephyrin clusters in fixed tissue (Figure 3—figure supplement 5). DARPin-hFc 27G2 detects previously overlooked gephyrin clusters in brain tissue Antibody-based identification of gephyrin clusters in the brain is widely used to identify inhibitory synaptic sites, but current reagents may only capture a subset of synaptic gephyrin clusters, namely, those with gephyrin significantly phosphorylated at S270. Therefore, we extended our analysis of postsynaptic gephyrin clusters using DARPin-hFc 27G2 and the phospho-S270-specific antibody Ab7a to mouse brain tissue using the hippocampal CA1 area as a model. The hippocampus is organized in a layered structure, stratifying somatic from dendritic compartments, with compartment-specific GABAergic interneuron innervation patterns well described (Pelkey et al., 2017). We found lamina-specific variability in relative gephyrin phosphorylation at S270, which was significantly elevated in the stratum oriens and stratum lacunosum moleculare compared to other layers (stratum pyramidale and radiatum) (Figure 4A–C). Within the stratum pyramidale, we noticed a population of large, relatively hypophosphorylated clusters (Figure 4D, Figure 4—figure supplement 1) reminiscent of A.I.S. synapses (Figure 4E). Indeed, while DARPin-hFc 27G2 labels large gephyrin clusters apposed to presynaptic VGAT terminals, Ab7a reactivity within the A.I.S. is relatively weak (Figure 4F). These hypophosphorylated clusters colocalize with the α2 GABAA receptor subunit thought to be enriched at the A.I.S. (Lorenz-Guertin and Jacob, 2018) and span the length of the A.I.S. as defined by the marker AnkG. Therefore, DARPin-hFc 27G2 can better assess postsynaptic gephyrin at the A.I.S. and at synapses where gephyrin phosphorylation is low. These data indicate that gephyrin clusters on the A.I.S. have likely gone un- or underreported in the literature, which is meaningful when considering that threshold-based detection of gephyrin is used as a proxy for inhibitory synapse presence and function (Micheva et al., 2010; Schneider Gasser et al., 2006). Figure 4 with 1 supplement see all Download asset Open asset DARPin-hFc 27G2 labeling of gephyrin clusters demonstrates laminar and axon-initial segment (A.I.S.)-specific S270 phosphorylation and phosphorylation-dependent cluster size regulation. (A) Left: the relative Ab7a to DARPin-hFc 27G2 fluorescence intensity in the mouse hippocampus area CA1 shows layer-specific variability. Right: colorized gephyrin puncta indicating relative S270 phosphorylation as seen from hotter (more red/yellow) coloration. (B) Distribution of relative gephyrin phosphorylated at S270 (p270) at puncta between hippocampal lamina. Data pooled between six adult mice, three sections analyzed per mouse encompassing 14,000–47,000 gephyrin puncta per layer. (C) Analysis of the median relative gephyrin pS270 ratio between hippocampal layers (data pooled between sections per mouse, n = 6 mice quantified). (D) Example distribution of gephyrin pS270 signal by puncta size in the CA1 stratum pyramidale, with a population of large, hypophosphorylated clusters outlined. (E) Representative image of s. pyramidale with hot colors indicating gephyrin clusters with elevated phosphorylation; arrows indicate trains of large hypophosphorylated clusters. (F) Representative image showing large DARPin-identified gephyrin clusters apposed to presynaptic VGAT-containing terminals with corresponding low Ab7a antibody signal. (G) Representative image indicating gephyrin clusters on the A.I.S. (AnkG) colocalize with the α2 GABAA receptor subunit. (H) Representative images of gephyrin puncta identified using cluster analysis software in WT and S268A/S270A phospho-null mutant mice in the hippocampus using identical imaging parameters. (I) Violin plots indicating the distribution of gephyrin puncta sizes (14,000–47,000 puncta per group, pooled across 5–6 mice per group). (J) Analysis of the median puncta size between hippocampal layers and genotypes indicating larger gephyrin clusters in mutant mice. Statistics: (C) one-way ANOVA, (J) mixed-effects analysis comparing hippocampal lamina (horizontal bars) and genotypes (angled bars). All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Median and SD are presented. Figure 4—source data 1 Data and statistical analysis presented in Figure 4B–D, I, and J. https://cdn.elifesciences.org/articles/80895/elife-80895-fig4-data1-v3.xlsx Download elife-80895-fig4-data1-v3.xlsx While gephyrin phosphorylation at S268 and S270 is thought to reduce gephyrin cluster size (Tyagarajan et al., 2013), the phosphosensitivity of clone Ab7a has prevented our analysis of this relationship as this antibody does not react with dephosphorylated gephyrin (S270 phosphorylation is blocked in the mutant mouse). The" @default.
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• W4309300949 title "Author response: A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology" @default.
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• W4309300949 workType "peer-review" @default.