Distribution of MAP 2 in dendritic spines and its colocalization with actin

Summary The distribution of MAP2 and actin in dendritic spines of the visual and cerebellar cortices, dentate fascia, and hippocampus was determined by using immunogold electron microscopy. By this approach, we have confirmed the presence of MAP2 in dendritic spines and identified substructures within the spine compartment showing MAP2 immunoreactivity. MAP2 immunolabeling was mainly associated with filaments which reacted with a monoclonal anti-actin antibody. Also, by immunogold double-labeling we colocalized MAP2 with actin on the endomembranes of the spine apparatus, smooth endoplasmic reticulum, and in the postsynaptic density. Labeling was nearly absent in axons and axonal terminals. These results indicate that MAP2 is an actin-associated protein in dendritic spines. Thus, MAP2 may organize actin filaments in the spine and endow the actin network of the spine with dynamic properties that are necessary for synaptic plasticity.     

Investigating Sub-Spine Actin Dynamics in Rat Hippocampal Neurons with Super-Resolution Optical Imaging

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.     

Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

Cytoplasmic organization in cerebellar dendritic spines

Three sets of filamentous structures were found to be associated with synaptic junctions in slices of cerebellar tissue prepared by rapid- freezing and freeze-etch techniques. The electron-dense fuzz subjacent to postsynaptic membranes corresponds to a web of 4-6-nm-diam filaments that were clearly visualized in rapid-frozen, freeze-etched preparations. Purkinje cell dendritic spines are filled with a meshwork of 5-7-nm filaments that were found to contact the spine membrane everywhere except at the synaptic junction, and extend through the neck of the spine into the parent dendrite. In addition, 8-10-nm microfilaments, possibly actin, were seen to be associated with the postsynaptic web and to extend into the body and neck of the spine. The arrangements and attachments of the filamentous elements in the Purkinje cell dendritic spine may account for its shape.     

Immunocytochemical localization of actin in dendritic spines of the cerebral cortex using colloidal gold as a probe

Immunocytochemical localization of actin in rat cerebral cortex embedded in the resin LR White was performed using 5 nm colloidal gold as a probe. Antigenicity is maintained throughout the embedding procedure and the low electron opacity of LR White permits fine filamentous structures to be visualized. Control experiments included incubating the sections with normal goat serum or mouse IgG instead of the primary antibody, preadsorbing the antibody with actin from bovine muscle or liver acetone powder, and heat treating the primary antibody. Immunoreactive actin was identified primarily in dendritic spines, particularly in the postsynaptic density (PSD), the subsynaptic web, and the spine apparatus and endothelial and smooth muscle cells of blood vessels. Within dendritic spines, actin which is labeled in the PSD is in continuity with the filaments of the subsynaptic web. These filaments, in turn, are in continuity with the spine apparatus and/or the spine membranes adjacent to the PSD. The PSD may therefore function like other submembranous filamentous arrays which communicate events occurring at the membrane, in this case, the postsynaptic membrane, to the underlying cytoskeletal network, i.e., the subsynaptic web of the spine. It is also suggested that the actin present in the spine may play a role in changes in spine shape and synaptic curvature. Some actin was also seen in the presynaptic process in association with synaptic vesicles, the filamentous network that is contiguous with the synaptic vesicle membrane, and the presynaptic dense projections. Actin may be involved in dynamic processes in the presynaptic ending which include vesicle translocation.     

Actin filaments and microtubules in dendritic spines

Dendritic spines are small protrusions emerging from their parent dendrites, and their morphological changes are involved in synaptic plasticity. These tiny structures are composed of thousands of different proteins belonging to several subfamilies such as membrane receptors, scaffold proteins, signal transduction proteins, and cytoskeletal proteins. Actin filaments in dendritic spines consist of double helix of actin protomers decorated with drebrin and ADF/cofilin, and the balance of the two is closely related to the actin dynamics, which may govern morphological and functional synaptic plasticity. During development, the accumulation of drebrin‐binding type actin filaments is one of the initial events occurring at the nascent excitatory postsynaptic site, and plays a pivotal role in spine formation as well as small GTPases. It has been recently reported that microtubules transiently appear in dendritic spines in correlation with synaptic activity. Interestingly, it is suggested that microtubule dynamics might couple with actin dynamics. In this review, we will summarize the contribution of both actin filaments and microtubules to the formation and regulation of dendritic spines, and further discuss the role of cytoskeletal deregulation in neurological disorders.     

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.     

Loss of the actin regulator cyclase-associated protein 1 (CAP1) modestly affects dendritic spine remodeling during synaptic plasticity

Dendritic spines form the postsynaptic compartment of most excitatory synapses in the vertebrate brain. Morphological changes of dendritic spines contribute to major forms of synaptic plasticity such as long-term potentiation (LTP) or depression (LTD). Synaptic plasticity underlies learning and memory, and defects in synaptic plasticity contribute to the pathogeneses of human brain disorders. Hence, deciphering the molecules that drive spine remodeling during synaptic plasticity is critical for understanding the neuronal basis of physiological and pathological brain function. Since actin filaments (F-actin) define dendritic spine morphology, actin-binding proteins (ABP) that accelerate dis-/assembly of F-actin moved into the focus as critical regulators of synaptic plasticity. We recently identified cyclase-associated protein 1 (CAP1) as a novel actin regulator in neurons that cooperates with cofilin1, an ABP relevant for synaptic plasticity. We therefore hypothesized a crucial role for CAP1 in structural synaptic plasticity. By exploiting mouse hippocampal neurons, we tested this hypothesis in the present study. We found that induction of both forms of synaptic plasticity oppositely altered concentration of exogenous, myc-tagged CAP1 in dendritic spines, with chemical LTP (cLTP) decreasing and chemical LTD (cLTD) increasing it. cLTP induced spine enlargement in CAP1-deficient neurons. However, it did not increase the density of large spines, different from control neurons. cLTD induced spine retraction and spine size reduction in control neurons, but not in CAP1-KO neurons. Together, we report that postsynaptic myc-CAP1 concentration oppositely changed during cLTP and cTLD and that CAP1 inactivation modestly affected structural plasticity.     

Surface spines of human blood flukes (Schistosoma mansoni) contain bundles of actin filaments having identical polarity

The spines of Schistosoma mansoni have crystalline structures that have been suggested to consist of actin filaments. In this ultrastructural study, binding of heavy meromyosin to the actin filament spines strongly supports this view. Moreover, we reveal that all the packed actin filaments in the spines have the same polarity pointing away from the apical plasma membrane toward the basal membrane of the surface syncytial epithelium of the parasites and that the spine filaments interact indirectly with both the apical and basal membranes.     

Cytoplasmic actin in neuronal processes as a possible mediator of synaptic plasticity

We have demonstrated that, after permeation with saponin and decoration with S-1 myosin subfragment, the cytoplasmic actin is organized in filaments in dendritic spines, dendrites, and axon terminals of the dentate molecular layer. The filaments are associated with the plasma membrane and the postsynaptic density with their barbed ends and also in parallel with periodical cross bridges. In the spine stalks and dendrites, the actin filaments are organized in long strands. Given the contractile properties of actin, these results suggest that the cytoplasmic actin may be involved in various forms of experimentally induced synaptic plasticity by changing the shape or volume of the pre- and postsynaptic side and by retracting and sprouting synapses.     

The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines

Synapse function and plasticity depend on the physical structure of dendritic spines as determined by the actin cytoskeleton. We have investigated the organization of filamentous (F-) actin within individual spines on CA1 pyramidal neurons in rat hippocampal slices. Using two-photon photoactivation of green fluorescent protein fused to beta-actin, we found that a dynamic pool of F-actin at the tip of the spine quickly treadmilled to generate an expansive force. The size of a stable F-actin pool at the base of the spine depended on spine volume. Repeated two-photon uncaging of glutamate formed a third pool of F-actin and enlarged the spine. The spine often released this "enlargement pool" into the dendritic shaft, but the pool had to be physically confined by a spine neck for the enlargement to be long-lasting. Ca2+/calmodulin-dependent protein kinase II regulated this confinement. Thus, spines have an elaborate mechanical nature that is regulated by actin fibers.     

Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. We show here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we have established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity.     

Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.     

Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.     

The actin binding domain of βI-spectrin regulates the morphological and functional dynamics of dendritic spines

Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.     

The Guanine Nucleotide Exchange Factor (GEF) Asef2 Promotes Dendritic Spine Formation via Rac Activation and Spinophilin-dependent Targeting

Dendritic spines are actin-rich protrusions that establish excitatory synaptic contacts with surrounding neurons. Reorganization of the actin cytoskeleton is critical for the development and plasticity of dendritic spines, which is the basis for learning and memory. Rho family GTPases are emerging as important modulators of spines and synapses, predominantly through their ability to regulate actin dynamics. Much less is known, however, about the function of guanine nucleotide exchange factors (GEFs), which activate these GTPases, in spine and synapse development. In this study we show that the Rho family GEF Asef2 is found at synaptic sites, where it promotes dendritic spine and synapse formation. Knockdown of endogenous Asef2 with shRNAs impairs spine and synapse formation, whereas exogenous expression of Asef2 causes an increase in spine and synapse density. This effect of Asef2 on spines and synapses is abrogated by expression of GEF activity-deficient Asef2 mutants or by knockdown of Rac, suggesting that Asef2-Rac signaling mediates spine development. Because Asef2 interacts with the F-actin-binding protein spinophilin, which localizes to spines, we investigated the role of spinophilin in Asef2-promoted spine formation. Spinophilin recruits Asef2 to spines, and knockdown of spinophilin hinders spine and synapse formation in Asef2-expressing neurons. Furthermore, inhibition of N-methyl-d-aspartate receptor (NMDA) activity blocks spinophilin-mediated localization of Asef2 to spines. These results collectively point to spinophilin-Asef2-Rac signaling as a novel mechanism for the development of dendritic spines and synapses.     

Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth

Two-photon fluorescence correlation spectroscopy (2P-FCS) within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA), a protocol previously used for the chemical induction of long-term potentiation (cLTP) strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.     

Actin Out: Regulation of the Synaptic Cytoskeleton

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.