Cytolinker Gas2L1 regulates axon morphology through microtubule‐modulated actin stabilization
2019; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês
10.15252/embr.201947732
ISSN1469-3178
AutoresDieudonnée van de Willige, J.J.A. Hummel, Celine Alkemade, Olga I. Kahn, Franco K.C. Au, Robert Z. Qi, Marileen Dogterom, Gijsje H. Koenderink, Casper C. Hoogenraad, Anna Akhmanova,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoArticle5 September 2019Open Access Transparent process Cytolinker Gas2L1 regulates axon morphology through microtubule-modulated actin stabilization Dieudonnée van de Willige Dieudonnée van de Willige Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jessica JA Hummel Jessica JA Hummel Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Celine Alkemade Celine Alkemade Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Living Matter Department, AMOLF, Amsterdam, The Netherlands Search for more papers by this author Olga I Kahn Olga I Kahn Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Franco KC Au Franco KC Au Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China Search for more papers by this author Robert Z Qi Robert Z Qi Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China Search for more papers by this author Marileen Dogterom Marileen Dogterom Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Gijsje H Koenderink Corresponding Author Gijsje H Koenderink [email protected] orcid.org/0000-0002-7823-8807 Living Matter Department, AMOLF, Amsterdam, The Netherlands Search for more papers by this author Casper C Hoogenraad Corresponding Author Casper C Hoogenraad [email protected] orcid.org/0000-0002-2666-0758 Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Anna Akhmanova Corresponding Author Anna Akhmanova [email protected] orcid.org/0000-0002-9048-8614 Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Dieudonnée van de Willige Dieudonnée van de Willige Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Jessica JA Hummel Jessica JA Hummel Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Celine Alkemade Celine Alkemade Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Living Matter Department, AMOLF, Amsterdam, The Netherlands Search for more papers by this author Olga I Kahn Olga I Kahn Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Franco KC Au Franco KC Au Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China Search for more papers by this author Robert Z Qi Robert Z Qi Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China Search for more papers by this author Marileen Dogterom Marileen Dogterom Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Gijsje H Koenderink Corresponding Author Gijsje H Koenderink [email protected] orcid.org/0000-0002-7823-8807 Living Matter Department, AMOLF, Amsterdam, The Netherlands Search for more papers by this author Casper C Hoogenraad Corresponding Author Casper C Hoogenraad [email protected] orcid.org/0000-0002-2666-0758 Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Anna Akhmanova Corresponding Author Anna Akhmanova [email protected] orcid.org/0000-0002-9048-8614 Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Author Information Dieudonnée Willige1,‡, Jessica JA Hummel1,‡, Celine Alkemade2,3,‡, Olga I Kahn1, Franco KC Au4, Robert Z Qi4, Marileen Dogterom2, Gijsje H Koenderink *,3,5, Casper C Hoogenraad *,1 and Anna Akhmanova *,1 1Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands 2Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands 3Living Matter Department, AMOLF, Amsterdam, The Netherlands 4Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China 5Present address: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands ‡These authors contributed equally to this work *Corresponding author. Tel: +31 015 27 89806; E-mail: [email protected] *Corresponding author. Tel: +31 30 2534585; E-mail: [email protected] *Corresponding author. Tel: +31 302532328; E-mail: [email protected] EMBO Reports (2019)20:e47732https://doi.org/10.15252/embr.201947732 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can directly bind to actin, microtubules and microtubule plus-end-tracking end binding proteins. We found that Gas2L1 promotes axon branching, but restricts axon elongation in cultured rat hippocampal neurons. Using pull-down experiments and in vitro reconstitution assays, in which purified Gas2L1 was combined with actin and dynamic microtubules, we demonstrated that Gas2L1 is autoinhibited. This autoinhibition is relieved by simultaneous binding to actin filaments and microtubules. In neurons, Gas2L1 primarily localizes to the actin cytoskeleton and functions as an actin stabilizer. The microtubule-binding tail region of Gas2L1 directs its actin-stabilizing activity towards the axon. We propose that Gas2L1 acts as an actin regulator, the function of which is spatially modulated by microtubules. Synopsis The actin-microtubule crosslinking protein Gas2L1 stimulates axon branching and reduces axon outgrowth through microtubule-modulated actin stabilization. Cytolinker Gas2L1 stabilizes actin, stimulates axon branching and reduces axon outgrowth in rat primary hippocampal neurons. Gas2L1 is autoinhibited by an interaction between its actin- and microtubule-binding domains. In vitro, autoinhibition of Gas2L1 is relieved by reciprocal binding to actin filaments and microtubules. The microtubule-binding tail region of Gas2L1 targets its actin-stabilizing activity towards the axon. Introduction The cytoskeleton is a key player in cellular morphogenesis, as it provides cells with structural support and acts as a scaffold for organelle positioning. An example of a process where cytoskeletal filaments play intricate roles is neuronal development. Neurons have complex morphologies that allow them to form elaborate networks and propagate signals in the brain. Developing neurons undergo extensive cell shape changes, which are coordinated by guidance cues relayed to the actin and microtubule (MT) cytoskeletons (reviewed in Ref. 1). In particular, the crosstalk between MTs and actin plays an essential role during axon maturation (reviewed in Refs 2-4). At the tips of axonal processes, specialized structures called growth cones determine the direction and rate of axon advance. Growth cones contain a central dynamic MT array which probes the actin-rich periphery. Axon outgrowth is preceded by MT stabilization in filopodia at the tip of the growth cone, and conversely, repellent cues restrict peripheral MT entry. In a similar process, axon branching is believed to start with the formation of an actin patch along the axon, either de novo or as a remnant of a pausing growth cone (reviewed in Refs 5, 6). At the site of branch formation, newly generated dynamic MT plus ends are stabilized on the actin patch to initiate a new branch. Actin-MT crosslinking proteins, also referred to as cytolinkers, are obvious candidates to regulate cytoskeletal crosstalk during axon development (reviewed in Ref. 7). Most studies in this context have focussed on spectraplakins, a family of large proteins, which directly bind both to actin filaments and to MT shafts and indirectly associate with growing MT plus ends through end binding (EB) proteins 8-11. The actin-MT crosslinking abilities of ACF7 (a spectraplakin otherwise known as MACF1) and its Drosophila ortholog Short stop (Shot) are necessary for axon extension 12, 13. Other MT plus end-associated proteins, such as CLASP and APC, participate in regulating axon outgrowth, possibly also by coordinating actin-MT coupling 14, 15. Gas2L1 (growth arrest-specific 2-like 1) is a much smaller cytolinker with a domain composition similar to ACF7, but its role is less well understood. Like ACF7, Gas2L1 contains an N-terminal actin-binding calponin homology (CH) domain, a MT lattice-binding Gas2-related (GAR) domain and a C-terminal SxIP motif, which mediates the interaction with MT plus ends via EB proteins 16-18. The actin-MT crosslinking abilities of Gas2L1 and its Drosophila ortholog Pigs (Pickled eggs) have been previously demonstrated in cells 18, 19. So far, Gas2L1 was shown to regulate the distance between centrioles in cycling cells 20, and Pigs was identified as a cytoskeletal target of Notch signalling, which participates in Drosophila wing muscle development and oogenesis 21. However, Gas2L1 has not been studied in the context of neuronal development, although Gas2L1 mRNA is abundant in mammalian brain tissue 16. Here, we reveal that Gas2L1 participates in regulating axon outgrowth and branching in developing mammalian neurons. By combining data obtained in primary rat hippocampal neurons, in vitro reconstitution assays and biochemical experiments, we show that Gas2L1 is autoinhibited and requires the simultaneous binding of both actin filaments and MTs to fully relieve this autoinhibition. Our data suggest that Gas2L1 locally regulates the actin cytoskeleton during axon maturation by stabilizing actin in response to MT binding. Gas2L1 hereby promotes axon branching while tempering axon extension. Results Gas2L1 affects axon branching and outgrowth in developing neurons To determine whether Gas2L1 plays a role in neuronal development, we examined the effects of Gas2L1 depletion and overexpression on axon development in dissociated primary rat hippocampal neurons. Depletion resulted in a ~ 64% reduction of Gas2L1 mRNA as determined by qPCR, for which day in vitro (DIV) 0 neurons were electroporated with the empty vector or Gas2L1 shRNA-encoding plasmids and subjected to puromycin selection before mRNA isolation at DIV3 (Figs 1A, and EV1A and B). Figure 1. Gas2L1 balances axon outgrowth and branching in developing neurons A. qPCR experiments showing the decrease in Gas2L1 (G2L1) mRNA levels upon DIV0-DIV3 shRNA treatment of primary rat hippocampal neurons (A; n = 3 biological replicates, black crosses represent individual data points). Electroporated neurons were subjected to 48-h puromycin selection prior to mRNA isolation. B. Silhouettes (composite images from β-galactosidase fill, top panels) of DIV3 neurons treated with scrambled (Scr) or G2L1 shRNA and co-expressing HA-β-galactosidase, or overexpressing HA-G2L1 and HA-β-galactosidase for 3 days. Red asterisks indicate the position of the soma. Bottom panels show fill (red) combined with TRIM46 staining (green) of the neurons shown in the panels above. C–F. Quantifications of total axon length (C), primary axon length (D), average (non-primary) branch length (E) and the number of branches per 100 μm axon (F) for DIV3 neurons treated as described in (B). Vector = empty pSuper shRNA vector co-expressing HA-β-galactosidase. n = 50 neurons per condition from three independent experiments. G. Quantification of the growth cone area in DIV4 neurons transfected with GW1-EV (empty GW1 vector), G2L1 shRNA or HA-G2L1 and HA-β-galactosidase for 1 day. n = 130–159 growth cones from 37 to 43 neurons per condition (159 growth cones from 43 neurons for GW1-EV, 130 growth cones from 37 neurons for G2L1 shRNA, 142 growth cones from 41 neurons for HA-G2L1) from three independent experiments. H, I. Rescue experiments showing the number of branches per 100 μm axon in neurons co-expressing scrambled or G2L1 shRNA with GFP, or G2L1 shRNA with GFP-G2L1, and HA-β-galactosidase from DIV0 to DIV3. Silhouettes (composite images from β-galactosidase fill) are shown in (H). Red asterisks indicate the position of the soma. n = 34–43 neurons per condition (36 for Scr shRNA, 34 for G2L1 shRNA, 43 for G2L1 shRNA + GFP-G2L1) from two independent experiments. J. Localization of GFP-Gas2L1 (G2L1) in a DIV3 neuron stained for fascin (α-fascin), as well as merged images showing co-localization between GFP-Gas2L1 and fascin (left panels). Boxes indicate zoomed regions (right panels). Data information: Scale bars: 30 μm in (B, H, J). Data are displayed as means ± SEM. Mann–Whitney test, ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Experimental details of neuronal phenotype validation A, B. Representative melt curves of various samples amplified with Gas2L1 primers (A) and amplification curves of GAPDH or Gas2L1 cDNA amplified in control and shRNA samples (B) from qPCR experiments shown in Fig 1A. cDNA samples of control (empty shRNA vector, in duplo) and Gas2L1-shRNA-expressing neurons (G2L1 shRNA, in duplo) show one peak in the melt curve (A), demonstrating specific amplification of Gas2L1 cDNA; negative water control (MQ instead of cDNA template) shows no amplification with Gas2L1-specific primers; negative RNA control (RNA instead of cDNA as template) reveals minor unspecific product that is not amplified and therefore not interfering in the presence of cDNA. C. Fine details of axon morphology of a DIV3 neuron overexpressing HA-Gas2L1 as described in Fig 1B. Boxed regions (1 and 2) are enlarged below. Red asterisk indicates the soma. D. Example of axon tracings for morphology analysis, using the DIV3 neuron expressing scrambled shRNA as shown in Fig 1B as a template. Boxed region is enlarged to the right. Blue tracing denotes the primary axon; the longest possible uninterrupted tracing from the soma to the tip of an axon branch. Red tracings denote non-primary branches and black circle marks the position of the soma. E–G. Rescue experiments showing the total axon length (E), primary axon length (F) and average axon branch length (G) in neurons co-expressing scrambled (Scr) or Gas2L1 shRNA with GFP, or Gas2L1 shRNA with GFP-Gas2L1, and HA-β-galactosidase from DIV0 to DIV3. Data belong to the experiment shown in Fig 1H and I. n = 34–43 neurons per condition (36 for Scr shRNA, 34 for G2L1 shRNA, 43 for G2L1 shRNA + GFP-G2L1) from two independent experiments. H, I. DIV3 neurons overexpressing GFP-G2L1 stained for cortactin (H) or p34-Arc (I), as well as merged images showing co-localization between GFP-Gas2L1 and cortactin (H) or p34-Arc (I) (left panels). Boxes indicate zoomed regions (right panels). Data information: Scale bars: 25 μm in (C), 30 μm in (D, H, I). Data are displayed as means ± SEM. Mann–Whitney test, ns: not significant, ***P < 0.001. Download figure Download PowerPoint The depletion of Gas2L1 from DIV0 to DIV3 resulted in less complex axons (Fig 1B) characterized by an increased axon branch length and reduced branch density (the number of branches per unit of axon length; Fig 1C–F). We note that in this set of experiments, total axon length was slightly reduced, whereas other experiments showed no significant difference (Figs EV1E and 5G). The effects of Gas2L1 depletion on neuronal morphology could be rescued by co-expressing low levels of Gas2L1 (Figs 1H and I, and EV1E–G), indicating that this phenotype is specific. Importantly, neuronal polarity was not affected by the loss of Gas2L1, as evidenced by staining of the axon initial segment marker TRIM46 in neurons depleted of Gas2L1 (Fig 1B) 22. When Gas2L1 was overexpressed from DIV0 to DIV3, the length of the primary axon and axonal branches was decreased, whereas the branch density was increased (Fig 1C–F). These axon maturation phenotypes were opposite to those seen after Gas2L1 depletion (Fig 1C–F). Gas2L1 overexpression also led to enlargement of axonal growth cones (Fig 1G). We also noted the development of excessive filopodia (Fig EV1C), as evidenced by their co-localization with a filopodia marker fascin (Fig 1J). In addition, Gas2L1 neurons formed structures that resembled lamellipodia (Fig EV1C) and displayed some punctate staining with the lamellipodia markers cortactin and p34-Arc/ARPC2 (Fig EV1H and I). We note that these features were beyond the level of detail included in our morphological analyses, which were based on axon tracings (Fig EV1D). Despite the obvious morphological defects induced by overexpression, axons of Gas2L1-overexpressing neurons were still polarized, as TRIM46 staining appeared normal in these neurons (Fig 1B). We conclude that Gas2L1 stimulates formation of new axon branches but restricts their elongation. The latter effect is different from that of ACF7, a structurally related cytolinker that promotes both axon outgrowth and branching 12, 13. The ability of a protein to interact with both actin filaments and MTs can thus be associated with distinct neurodevelopmental functions. Gas2L1 specifically localizes to actin-MT overlaps in in vitro reconstitution assays To better understand how Gas2L1 mediates actin-MT crosstalk, we purified full-length Gas2L1, its actin-binding CH domain and its MT-binding C-terminal fragment, which was termed Tail (Figs 2A and EV2A). We studied the behaviour of these proteins in an in vitro reconstitution assay, which included dynamic MTs and actin filaments stabilized with phalloidin 23. We immobilized stable MT seeds on a glass surface, allowing dynamic MTs to grow in a solution containing tubulin dimers and unattached free-moving actin filaments (Fig 2B). Figure 2. In vitro reconstitution of the interaction of Gas2L1 with actin filaments and MTs A. Schematic depiction of the domain structure of Gas2L1 (G2L1) and Gas2L1 mutants used in this study. B. Schematic depiction of in vitro TIRF assays. Stable MT seeds are attached to the glass surface, from which dynamic MTs grow by addition of tubulin dimers (either with or without EB3 present). Actin filaments are free to move around. G2L1 links F-actin to MTs. C. In vitro reconstitution of full-length Gas2L1 in the presence of actin (0.5 μM) only. D. In vitro reconstitution of the Gas2L1 CH domain in the presence of actin (0.5 μM) only. The intensity range between the F-actin panels and Gas2L1/CH panels of (C) and (D) are equal. E. In vitro reconstitution of full-length Gas2L1 in the presence of MTs only. F. In vitro reconstitution of the Gas2L1 Tail domain in the presence of MTs only. G. In vitro reconstitution of full-length Gas2L1 in a composite assay with both actin (10 nM) and MTs. Gas2L1 only localizes at MT-actin overlaps. Gas2L1 accumulation (yellow arrows) occurs after an actin filament "lands" (blue arrows) and zippers (purple arrows) onto a MT. H. In vitro reconstitution of full-length Gas2L1 in a composite assay as in (G), but with higher actin concentration (1 μM). I. Still frames showing the dynamics of a Gas2L1-F-actin-MT bundle, from a composite in vitro reconstitution assay as described in (H). The red line indicates the (stable) MT seed. J. Kymographs showing the dynamics of the Gas2L1-F-actin-MT bundle seen in (I). K. Still frames taken from Movie EV1 at the indicated timestamps, showing the dispersion of actin filaments (yellow arrowheads) after Gas2L1 and the MT disappear from the Gas2L1-F-actin-MT bundle and renewed actin bundling and Gas2L1 accumulation upon MT regrowth. Experimental conditions are as described for (H). L. Model of Gas2L1 autoinhibition. Data information: Scale bars: 10 μm (C–F, H, I), 5 μm (G, K). For (J), vertical scale bar: 3 min, horizontal scale bar 5 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. In vitro reconstitution assays showing the influence of EB3 on the interaction of Gas2L1 with MTs and the relation between Gas2L1 and actin A. Coomassie-stained SDS–PAGE gels showing the purity of the purified Gas2L1 (G2L1) fusions used for in vitro reconstitutions. For GFP-CH, the additional band at 40 kDa was identified as co-purified actin by Western blotting. B. Intensity ratios of GFP signal over actin signal from Fig 2C (orange) and 2D (green). "Empty" (dark red) is from the channel with only F-actin and no GFP-protein present. This analysis reveals that there is no GFP-Gas2L1 (orange) accumulated on F-actin, since it has nearly the same intensity ratio as the empty channel (dark red). We do observe GFP-CH (green) binding to F-actin. TIRF images were captured on the same day using identical microscope settings. C, D. Kymographs of Gas2L1 (C) and Tail (D) from an in vitro reconstitution assay with MTs and EB3. Only the Tail fragment behaves as a plus-end tracking protein. E–G. TIRF images (E) and kymographs (F, G) showing specific Gas2L1 localization to MT-actin overlaps and absence of plus-end tracking in an in vitro reconstitution assay with Gas2L1, F-actin (1 μM), MTs and EB3. These data indicate that EB3 does not influence the localization of Gas2L1 in this system, even when EB3 tracks growing MT plus ends (G). H. Control in vitro reconstitution experiment showing no alignment of F-actin (1 μM) to MTs in the absence of Gas2L1. I. In vitro reconstitution experiments with F-actin (1 μM) without Gas2L1 (top) or with Gas2L1 added (bottom) observed at different time intervals after mixing. Gas2L1 enhances actin bundling and localizes to these bundles. No actin bundles appear at 10 min after flushing in the F-actin mix in the absence of Gas2L1 (top, middle panel), while a few Gas2L1-decorated bundles can be observed right after flushing in the mix in the presence of Gas2L1 (left, bottom panels). Over time, the number of bundles increases as a result of the densification of actin filaments due to the presence of methyl cellulose. Data information: Scale bars: 10 μm except for (C) and (F). For (C) and (F), horizontal scale bars 5 μm. All vertical (time) scale bars: 3 min, except for (G). For (G), vertical scale bar 5 min. Download figure Download PowerPoint As the first step, we tested the behaviour of Gas2L1 and mutants in the presence of dynamic MTs or stabilized actin filaments alone. Full-length Gas2L1 did not bind to single actin filaments in the absence of MTs (Fig 2C), whereas the CH domain did (Figs 2D and EV2B). Similarly, in an assay with MTs alone, full-length Gas2L1 did not bind MTs (Fig 2E). By contrast, the Tail domain was able to bind MTs in the absence of actin filaments (Fig 2F). Moreover, Gas2L1 did not track growing MT plus ends in the presence of EB3, whereas the Tail fragment did (Fig EV2C and D). These results indicate that under the tested conditions, individual domains of Gas2L1 are capable of binding actin filaments, the MT lattice and MT plus ends, but the full-length protein is not. Surprisingly, when we added Gas2L1 to a composite assay with both MTs and low concentrations of actin filaments, we observed that Gas2L1 exclusively accumulated at the sites where actin filaments and MTs overlapped (Fig 2G). Specific accumulation of Gas2L1 on actin-MT overlaps was especially obvious from events during which an actin filament gradually aligned with a growing MT: the appearance of the Gas2L1 signal along the MT coincided with the zippering of the unbound part of an actin filament along the MT (Fig 2G). Similar zippering of actin filaments and MTs was observed previously with an engineered cytolinker containing a CH domain targeting actin and an SxIP motif targeting EB proteins 24. In a composite assay where more actin filaments were available for binding, we observed MTs covered with multiple co-aligned actin filaments (Fig 2H), whereas no MT-actin co-alignment occurred without Gas2L1 (Fig EV2H). Time lapse analysis of actin-MT-Gas2L1 bundles revealed that MTs inside these bundles remained dynamic (Fig 2I and J). When a catastrophe occurred and the MT shrunk back, Gas2L1 did not remain bound to actin, but disappeared together with the MT (Fig 2I and J), and the actin filaments that were initially co-aligned with the MT dispersed (Movie EV1, Fig 2K). Once the MT repolymerized, the accumulation of Gas2L1 and actin along its shaft was restored (Fig 2K). The addition of EB3 to the assay did not alter Gas2L1-induced MT-actin co-alignment and did not induce any enrichment of Gas2L1 at growing MT plus ends, in spite of the fact that EB3 was able to track MT plus ends in the same assay (Fig EV2E–G). These data confirm that Gas2L1 does not behave as a canonical plus-end-tracking protein although it does contain an EB-binding SxIP motif. Our results confirm that Gas2L1 can crosslink actin filaments and MTs. However, while individual fragments of Gas2L1 are able to bind MTs or actin filaments, full-length Gas2L1 does not localize to either of these separate cytoskeletal components in vitro. Instead, Gas2L1 binds to MTs and actin filaments simultaneously, suggesting that the protein might be autoinhibited and that the interaction with both actin and MTs is required to relieve autoinhibition. Of note, Gas2L1 did not bind independently to MTs regardless of incubation time. However, over time Gas2L1 accumulated on slowly forming actin bundles (but never individual filaments) in the absence of MTs (Fig EV2I). Since there appears to be some interaction between Gas2L1 and actin in the absence of MTs, we propose that the binding of Gas2L1 to actin acts as the first step towards initiating actin-MT crosslinking (Fig 2L). Autoinhibition of Gas2L1 is a result of the interaction between the CH domain and MT-binding tail If Gas2L1 is indeed autoinhibited, its MT-binding tail should directly compete with actin for binding its CH domain. A similar intramolecular interaction was reported for Shot: its N-terminal tandem CH domains interact with the C-terminal EF-hand/GAR region 25. We mapped potential intramolecular interactions of Gas2L1 by testing whether its different deletion mutants (Fig 2A) interacted with the CH domain in a pull-down assay. Gas2L1 fragments were tagged with an N-terminal AviTag fused to GFP (bioGFP), which was biotinylated by co-expressing the biotin ligase BirA. Streptavidin beads were used to bind biotinylated Gas2L1 fragments and incubated with HA-tagged CH domains, which in case of interaction were retained on the beads after washing and could be detected by Western blotting. Full-length Gas2L1 and the MT-binding Tail fragment pulled down the CH domain, whereas the biotinylated GFP, used as a negative control, displayed no binding (Fig 3A). Moreover, a Tail mutant harbouring a mutated SxIP motif (Tail-SxAA), which was previously shown to abolish Gas2L1-EB interaction 20, still bound to the CH domain. When the Tail domain was split into smaller fragments, the binding to the CH domain was lost (Fig 3A). These data suggest that the interaction of the CH domain of Gas2L1 with the MT-binding C-terminal part of the protein requires both the GAR domain and the unstructured region, and occurs independently from the association with EB proteins. Figure 3. Intramolecular interaction between Gas2L1 domains A. Pull-down experiment showing the binding of Gas2L1's CH domain (HA-CH) to full-length Gas2L1 (FL) and both Tail and Tail-SxAA mutants in HEK293 cell lysates, as well as co-precipitation of actin with full-length Gas2L1 bound to HA-CH. B. Pull-down experiment showing co-precipitation of actin with Gas2L1's CH domain in HEK293 cell lysates and full-length Gas2L1 only when HA-CH is co-expressed. C. Model of increased co-precipitation of actin with Gas2L1 in the presence of HA-CH. D. Schematic depiction of the domain structure of Gas2L1_1-304. E. Pull-down experiment showing the co-precipitation of actin with Gas2L1's CH domain (CH), but not with full-length Gas2L1 (FL) or Gas2L1-1-304 (1–304) in HEK293 cell lysates. Data information: Dotted lines separate marker lanes from sample lanes on the same blots (top panels). For bottom panel blots, markers were detected on the same blot, but in a different channel than the one used for signal detection that are shown here. Download figure Download PowerPoint Interestingly, consistent with the pattern of in vitro reconstitution experiments, the CH domain of Gas2L1 pulled do
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