An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate Signaling.

Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts.

Drebrin Regulates Collateral Axon Branching in Cortical Layer II/III Somatosensory Neurons.

Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral branches in a laminar-specific manner that dictates synaptic partners and overall circuit organization. Here, we leverage both male and female mouse models, single-cell labeling and imaging approaches to identify intrinsic regulators of laminar-specific collateral, also termed interstitial, axon branching. We developed new approaches for the robust, sparse, labeling of Layer II/III pyramidal neurons to obtain single-cell quantitative assessment of axon branch morphologies. We combined these approaches with cell-autonomous loss-of-function (LOF) and overexpression (OE) manipulations in an in vivo candidate screen to identify regulators of cortical neuron axon branch lamination. We identify a role for the cytoskeletal binding protein drebrin (Dbn1) in regulating Layer II/III cortical projection neuron (CPN) collateral axon branching in vitro LOF experiments show that Dbn1 is necessary to suppress the elongation of Layer II/III CPN collateral axon branches within Layer IV, where axon branching by Layer II/III CPNs is normally absent. Conversely, Dbn1 OE produces excess short axonal protrusions reminiscent of nascent axon collaterals that fail to elongate. Structure-function analyses implicate Dbn1S142 phosphorylation and Dbn1 protein domains known to mediate F-actin bundling and microtubule (MT) coupling as necessary for collateral branch initiation upon Dbn1 OE. Taken together, these results contribute to our understanding of the molecular mechanisms that regulate collateral axon branching in excitatory CPNs, a key process in the elaboration of neocortical circuit formation.SIGNIFICANCE STATEMENT Laminar-specific axon targeting is essential for cortical circuit formation. Here, we show that the cytoskeletal protein drebrin (Dbn1) regulates excitatory Layer II/III cortical projection neuron (CPN) collateral axon branching, lending insight into the molecular mechanisms that underlie neocortical laminar-specific innervation. To identify branching patterns of single cortical neurons in vivo, we have developed tools that allow us to obtain detailed images of individual CPN morphologies throughout postnatal development and to manipulate gene expression in these same neurons. Our results showing that Dbn1 regulates CPN interstitial axon branching both in vivo and in vitro may aid in our understanding of how aberrant cortical neuron morphology contributes to dysfunctions observed in autism spectrum disorder and epilepsy.

Drebrin regulates cytoskeleton dynamics in migrating neurons through interaction with CXCR4.

Stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR4) are regulators of neuronal migration (e.g., GnRH neurons, cortical neurons, and hippocampal granule cells). However, how SDF-1/CXCR4 alters cytoskeletal components remains unclear. Developmentally regulated brain protein (drebrin) stabilizes actin polymerization, interacts with microtubule plus ends, and has been proposed to directly interact with CXCR4 in T cells. The current study examined, in mice, whether CXCR4 under SDF-1 stimulation interacts with drebrin to facilitate neuronal migration. Bioinformatic prediction of protein-protein interaction highlighted binding sites between drebrin and crystallized CXCR4. In migrating GnRH neurons, drebrin, CXCR4, and the microtubule plus-end binding protein EB1 were localized close to the cell membrane. Coimmunoprecipitation (co-IP) confirmed a direct interaction between drebrin and CXCR4 using wild-type E14.5 whole head and a GnRH cell line. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain. A decrease in hippocampal granule cells was also detected, and co-IP confirmed a direct interaction between drebrin and CXCR4 in PN4 hippocampi. Migration assays on primary neurons established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1 on neuronal movement. Bioinformatic prediction then identified binding sites between drebrin and the microtubule plus end protein, EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during central nervous system development.

Individual differences in the positive outcome from adolescent ketamine treatment in a female mouse model of anorexia nervosa involve drebrin A at excitatory synapses of the medial prefrontal cortex.

Anorexia nervosa (AN) is a mental illness with the highest rates of mortality and relapse, and no approved pharmacological treatment. Using an animal model of AN, called activity-based anorexia (ABA), we showed earlier that a single intraperitoneal injection of ketamine at a dose of 30 mg/kg (30mgKET), but not 3 mg/kg (3mgKET), has a long-lasting effect upon adolescent females of ameliorating anorexia-like symptoms through the following changes: enhanced food consumption and body weight; reduced running and anxiety-like behavior. However, there were also individual differences in the drug's efficacy. We hypothesized that individual differences in ketamine's ameliorative effects involve drebrin A, an F-actin-binding protein known to be required for the activity-dependent trafficking of NMDA receptors (NMDARs). We tested this hypothesis by electron microscopic quantifications of drebrin A immunoreactivity at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (GABA-IN) in deep layer 1 of prefrontal cortex (PFC) of these mice. Results reveal that (1) the areal density of excitatory synapses on GABA-IN is greater for the 30mgKET group than the 3mgKET group; (2) the proportion of drebrin A+ excitatory synapses is greater for both PN and GABA-IN of 30mgKET than 3mgKET group. Correlation analyses with behavioral measurements revealed that (3) 30mgKET's protection is associated with reduced levels of drebrin A in the cytoplasm of GABA-IN and higher levels at extrasynaptic membranous sites of PN and GABA-IN; (5) altogether pointing to 30mgKET-induced homeostatic plasticity that engages drebrin A at excitatory synapses of both PN and GABA-IN.

The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Drebrin promotes lung adenocarcinoma cell migration through inducing integrin ß1 endocytosis.

Lung adenocarcinoma (LUAD) is the most common type of lung cancers, which remains the leading cause of cancer-related death worldwide. Drebrin can promote cell migration and invasion with poor prognosis, but its roes in LUAD tumor progression remains unknown. We showed that the expression of Drebrin was upregulated in clinical LUAD samples. A Kaplan-Meier survival analysis showed that a high expression of Drebrin predicated poor prognosis in LUAD. In vitro, Drebrin promoted anchorage-independent growth and migration of LUAD cells. Drebrin interacted with dynamin through CT domain, and served as an adaptor to promote LUAD cell migration through inducing integrin ß1 endocytosis. Thus, this study demonstrated the critical role of Drebrin in LUAD and associated mechanism.

The Difference in the Cytoskeletal Machinery of Growth Cones of Growing Axons and Leading Processes.

Neuronal migration and axon elongation in the developing brain are essential events for neural network formation. Leading processes of migrating neurons and elongating axons have growth cones at their tips. Cytoskeletal machinery for advance of growth cones of the two processes has been thought the same. In this study, we compared axonal-elongating growth cones and leading-process growth cones in the same conditions that manipulated filopodia, lamellipodia, and drebrin, the latter mediates actin filament-microtubule interaction. Cerebral cortex (CX) neurons and medial ganglionic eminence (MGE) neurons from embryonic mice were cultured on less-adhesive cover glasses. Inhibition of filopodia formation by triple knockdown of mammalian-enabled, Ena-VASP-like, and vasodilator-stimulated phosphoprotein or double knockdown of Daam1 and fascin affected axon formation of CX neurons but did not affect the morphology of leading process of MGE neurons. On the other hand, treatment with CK666, to inhibit lamellipodia formation, did not affect axons but destroyed the leading-process growth cones. When drebrin was knocked down, the morphology of CX neurons remained unchanged, but the leading processes of MGE neurons became shorter. In vivo assay of radial migration of CX neurons revealed that drebrin knockdown inhibited migration, while it did not affect axon elongation. These results showed that the filopodia-microtubule system is the main driving machinery in elongating growth cones, while the lamellipodia-drebrin-microtubule system is the main system in leading-process growth cones of migrating neurons.

Effects of neuronal drebrin on actin dynamics.

Drebrin is a key regulator of actin cytoskeleton in neuronal cells which is critical for synaptic plasticity, neuritogenesis, and neuronal migration. It is also known to orchestrate a cross-talk between actin and microtubules. Decreased level of drebrin is a hallmark of multiple neurodegenerative disorders such as Alzheimer's disease. Despite its established importance in health and disease, we still have a lot to learn about drebrin's interactome and its effects on cytoskeletal dynamics. This review aims to summarize the recently reported novel effects of drebrin on actin and its regulators. Here I will also reflect on the most recent progress made in understanding of the role of drebrin isoforms and posttranslational modifications on its functionality.

Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery.

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex "pretzel-like" structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.

Drebrin is induced during myofibroblast differentiation and enhances the production of fibrosis-related genes.

Fibrosis is attributed to excess deposition of extracellular matrix (ECM) proteins including collagen and is associated with various organ dysfunction. This excessive ECM is produced by myofibroblasts, which are differentiated from various cells by a variety of stimuli, represented by TGF-ß. However, molecular mechanisms for the regulation of ECM production in myofibroblasts remain obscure. In this study, we demonstrate that the expression of drebrin, which binds to and increases the stability of actin filament in neurons, is increased in mouse hearts and lungs upon fibrosis. Drebrin is mainly expressed in myofibroblasts in the fibrotic hearts and lungs and promotes the expression of fibrosis-related genes, such as Acta2 and Col1a1. Taken together, our study identifies drebrin as a molecule that promotes the production of fibrosis-related genes in myofibroblasts.

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis.

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

Long-Range and Directional Allostery of Actin Filaments Plays Important Roles in Various Cellular Activities.

A wide variety of uniquely localized actin-binding proteins (ABPs) are involved in various cellular activities, such as cytokinesis, migration, adhesion, morphogenesis, and intracellular transport. In a micrometer-scale space such as the inside of cells, protein molecules diffuse throughout the cell interior within seconds. In this condition, how can ABPs selectively bind to particular actin filaments when there is an abundance of actin filaments in the cytoplasm? In recent years, several ABPs have been reported to induce cooperative conformational changes to actin filaments allowing structural changes to propagate along the filament cables uni- or bidirectionally, thereby regulating the subsequent binding of ABPs. Such propagation of ABP-induced cooperative conformational changes in actin filaments may be advantageous for the elaborate regulation of cellular activities driven by actin-based machineries in the intracellular space, which is dominated by diffusion. In this review, we focus on long-range allosteric regulation driven by cooperative conformational changes of actin filaments that are evoked by binding of ABPs, and discuss roles of allostery of actin filaments in narrow intracellular spaces.

Phosphorylation of Cx43 residue Y313 by Src contributes to blocking the interaction with Drebrin and disassembling gap junctions.

Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Y → F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.

CaMKIIß is localized in dendritic spines as both drebrin-dependent and drebrin-independent pools.

Drebrin is a major F-actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel drebrin-binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca2+ /calmodulin-dependent protein kinase II (CaMKIIß) as a drebrin-binding protein using a yeast two-hybrid system, and investigated the drebrin-CaMKIIß relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIß in dendrites during the resting state, suggesting that drebrin is involved in the accumulation of CaMKIIß in dendritic spines. Fluorescence recovery after photobleaching analysis showed that drebrin knockdown increased the stable fraction of CaMKIIß, indicating the presence of drebrin-independent, more stable CaMKIIß. NMDA receptor activation also increased the stable fraction in parallel with drebrin exodus from dendritic spines. These findings suggest that CaMKIIß can be classified into distinct pools: CaMKIIß associated with drebrin, CaMKIIß associated with post-synaptic density (PSD), and CaMKIIß free from PSD and drebrin. CaMKIIß appears to be anchored to a protein complex composed of drebrin-binding F-actin during the resting state. NMDA receptor activation releases CaMKIIß from drebrin resulting in CaMKIIß association with PSD.

Early growth response-1-mediated down-regulation of drebrin correlates with loss of dendritic spines.

Post-synaptic dendritic spines are structurally composed of actin cytoskeleton, which undergoes dynamic morphological changes to accommodate incoming synaptic activity. Drebrin is an actin-binding protein highly expressed in dendritic spines that serves an important role in regulating spine morphology. Functionally, loss of drebrin directly correlates with deficits in learning and memory, as is the case observed in Alzheimer's disease. Despite these findings, the regulatory factor responsible for drebrin loss remains unclear. Here, we show that early growth response-1 (Egr-1), an inducible zinc finger transcription factor, down-regulates drebrin expression. Chromatin immunoprecipitation analyses identified Egr-1 binding sites upstream of the drebrin start site in neuronal cells. Over-expression of Egr-1 in vitro in primary hippocampal neurons or in vivo in homogenates prepared from the hippocampi of an inducible mouse model of Egr-1 show reduced drebrin mRNA and protein levels. Conversely, increased drebrin was detected in hippocampal samples isolated from Egr-1-deficient brain. These data demonstrate that Egr-1 interacts with the drebrin promoter and negatively regulates drebrin expression. Furthermore, immunocytochemical and Golgi staining analyses revealed reduced drebrin protein and dendritic spine density as well as reduced expression of synaptic markers in in vitro hippocampal neurons over-expressing Egr-1 and in vivo inducible mouse model of Egr-1. In contrast, increased drebrin expression correlated with increased dendritic spine density was detected in samples from Egr-1-deficient mice. These data provide evidence that Egr-1 is a novel regulator of drebrin expression, which is linked to changes in dendritic spine density.

60th Anniversary of the Japanese Society for Neurochemistry.

To welcome the 60th anniversary of the Japanese Society for Neurochemistry (JSN), in this issue, we publish five articles from leading groups of the JSN in the Journal of Neurochemistry. These five reviews are regarding molecular base-investigation of neuronal regulation, and the research styles and its destination are exhibiting the characteristics that identify our society. Here, we introduce what we have archived in the neurochemical fields of research, including Ca2+ neurobiology, synaptic plasticity, neurogenesis and neuroregeneration. With the achievements in the past decades in mind, we will continue to contribute to the development of neurochemistry from now on too. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

The role of drebrin in neurons.

Drebrin is an actin-binding protein that changes the helical pitch of actin filaments (F-actin), and drebrin-decorated F-actin shows slow treadmilling and decreased rate of depolymerization. Moreover, the characteristic morphology of drebrin-decorated F-actin enables it to respond differently to the same signals from other actin cytoskeletons. Drebrin consists of two major isoforms, drebrin E and drebrin A. In the developing brain, drebrin E appears in migrating neurons and accumulates in the growth cones of axons and dendrites. Drebrin E-decorated F-actin links lamellipodium F-actin to microtubules in the growth cones. Then drebrin A appears at nascent synapses and drebrin A-decorated F-actin facilitates postsynaptic molecular assembly. In the adult brain, drebrin A-decorated F-actin is concentrated in the central region of dendritic spines. During long-term potentiation initiation, NMDA receptor-mediated Ca2+ influx induces the transient exodus of drebrin A-decorated F-actin via myosin II ATPase activation. Because of the unique physical characteristics of drebrin A-decorated F-actin, this exodus likely contributes to the facilitation of F-actin polymerization and spine enlargement. Additionally, drebrin reaccumulation in dendritic spines is observed after the exodus. In our drebrin exodus model of structure-based synaptic plasticity, reestablishment of drebrin A-decorated F-actin is necessary to keep the enlarged spine size during long-term potentiation maintenance. In this review, we introduce the genetic and biochemical properties of drebrin and the roles of drebrin in early stage of brain development, synaptic formation and synaptic plasticity. Further, we discuss the pathological relevance of drebrin loss in Alzheimer's disease. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

The Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia.

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.

The Role of Drebrin in Cancer Cell Invasion.

Cancer progression is characterized by the capacity of malignant cells to exploit an innate migratory ability in order to invade adjacent tissues, enter the vasculature and eventually metastasize to secondary organs. It is this spread of cancer cells that is the major cause of death in cancer patients. Understanding the basic biology of how cancer cells generate an invasive phenotype will be crucial to the identification of drug targets with the aim of impeding tumour dissemination. Ten years on from its initial description in neuronal cells, drebrin expression was found in a wide variety of non-neuronal cells that importantly included cancer cell lines. Since then mounting evidence suggests that drebrin may be a key player in the advancement of several diverse cancer types where its expression is frequently upregulated. Cancer cell motility and invasion are crucial elements in the metastatic cascade and involve dramatic changes in cellular morphology that are associated with dynamic remodelling of the cytoskeleton. Interestingly, it now appears that drebrin could deliver this role during cancer development.

Juxtanuclear Drebrin-Enriched Zone.

Drebrin E contributes to remodeling of the actin cytoskeleton and formation of cell processes. Therefore, its role in cell migration was studied in prototypes of motile cells with prominent lamellipodia such as murine B16F1 melanoma and Swiss 3T3 cells and in human SV80 fibroblasts. Confocal microscopy revealed absence of drebrin from the tips of lamellipodia but enrichment in the tail of the cells, in retraction zones and in a specific juxtanuclear actin filament compartment, named "drebrin-enriched zone." A similar subset of juxtanuclear actin filaments is characterized by the actin-binding protein SWAP-70, but drebrin and SWAP-70 localized to different compartments, suggesting the existence of novel distinct subdomains within the actin filament system. In cells overexpressing drebrin-EGFP, numerous long, branched cell processes were formed which slowly retracted and extended. However, in stable transfectants containing lower amounts of the fusion protein, drebrin-EGFP was recruited to the same sites as the endogenous protein during cell migration, i.e., to retracting membrane domains and into the juxtanuclear drebrin-enriched zone. In the leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was concentrated along posterior portions of the microspikes, together with tropomyosin, with which it competes for actin binding. Drebrin knockdown by siRNA did not impact forward migration or ruffling. Taken together, these findings suggest that during cell migration drebrin is involved in retraction processes but not in lamellipodia formation. The novel, sizable juxtanuclear drebrin-enriched zone remains to be characterized in detail with respect to its molecular assembly and functions.