Author response: Palmitoylation of LIM Kinase-1 ensures spine-specific actin polymerization and morphological plasticity

Field: Neuroscience (Life Sciences)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Precise regulation of the dendritic spine actin cytoskeleton is critical for neurodevelopment and neuronal plasticity, but how neurons spatially control actin dynamics is not well defined. Here, we identify direct palmitoylation of the actin regulator LIM kinase-1 (LIMK1) as a novel mechanism to control spine-specific actin dynamics. A conserved palmitoyl-motif is necessary and sufficient to target LIMK1 to spines and to anchor LIMK1 in spines. ShRNA knockdown/rescue experiments reveal that LIMK1 palmitoylation is essential for normal spine actin polymerization, for spine-specific structural plasticity and for long-term spine stability. Palmitoylation is critical for LIMK1 function because this modification not only controls LIMK1 targeting, but is also essential for LIMK1 activation by its membrane-localized upstream activator PAK. These novel roles for palmitoylation in the spatial control of actin dynamics and kinase signaling provide new insights into structural plasticity mechanisms and strengthen links between dendritic spine impairments and neuropathological conditions. https://doi.org/10.7554/eLife.06327.001 eLife digest Neurons transmit information from one cell to the next by passing signals across junctions called synapses. For the neurons that receive these signals, these junctions are found on fine branch-like structures called dendrites that stick out of the cell. Dendrites themselves are decorated with smaller structures called dendritic spines, which typically receive information from one other neuron via a single synapse. Dendritic spines form in response to the signaling activity of the neuron, and problems with forming these spines have been linked to conditions such as autism and schizophrenia. Dendritic spines are created by the cell's cytoskeleton—a network of proteins that creates a constantly changing internal scaffold that shapes cells. One cytoskeleton protein called actin exists as thin filaments that can be extended or broken up by other proteins. It is not fully understood how actin is regulated in the dendritic spines. However, some researchers thought that the proteins that control the formation of the actin filaments would need to be localized to the dendritic spines to ensure that the spines form correctly. Some proteins can be made to localize to cell membranes by attaching a molecule called palmitic acid to them. Previous research has suggested that this ‘palmitoylation’ process is particularly important in neurons. Through a combination of experimental techniques, George et al. now show that palmitoylation is required to localize a protein called LIMK1, which regulates the construction of actin filaments, to the tips of dendritic spines. Further experiments showed that blocking the palmitoylation of LIMK1 alters how actin filaments form, makes spines unstable and causes synapses to be lost. George et al. also discovered that palmitoylation is necessary for LIMK1 to be activated by another protein that is found at dendritic spine membranes. This ‘dual-control’ mechanism makes it possible to precisely control where actin filaments form within dendritic spines. In addition to LIMK1, several other enzymes are also modified by palmitoylation. It will therefore be interesting to determine whether this dual control mechanism is broadly used by neurons to precisely regulate the structure and function of individual spines and synapses. https://doi.org/10.7554/eLife.06327.002 Introduction Most excitatory synapses are formed on dendritic spines—small protrusions that decorate the shaft of neuronal dendrites (Bourne and Harris, 2008; Hotulainen and Hoogenraad, 2010). Changes in the size and shape of individual spines are closely associated with Long-term potentiation (LTP), a cellular correlate of learning and memory (Fifková and Van Harreveld, 1977; Yuste and Bonhoeffer, 2001; Matsuzaki et al., 2004; Bosch and Hayashi, 2012; Murakoshi and Yasuda, 2012). Moreover, abnormal spine morphology and/or density are hallmarks of Intellectual Disability and other cognitive dysfunctions, including Autism-Spectrum Disorders and schizophrenia (Fiala et al., 2002; Penzes et al., 2011). These findings suggest that precise regulation of dendritic spine morphology and number is critical for normal cognition. Spines are highly enriched in actin filaments, and dynamic modulation of the actin cytoskeleton is crucial for controlling not only spine formation and elimination, but also modifications of the size, shape and motility of existing spines (Hotulainen and Hoogenraad, 2010; Bosch and Hayashi, 2012). In response to local synaptic cues, neurons can rapidly alter the morphology of individual spines (Matsuzaki et al., 2004; Okamoto et al., 2004; Honkura et al., 2008; Murakoshi et al., 2011). This process likely requires precise spatial regulation of proteins that increase actin polymerization and those that sever/disassemble actin filaments in a given spine. However, many actin regulators are predicted to be soluble, diffusible proteins, which appear poorly suited to operate with the necessary spatial specificity. This raises the question of how spatially precise, spine-specific actin regulation is achieved. We hypothesized that neurons must possess mechanisms to localize and/or confine certain actin regulatory proteins within dendritic spines, not only to ensure spine-specific regulation but perhaps also to control actin polymerization/depolymerization at the subspine level. One mechanism to control protein localization is palmitoylation, a protein-lipid modification that targets cytosolic proteins to specific membranes (Fukata and Fukata, 2010; Thomas and Huganir, 2013). Palmitoylation occurs in all eukaryotic cells but appears to be especially important in neurons because human genetic mutations and mouse knockouts of Palmitoyl acyltransferases (PATs, which catalyze palmitoylation) frequently lead to neurological and/or cognitive deficits (Fukata and Fukata, 2010; Greaves and Chamberlain, 2011). We therefore hypothesized that palmitoylation might modulate actin regulators to ensure spatially restricted signaling in spines. Two important actin regulatory proteins are the LIM kinases (LIMK1 and LIMK2 [Tada and Sheng, 2006]), which phosphorylate and inactivate the actin severing protein cofilin, thus promoting actin polymerization (Mizuno et al., 1994; Arber et al., 1998; Yang et al., 1998). Interestingly, LIMK1 appears particularly important for actin regulation in spines, because LIMK1 knockout in mice or genetic mutation in humans is associated with spine abnormalities and cognitive impairments (Frangiskakis et al., 1996; Tassabehji et al., 1996; Meng et al., 2002). Moreover, although LIMK1's upstream activator in neurons is unclear, spine abnormalities and intellectual impairments are also linked to mutations in PAK3 (Allen et al., 1998; Boda et al., 2004), a member of the p21-activated kinase (PAK) family that phosphorylates and activates LIMK1 in non-neuronal cells (Edwards et al., 1999). These findings link PAK/LIMK1-dependent regulation of actin polymerization to the control of spine morphology and higher brain function. However, PAKs and LIMKs are predicted soluble, cytosolic proteins. How, then, might they regulate actin with the necessary spatial precision? Here we report that palmitoylation of LIMK1 at a specific N-terminal motif is necessary and sufficient to target LIMK1 to spines. Palmitoyl-LIMK1 is essential for normal spine actin turnover, activity-dependent morphological plasticity and long-term spine stability. Strikingly, palmitoylation controls not only LIMK1 localization but also its activation by PAK in neurons. This novel ‘dual-control’ mechanism ensures spatially precise actin regulation at the single spine and potentially also the subspine levels. Results A conserved palmitoyl-motif in LIMK1 To address whether palmitoylation of actin regulators facilitates spatial control of actin polymerization in spines, we performed two bioinformatic searches for potential palmitoyl-motifs in actin regulatory proteins. LIMK1 was a prominent hit in both searches and adjacent cysteine residues (Cys7, Cys8, conserved in all vertebrate LIMK1 orthologs) were predicted to be palmitoylated (Figure 1A). Figure 1 with 2 supplements see all Download asset Open asset Palmitoylation at a unique di-cysteine motif targets LIMK1 to dendritic spines. (A). Upper panel: LIMK1 schematic, showing predicted palmitoyl-motif (CC, red) and LIM, PDZ and kinase domains. Lower panel: Multiple sequence alignment of the N-terminal region of LIMK1 orthologs from the indicated species. CC palmitoylation motif (highlighted with asterisks) is conserved in vertebrates. (B) HEK293T cells were transfected with C-terminal myc-tagged LIMK1wt (wt-LIMK1-myc) or CCSS-LIMK1-myc (cys 7, 8 mutated to Ser). ABE fractions prepared from lysates were blotted to detect palmitoyl-LIMK1 (top panel). Lysates were blotted to detect total LIMK1 expression (bottom panel). CCSS mutation eliminates LIMK1 palmitoylation. (C) LIMK1 palmitoylation increases, coincident with spine maturation and synapse formation. Hippocampal neurons, cultured for the indicated number of Days in vitro (DIV), were lysed and identical amounts of total protein were subjected to ABE to detect palmitoyl-LIMK1. (D) Homogenate and palmitoylated (ABE) fractions from rat forebrain were blotted to detect LIMK1. (E) Palmitoylation targets LIMK1 to dendritic spines. Representative images of hippocampal neurons (DIV18), transfected to express GFP plus Wt-LIMK1-myc or CCSS-LIMK1-myc and immunostained with the indicated antibodies. Scale bar: 20 μm. Lower panels show magnified images of single dendrites (scale bar: 1 μm). (F) Quantified spine targeting ratio (signal intensity in dendritic spines compared to adjacent dendritic shaft) for each construct from E. Data are mean + SEM for n = 30 neurons for each condition. *p < 0.05, ANOVA with Dunnett's post hoc correction. https://doi.org/10.7554/eLife.06327.003 To test whether the LIMK1 Cys7/Cys8 (CC) motif is indeed palmitoylated, we transfected HEK293T cells with C-terminal myc-tagged wild type LIMK1 (wt-LIMK1-myc) or CCSS-LIMK1-myc (Cys7/Cys8 mutated to non-palmitoylatable Ser). To isolate palmitoyl-proteins, we subjected lysates to Acyl-biotin exchange (ABE). ABE uses an exchange of thioester-linked acyl modifications (i.e., palmitoylation), for biotin, with the resultant biotinylated proteins being affinity-purified using neutravidin-conjugated beads (Wan et al., 2007; Thomas et al., 2012). Wt-LIMK1-myc was clearly detected in ABE fractions, indicative of palmitoylation, but was not detected in control purifications in which the essential ABE reagent hydroxylamine (NH2OH) was omitted (Figure 1B). In contrast, CCSS-LIMK1-myc was not detected in ABE fractions (Figure 1B), suggesting that LIMK1 is palmitoylated at Cys7/Cys8. Palmitoylation is necessary to target and confine LIMK1 to dendritic spines To investigate the neuronal role of palmitoyl-LIMK1, we examined LIMK1 palmitoylation in cultured hippocampal neurons, focusing on four developmental stages: 4 days in vitro (DIV4), when neurites are extending; DIV8, when dendrites elaborate and undergo branching; DIV12, when dendritic spines first appear and DIV20, when spines and synapses are mature (a time course similar to previous reports [Kaech and Banker, 2006; Beaudoin et al., 2012]). Interestingly, despite similar LIMK1 protein expression across these developmental stages, palmitoyl-LIMK1 was not detected in ABE fractions from DIV4 neurons (Figure 1C). However, palmitoyl-LIMK1 became detectable at DIV8, increased markedly at DIV12 and remained prominent at DIV20 (Figure 1C). These findings suggest that LIMK1 is palmitoylated in hippocampal neurons and that its palmitoylation coincides with spine development and maturation. This conclusion is consistent with the robust palmitoylation of LIMK1 in adult rat forebrain homogenates (Figure 1D). The temporal correlation of LIMK1 palmitoylation and spine formation (Figure 1C), and the altered spine morphology in LIMK1 knockout mice (Meng et al., 2002), suggested that palmitoyl-LIMK1 might play a specific role in spines. In support of this hypothesis, wt-LIMK1 was highly enriched in spines but CCSS-LIMK1 was not (Figure 1E,F). We reasoned that palmitoylation might anchor LIMK1 in spines to limit its diffusion, thus enhancing spatial control of actin regulation. We therefore transfected neurons with GFP-tagged LIMK1 and monitored its diffusibility by Fluorescence Recovery After Photobleaching (FRAP) using time-lapse imaging. Prominent FRAP, indicative of diffusion of the tagged protein, was observed with CCSS-LIMK1-GFP but not with wt-LIMK1-GFP (Figure 1—figure supplement 1). Quantitative analysis of FRAP data confirmed that CCSS mutation decreased the stable fraction of LIMK1-GFP in spines (Figure 1—figure supplement 1). We note that the time course of LIMK1-GFP FRAP, irrespective of its palmitoylation status, is markedly slower than previously characterized soluble proteins (Star et al., 2002; Bingol et al., 2010; Zheng et al., 2010). This suggests that other factors, most likely protein–protein interactions, are important to constrain LIMK1 in spines. However, our FRAP results suggest that palmitoylation, in addition to targeting LIMK1 to spines, also increases the fraction of LIMK1 that is stably anchored in spine heads. As an additional line of evidence that the impaired spine targeting caused by Cys7,8 mutation is palmitoylation-specific, we used a pharmacological approach. The palmitoylation inhibitor 2-Bromopalmitate (2-Br; [Jennings et al., 2009]) did not affect endogenous LIMK1 protein levels, but greatly reduced LIMK1 palmitoylation in neurons (Figure 1—figure supplement 2), reinforcing the conclusion that LIMK1 is palmitoylated. In parallel experiments, 2-Br greatly reduced wt-LIMK1 spine targeting without affecting overall spine numbers (Figure 1—figure supplement 2). The similar effects of CCSS mutation and 2-Br treatment strongly suggest that Cys7/Cys8 palmitoylation is required for LIMK1 spine targeting. LIMK1's dual palmitoylation motif is sufficient for spine targeting We next asked whether LIMK1's palmitoylation motif is sufficient for spine targeting. LIMK1-GFP deletion mutants lacking the kinase domain (LIMK1-1-258) or lacking the kinase and PDZ domains (LIMK1 1-137) localized to spines as effectively as full-length LIMK1 (Figure 2A–C). Remarkably, a minimal palmitoylation motif construct lacking the kinase, PDZ and LIM domains (LIMK1 [1–15]-GFP; Figure 2A–C) was sufficient to target GFP to spines. In contrast, cytosolic GFP was not enriched in spines (Figure 2A–C). These findings indicate that LIMK1's N-terminal 15 amino acid sequence is a specific spine targeting motif. Figure 2 with 2 supplements see all Download asset Open asset LIMK1's palmitoyl-motif is a minimal spine targeting sequence. (A) Schematic of LIMK1 deletion mutants. (B) LIMK1's palmitoyl-motif is sufficient for spine targeting. Representative images of hippopcampal neurons (DIV18), transfected to express mCherry plus the indicated GFP-tagged LIMK1 deletion mutants from A and immunostained with the indicated antibodies (scale bar: 20 μm). Lower panels show magnified images of single dendrites (scale bar: 1 μm). (C) Spine targeting ratio (mean ± SEM ) for n = 5–15 neurons per condition from B. n.s.; p > 0.05 compared to wt-LIMK1-GFP. *; p < 0.05 compared to GFP. ANOVA with Tukey post hoc test. https://doi.org/10.7554/eLife.06327.006 Other post-synaptic palmitoyl-proteins contain di-cysteine motifs (Kang et al., 2008; Fukata and Fukata, 2010; Brigidi et al., 2014), raising the possibility that LIMK1 must be dually palmitoylated to localize to spines. Consistent with this notion, C7S- and C8S-LIMK1 mutants were not targeted to spines (Figure 2—figure supplement 1). Moreover, a CCSS-LIMK1 mutant carrying a sequence that directs addition of the lipid myristate (Myr-CCSS-LIMK1), which can mimic a single palmitoylation event (Thomas et al., 2012), was not targeted to spines (Figure 2—figure supplement 1). Lastly, we examined the spine targeting of N-terminal mutants of LIMK1 that are predicted to be doubly lipid-modified with myristate plus single palmitate (Myr-SC-LIMK1, Myr-CS-LIMK1). Neither of these mutants was enriched in spines (Figure 2—figure supplement 2). Taken together, these results suggest that an intact CC palmitoyl-motif is critical for spine LIMK1 targeting. LIMK1's palmitoyl-motif accounts for differential localization of LIMK1 and LIMK2 Interestingly, LIMK1 genetic loss or mutation affects spine structure and cognition, despite the presence of LIMK2, which is also expressed in hippocampal neurons (Cajigas et al., 2012). However, we hypothesized that LIMK2's lacks of a CC palmitoyl-motif (Figure 3A) might render LIMK2 unable to localize to spines and thus unable to compensate for loss of LIMK1. Consistent with this notion, LIMK1 was detected in rat brain Post-Synaptic Density (PSD) fractions, including the PSDIII fraction, which is enriched in spine-associated proteins. In contrast, LIMK2 was absent from PSD fractions (Figure 3B, Figure 3—figure supplement 1) and was not enriched in spines (Figure 3C,D). Biochemical and immunocytochemical assays thus suggest that LIMK1 is targeted to spines but LIMK2 is not. Strikingly, however, a chimeric protein consisting of LIMK1's minimal spine targeting sequence fused to LIMK2 was readily detected in spines (Figure 3C,D). This result suggests that LIMK1's palmitoyl-motif accounts for differential localization of the two LIMKs, potentially explaining why loss of LIMK1 causes spine-specific phenotypes. Figure 3 with 1 supplement see all Download asset Open asset LIMK1's palmitoyl-motif accounts for differential localization of LIMK1 and LIMK2. (A) Schematic of LIMK1 (top) and LIMK2 (middle), showing similar overall domain arrangement. Rat LIMK1 and LIMK2 kinase domains are 70% identical, 84% similar (NCBI BLAST). Expanded N-terminal sequences show LIMK1's unique CC motif. Lower schematic shows a chimeric protein in which the N-terminal 15 amino acids of LIMK1 are fused to LIMK2 (‘Chimera LIMK2’). (B) The indicated rat forebrain subcellular fractions were blotted to detect endogenous LIMK1 and LIMK2. LIMK1 is seen in Post-synaptic Density (PSD) fractions, consistent with a previous study, but LIMK2 is not. Fidelity of the preparation and antibody specificity is confirmed in Figure 3—figure supplement 1. (C) Hippocampal neurons (DIV18) were transfected to express GFP plus the indicated myc-tagged LIMK variants. Representative images of single neurons immunostained with the indicated antibodies are shown (scale bar: 20 μm). Lower panels show magnified images of single dendrites. (D) Spine targeting ratio (mean ± SEM) for n = 30 neurons per condition from C. * p < 0.05; n.s.: p > 0.05, ANOVA with Dunnett's post hoc correction. https://doi.org/10.7554/eLife.06327.009 Loss of palmitoyl-LIMK1 impacts spine actin turnover The striking effects of palmitoylation on LIMK1 targeting to, and anchoring in, dendritic spines (Figure 1, Figure 1—figure supplement 1) suggested that palmitoylation is critical for LIMK1-dependent control of actin polymerization (Arber et al., 1998; Yang et al., 1998) in spines. To examine this possibility, while circumventing possible issues arising from LIMK1's neurodevelopmental roles (Meng et al., 2002; Rosso et al., 2004), we used small hairpin RNA (shRNA) knockdown/rescue to replace endogenous LIMK1 with the CCSS-LIMK1 mutant in mature neurons. We first identified an shRNA that potently reduced levels of cotransfected rat LIMK1-myc (Figure 4—figure supplement 1). The same shRNA greatly reduced endogenous LIMK1 levels when packaged into lentiviral particles and used to infect hippocampal neurons (Figure 4—figure supplement 1). shRNA-resistant (shr) ‘rescue’ forms of wt-LIMK1 (shr-wt-LIMK1-myc) and CCSS-LIMK1 (shr-CCSS-LIMK1-myc) were insensitive to LIMK1 shRNA (Figure 4—figure supplement 1). To address palmitoyl-LIMK1's role in spine actin regulation, we used FRAP to monitor the dynamics of GFP-tagged beta actin (GFP-actin) in single spines (Star et al., 2002; Okamoto et al., 2004; Hotulainen et al., 2009). Actin filaments in spines normally turn over rapidly, due to treadmilling, while actin monomers exchange bidirectionally between spines and the adjacent dendritic shaft ([Star et al., 2002; Bosch and Hayashi, 2012]; Figure 4A). If GFP-actin in a spine is photobleached, new fluorescent GFP-actin monomers normally diffuse into the spine and are incorporated into the barbed end of bleached filaments, predominantly in the juxtamembrane (‘shell’) region of the spine head (Hotulainen et al., 2009; Frost et al., 2010). Concomitantly, bleached actin molecules are severed from filament pointed ends (closer to the spine ‘core’) and exchange out of the spine, which recovers its fluorescence. However, if turnover is impaired, bleached GFP-actin remains trapped within filaments and FRAP is attenuated. The fraction of actin filaments in the spine that are undergoing rapid turnover can thus be determined from the extent of FRAP (Star et al., 2002). Figure 4 with 2 supplements see all Download asset Open asset Acute loss of palmitoyl-LIMK1 impairs Fluorescence Recovery After Photobleaching (FRAP) of GFP-actin in dendritic spines. (A) Schematic of FRAP assay. GFP-actin is photobleached in a single spine. When actin filament turnover is normal, fluorescence recovers as new fluorescent actin molecules are incorporated at the barbed ends of filaments, while bleached actin is released from pointed ends. (B) Hippocampal neurons (DIV17) were transfected to coexpress GFP-actin and mCherry with or without LIMK1 shRNA, plus shr-LIMK1 rescue constructs as indicated. Dual color live images of individual dendrites are shown for each condition. (C) Images of baseline GFP-actin signal from the same dendritic regions shown in B (left column, t = 0 s, Pre-bleach). A Region of Interest (ROI, yellow circle) was photobleached and the dendrite was imaged immediately thereafter (second column images, ‘Bleach’) and at the indicated times post-bleach (D) FRAP to from for each condition from C. are mean ± n = spines per condition. (E) of the stable fraction of GFP-actin (mean ± SEM) between t = and t = s, for each individual FRAP used to the data in ± LIMK1 ± ± ± p < 0.05 compared to control n.s.; not ANOVA with Dunnett's post hoc correction. of GFP-actin and cotransfected mCherry in hippocampal neurons mature or spines GFP-actin signals (Figure of single spines rapidly reduced GFP-actin which to an extent similar to previous reports (Figure et al., LIMK1 neurons mCherry and LIMK1 shRNA in spine number or morphology in the post Figure 4—figure supplement 1). However, the of GFP-actin fluorescence in LIMK1 neurons was compared to control neurons. This was in of the data (Figure or of all individual FRAP (Figure 4—figure supplement and is consistent with an increase in the of stable Two methods confirmed that the fraction of stable actin is indeed increased in LIMK1 spines (Figure Figure 4—figure supplement 2), see also et al., Strikingly, the increase of stable GFP-actin caused by LIMK1 was to control levels by but not by (Figure Figure 4—figure supplement 2). In to these effects on the of stable the of GFP-actin fluorescence was not condition examined (Figure 4—figure supplement 2). Taken together, these data suggest that the of loss of palmitoyl-LIMK1 on GFP-actin turnover is to the of actin in spines. This is because LIMK1 is as a regulator of cofilin, and increased activity in the of palmitoyl-LIMK1 might be to increase actin We possible for this in the but together, these results suggest that palmitoyl-LIMK1 is essential for normal actin turnover in spines. Palmitoyl-LIMK1 is critical for activity-dependent spine We next to identify of impaired spine actin turnover, caused by loss of palmitoyl-LIMK1. Spine morphological plasticity requires actin polymerization (Matsuzaki et al., 2004; Okamoto et al., 2004), and thus potentially also palmitoyl-LIMK1. To address this possibility, we used shRNA knockdown/rescue in hippocampal and examined spine-specific morphological plasticity activation of by of (Matsuzaki et al., 2004; and In transfected neurons the morphology of on spines a increase of the spine, similar to results ± of Figure Figure supplement 1). (i.e., to 0 in to a increase in the of the spine that was by a spine-specific increase in the of Figure supplement 2). Figure with 2 supplements see all Download asset Open asset Palmitoyl-LIMK1 is required for spine-specific activity-dependent morphological (A) images of individual neurons in hippocampal mCherry with or without LIMK1 images of individual dendrites were at the indicated times to and of on the head of the indicated spine Scale 20 2 μm. (B) course of (mean ± SEM) of and spines spines, LIMK1 spines, spine was and used for p < 0.05, in Images and time as in B for neurons mCherry spines, or LIMK1 shRNA and + spines, 8 Images and time as in B for neurons mCherry spines, or LIMK1 shRNA and CCSS spines, 8 In experiments we on activity-dependent spine and first the for LIMK1 in this In neurons mCherry plus LIMK1 shRNA, activity-dependent spine was ± of p = control neurons, Figure Figure supplement 1). This result suggests that LIMK1 is required for activity-dependent spine consistent with the of actin polymerization in this process (Matsuzaki et al., 2004; Okamoto et al., Strikingly, activity-dependent spine be by ± wt-LIMK1 ± p = but not by ± ± p = Figure Figure supplement 1). These findings suggest that palmitoyl-LIMK1 is required for activity-dependent spine loss of palmitoyl-LIMK1 causes spine and synapse Acute loss of palmitoyl-LIMK1 markedly impaired spine actin turnover without affecting dendritic spine number (Figure Figure 4—figure supplement 1). However, we hypothesized that impaired actin turnover and activity-dependent plasticity caused by of palmitoyl-LIMK1 might spine stability. To address this possibility, we transfected mature neurons to