Knockdown Of Drebrin Research Articles

Super-resolution imaging reveals the relationship between CaMKIIβ and drebrin within dendritic spines

Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIβ subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIβ binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIβ within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIβ co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIβ and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIβ was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIβ in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIβ is in a standby state before its activation.

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.

A novel role for the actin-binding protein drebrin in regulating opiate addiction

Persistent transcriptional and morphological events in the nucleus accumbens (NAc) and other brain reward regions contribute to the long-lasting behavioral adaptations that characterize drug addiction. Opiate exposure reduces the density of dendritic spines on medium spiny neurons of the NAc; however, the underlying transcriptional and cellular events mediating this remain unknown. We show that heroin self-administration negatively regulates the actin-binding protein drebrin in the NAc. Using virus-mediated gene transfer, we show that drebrin overexpression in the NAc is sufficient to decrease drug seeking and increase dendritic spine density, whereas drebrin knockdown potentiates these effects. We demonstrate that drebrin is transcriptionally repressed by the histone modifier HDAC2, which is relieved by pharmacological inhibition of histone deacetylases. Importantly, we demonstrate that heroin-induced adaptations occur only in the D1+ subset of medium spiny neurons. These findings establish an essential role for drebrin, and upstream transcriptional regulator HDAC2, in opiate-induced plasticity in the NAc.

CaMKIIβ 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 (CaMKIIβ) as a drebrin‐binding protein using a yeast two‐hybrid system, and investigated the drebrin–CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of drebrin‐independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with drebrin, CaMKIIβ associated with post‐synaptic density (PSD), and CaMKIIβ free from PSD and drebrin. CaMKIIβ appears to be anchored to a protein complex composed of drebrin‐binding F‐actin during the resting state. NMDA receptor activation releases CaMKIIβ from drebrin resulting in CaMKIIβ association with PSD.

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.

Drebrin's Role in the Maintenance of Endothelial Integrity.

The human endothelium forms a permeable barrier between the blood stream and surrounding tissues, strictly governing the passage of immune cells, fluids and metabolites. The regulation of cell-cell contact dynamics between endothelial cells is essential for this function and thus for the maintenance of vascular integrity. Intercellular adhesion within the endothelium is mainly dependent on adherens junctions, composed of cell-cell adhesion proteins such as VE-cadherin and nectin, and their associated proteins. Recent research points to a critical role of the actin cytoskeleton in endothelial integrity, by providing anchorage of adhesion complexes to the cell cortex. We could show that the F-actin-binding protein drebrin is a critical regulator of endothelial integrity, by linking nectin to the cortical actin cytoskeleton. In particular, the knockdown of drebrin leads to functional impairment of endothelial cells, characterized by rupturing of endothelial monolayers cultured under conditions mimicking vascular flow. This weakening of cell-cell contacts upon drebrin depletion is based on the destabilization of nectin at adherens junctions, followed by internalization and degradation in lysosomes. Conducting interaction studies, we showed that drebrin binds to nectin's interaction partner afadin, thus linking the nectin/afadin system to the cortical F-actin network. Drebrin, containing binding sites for both afadin and F-actin, is thus uniquely equipped to stabilize nectin at adherens junctions, thereby preserving endothelial integrity. Collectively, these results contribute to the current understanding of cell-cell junction regulation, introducing a new function of drebrin as a stabilizer of endothelial integrity.

Drebrin preserves endothelial integrity by stabilizing nectin at adherens junctions

Regulation of cell-cell contacts is essential for integrity of the vascular endothelium. Here, a critical role of the F-actin-binding protein drebrin in maintaining endothelial integrity is revealed under conditions mimicking vascular flow. Drebrin knockdown leads to weakening of cell-cell contacts, characterized by loss of nectin from adherens junctions and its subsequent lysosomal degradation. Immunoprecipitation, FRAP and mitochondrial re-targeting experiments show that nectin stabilization occurs through a chain of interactions: drebrin binding to F-actin, interaction of drebrin and afadin through their polyproline and PR1-2 regions, and recruitment of nectin through the PDZ region of afadin. Key elements are modules in drebrin that confer binding to afadin and F-actin. Evidence for this was obtained using constructs containing the PDZ region of afadin coupled to the F-actin-binding region of drebrin or to lifeact, which restore junctional nectin under knockdown of drebrin or of both drebrin and afadin. Drebrin, containing binding sites for both afadin and F-actin, is thus uniquely equipped to stabilize nectin at endothelial junctions and to preserve endothelial integrity under vascular flow.

The role of drebrin in glioma migration and invasion

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet been fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility.

Regulation of myotube formation by the actin-binding factor drebrin

BackgroundMyogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.MethodsRNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.ResultsIn a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.ConclusionsOur findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.

F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse

The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

Drebrin E is involved in the regulation of axonal growth through actin–myosin interactions

Drebrin is a well-known side-binding protein of F-actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F-actins and microtubules overlapped. Over-expression of drebrin E caused drebrin E and F-actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F-actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over-expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin-myosin interactions in the transitional zone of axonal growth cones.

Interactions between drebrin and Ras regulate dendritic spine plasticity

Dendritic spines are major sites of morphological plasticity in the CNS, but the molecular mechanisms that regulate their dynamics remain poorly understood. Here we show that the association of drebrin with actin filaments plays a major role in regulating dendritic spine stability and plasticity. Overexpressing drebrin or the internal actin-binding site of drebrin in rat hippocampal neurons destabilized mature dendritic spines so that they lost synaptic contacts and came to resemble immature dendritic filopodia. Drebrin-induced spine destabilization was dependent on Ras activation: expression of constitutively active Ras destabilized spine morphology whereas drebrin-induced spine destabilization was rescued by co-expressing dominant negative Ras. Conversely, RNAi-mediated drebrin knockdown prevented Ras-induced destabilization and promoted spine maturation in developing neurons. Together these data demonstrate a novel mechanism in which the balance between stability and plasticity in dendritic spines depends on binding of drebrin to actin filaments in a manner that is regulated by Ras.

Dynamics of the actin-binding protein drebrin in motile cells and definition of a juxtanuclear drebrin-enriched zone

The actin-binding protein (ABP) drebrin, isoform E2, is involved in remodelling of the actin cytoskeleton and in formation of cell processes, but its role in cell migration has not yet been investigated. Therefore, we have studied the organization of drebrin in motile cultured cells such as murine B16F1 melanoma and human SV80 fibroblast cells, using live cell confocal microscopy. In cells overexpressing DNA constructs encoding drebrin linked to EGFP, numerous long, branched cell processes were formed which slowly retracted and extended, whereas forward movement was halted. In contrast, stably transfected B16F1 cells containing drebrin-EGFP at physiological levels displayed lamellipodia and were able to migrate on laminin. Surprisingly, in such cells, drebrin was absent from anterior lamellipodia but was enriched in a specific juxtanuclear zone, the “drebrin-enriched zone” (DZ), and in the tail. In leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was specifically enriched along posterior portions of the microspikes, together with tropomyosin. Drebrin knock-down by small interfering RNAs did not impair movements of SV80 cells. Our results confirm the role of drebrin E2 in the formation of branching processes and further indicate that during cell migration, the protein contributes to retraction of the cell body and the tail but not to lamellipodia formation. In particular, the novel, sizable juxtanuclear DZ structure will have to be characterized in future experiments with respect to its molecular assembly and cell biological functions.