Instruct: Given a scientific claim, retrieve documents that support or refute the claim.
Query: MICAL redox enzymes regulate actin dynamics.
The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.
Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, "cryo-positive stain" electron microscopy. The dynein molecules in the complex were shown to be active in in vitro motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP.vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).
Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
The actin cytoskeleton and adhesion junctions are physically and functionally coupled at the cell-cell interface between epithelial cells. The actin regulatory complex Arp2/3 has an established role in the turnover of junctional actin; however, the role of formins, the largest group of actin regulators, is less clear. Formins dynamically shape the actin cytoskeleton and have various functions within cells. In this review we describe recent progress on how formins regulate actin dynamics at cell-cell contacts and highlight formin functions during polarized protein traffic necessary for epithelialization.
The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.
We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.
Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.
The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.
CaMKII is activated by oxidation of methionine residues residing in the regulatory domain. Oxidized CaMKII (ox-CaMKII) is now thought to participate in cardiovascular and pulmonary diseases and cancer. This invited review summarizes current evidence for the role of ox-CaMKII in disease, considers critical knowledge gaps and suggests new areas for inquiry.
Cytoplasmic dynein is a microtubule-based motor protein that is responsible for most intracellular retrograde transports along microtubule filaments. The motor domain of dynein contains six tandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first four containing predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiple nucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains in which mutations were introduced to block nucleotide binding at each of the four AAA modules, and then examined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state even in the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity for microtubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, but retained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-fold slower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparent binding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubule sliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essential for the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysis sites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but is critical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.
Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.
CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.
WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2–Abi1–Nap1–PIR121 complex. The WAVE2–Abi1–Nap1–PIR121 complex is as active as the WAVE2–Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.
The dynein motor domain is composed of a tail, head, and stalk and is thought to generate a force to microtubules by swinging the tail against the head during its ATPase cycle. For this "power stroke," dynein has to coordinate the tail swing with microtubule association/dissociation at the tip of the stalk. Although a detailed picture of the former process is emerging, the latter process remains to be elucidated. By using the single-headed recombinant motor domain of Dictyostelium cytoplasmic dynein, we address the questions of how the interaction of the motor domain with a microtubule is modulated by ATPase steps, how the two mechanical cycles (the microtubule association/dissociation and tail swing) are coordinated, and which ATPase site among the multiple sites in the motor domain regulates the coordination. Based on steady-state and pre-steady-state measurements, we demonstrate that the two mechanical cycles proceed synchronously at most of the intermediate states in the ATPase cycle: the motor domain in the poststroke state binds strongly to the microtubule with a K(d) of approximately 0.2 microM, whereas most of the motor domains in the prestroke state bind weakly to the microtubule with a K(d) of >10 microM. However, our results suggest that the timings of the microtubule affinity change and tail swing are staggered at the recovery stroke step in which the tail swings from the poststroke to the prestroke position. The ATPase site in the AAA1 module of the motor domain was found to be responsible for the coordination of these two mechanical processes.
The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.
Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.
No direct evidence has been reported whether the spatial organization of ICAM-1 on the cell surface is linked to its physiological function in terms of leukocyte adhesion and transendothelial migration (TEM). Here we observed that ICAM-1 by itself directly regulates the de novo elongation of microvilli and is thereby clustered on the microvilli. However, truncation of the intracellular domain resulted in uniform cell surface distribution of ICAM-1. Mutation analysis revealed that the C-terminal 21 amino acids are dispensable, whereas a segment of 5 amino acids ((507)RKIKK(511)) in the NH-terminal third of intracellular domain, is required for the proper localization and dynamic distribution of ICAM-1 and the association of ICAM-1 with F-actin, ezrin, and moesin. Importantly, deletion of the (507)RKIKK(511) significantly delayed the LFA-1-dependent membrane projection and decreased leukocyte adhesion and subsequent TEM. Endothelial cells treated with cell-permeant penetratin-ICAM-1 peptides comprising ICAM-1 RKIKK sequences inhibited leukocyte TEM. Collectively, these findings demonstrate that (507)RKIKK(511) is an essential motif for the microvillus ICAM-1 presentation and further suggest a novel regulatory role for ICAM-1 topography in leukocyte TEM.
How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.
Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.
A model for the unidirectional movement of dynein is presented based on structural observations and biochemical experimental results available. In this model, the binding affinity of dynein for microtubule is independent of its nucleotide state and the change between strong and weak microtubule-binding is determined naturally by the variation of relative orientation between the stalk and microtubule as the stalk rotates following nucleotide-state transition. Thus the enigmatic communication from the ATP binding site in the globular domain to the far MT-binding site in the tip of the stalk, which is prerequisite in conventional models, is not required. Using the present model, the previous experimental results such as the effect of ATP and ADP bindings on dissociation of dynein from microtubule, the processive movement of single-headed axonemal dyneins at saturating ATP concentration, the load dependence of step size for the processive movement of two-headed cytoplasmic dyneins and the dependence of stall force on ATP concentration can be well explained.
The synapse is a highly organized cellular specialization whose structure and composition are reorganized, both positively and negatively, depending on the strength of input signals. The mechanisms orchestrating these changes are not well understood. A plausible locus for the reorganization of synapse components and structure is actin, because it serves as both cytoskeleton and scaffold for synapses and exists in a dynamic equilibrium between F-actin and G-actin that is modulated bidirectionally by cellular signaling. Using a new FRET-based imaging technique to monitor F-actin/G-actin equilibrium, we show here that tetanic stimulation causes a rapid, persistent shift of actin equilibrium toward F-actin in the dendritic spines of rat hippocampal neurons. This enlarges the spines and increases postsynaptic binding capacity. In contrast, prolonged low-frequency stimulation shifts the equilibrium toward G-actin, resulting in a loss of postsynaptic actin and of structure. This bidirectional regulation of actin is actively involved in protein assembly and disassembly and provides a substrate for bidirectional synaptic plasticity.
Dynein at the cortex contributes to microtubule-based positioning processes such as spindle positioning during embryonic cell division and centrosome positioning during fibroblast migration. To investigate how cortical dynein interacts with microtubule ends to generate force and how this functional association impacts positioning, we have reconstituted the 'cortical' interaction between dynein and dynamic microtubule ends in an in vitro system using microfabricated barriers. We show that barrier-attached dynein captures microtubule ends, inhibits growth, and triggers microtubule catastrophes, thereby controlling microtubule length. The subsequent interaction with shrinking microtubule ends generates pulling forces up to several pN. By combining experiments in microchambers with a theoretical description of aster mechanics, we show that dynein-mediated pulling forces lead to the reliable centering of microtubule asters in simple confining geometries. Our results demonstrate the intrinsic ability of cortical microtubule-dynein interactions to regulate microtubule dynamics and drive positioning processes in living cells.
The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GbetaL proteins to form a complex that is the target of rapamycin. Here, we demonstrate that mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GbetaL but not raptor and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. We find that the rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCalpha) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals.
Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.
The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain.