Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development.     

Polarization of actin cytoskeleton is reduced in dendritic protrusions during early spine development in hippocampal neuron

Dendritic spines are small protrusions that receive synaptic signals in neuronal networks. The actin cytoskeleton plays a key role in regulating spine morphogenesis, as well as in the function of synapses. Here we report the first quantitative measurement of F-actin retrograde flow rate in dendritic filopodia, the precursor of dendritic spines, and in newly formed spines, using a technique based on photoactivation localization microscopy. We found a fast F-actin retrograde flow in the dendritic filopodia but not in the spine necks. The quantification of F-actin flow rates, combined with fluorescence recovery after photobleaching measurements, allowed for a full quantification of spatially resolved kinetic rates of actin turnover, which was not previously feasible. Furthermore we provide evidences that myosin II regulates the actin flow in dendritic filopodia and translocates from the base to the tip of the protrusion upon spine formation. Rac1 inhibition led to mislocalization of myosin II, as well as to disruption of the F-actin flow. These results provide advances in the quantitative understanding of F-actin remodeling during spine formation.     

Role of actin cytoskeleton in dendritic spine morphogenesis

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses, and their morphological plasticity play a pivotal role in higher brain functions, such as learning and memory. The dynamics of spine morphology is due to the actin cytoskeleton concentrated highly in spines. Filopodia, which are thin and headless protrusions, are thought to be precursors of dendritic spines. Drebrin, a spine-resident side-binding protein of filamentous actin (F-actin), is responsible for recruiting F-actin and PSD-95 into filopodia, and is suggested to govern spine morphogenesis. Interestingly, some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction. In this review, to understand the importance of actin-binding proteins in spine morphogenesis, we first outline the well-established knowledge pertaining to the actin cytoskeleton in non-neuronal cells, such as the mechanism of regulation by small GTPases, the equilibrium between globular actin (G-actin) and F-actin, and the distinct roles of various actin-binding proteins. Then, we review the dynamic changes in the localization of drebrin during synaptogenesis and in response to glutamate receptor activation. Because side-binding proteins are located upstream of the regulatory pathway for actin organization via other actin-binding proteins, we discuss the significance of drebrin in the regulatory mechanism of spine morphology through the reorganization of the actin cytoskeleton. In addition, we discuss the possible involvement of an actin-myosin interaction in the morphological plasticity of spines.     

Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo

The dendritic spine is an important site of neuronal plasticity and contains extremely high levels of cytoskeletal actin. However, the dynamics of the actin cytoskeleton during synaptic plasticity and its in vivo function remain unclear. Here we used an in vivo dentate gyrus LTP model to show that LTP induction is associated with actin cytoskeletal reorganization characterized by a long-lasting increase in F-actin content within dendritic spines. This increase in F-actin content is dependent on NMDA receptor activation and involves the inactivation of actin depolymerizing factor/cofilin. Inhibition of actin polymerization with latrunculin A impaired late phase of LTP without affecting the initial amplitude and early maintenance of LTP. These observations suggest that mechanisms regulating the spine actin cytoskeleton contribute to the persistence of LTP.     

Synaptopodin, a molecule involved in the formation of the dendritic spine apparatus, is a dual actin/alpha-actinin binding protein

Synaptopodin (SYNPO) is a cytoskeletal protein that is preferentially located in mature dendritic spines, where it accumulates in the spine neck and closely associates with the spine apparatus. Formation of the spine apparatus critically depends on SYNPO. To further determine its molecular action, we screened for cellular binding partners. Using the yeast two-hybrid system and biochemical assays, SYNPO was found to associate with both F-actin and alpha-actinin. Ectopic expression of SYNPO in neuronal and non-neuronal cells induced actin aggregates, thus confirming a cytoplasmic interaction with the actin cytoskeleton. Whereas F-actin association is mediated by a central SYNPO motif, binding to alpha-actinin requires the C-terminal domain. Notably, the alpha-actinin binding domain is also essential for dendritic targeting and postsynaptic accumulation of SYNPO in primary neurons. Taken together, our data suggest that dendritic spine accumulation of SYNPO critically depends on its interaction with postsynaptic alpha-actinin and that SYNPO may regulate spine morphology, motility and function via its distinct modes of association with the actin cytoskeleton.     

Villin enhances hepatocyte growth factor-induced actin cytoskeleton remodeling in epithelial cells

Villin is an actin-binding protein localized to intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a calcium-dependent manner. Although villin is not necessary for the bundling of F-actin in vivo, it is important for the reorganization of the actin cytoskeleton elicited by stress during both physiological and pathological conditions (Ferrary et al., 1999). These data suggest that villin may be involved in actin cytoskeleton remodeling necessary for many processes requiring cellular plasticity. Here, we study the role of villin in hepatocyte growth factor (HGF)-induced epithelial cell motility and morphogenesis. For this purpose, we used primary cultures of enterocytes derived from wild-type and villin knock-out mice and Madin-Darby canine kidney cells, expressing villin in an inducible manner. In vitro, we show that epithelial cell lysates from villin-expressing cells induced dramatic, calcium-dependent severing of actin filaments. In cell culture, we found that villin-expressing cells exhibit enhanced cell motility and morphogenesis upon HGF stimulation. In addition, we show that the ability of villin to potentiate HGF-induced actin reorganization occurs through the HGF-activated phospholipase Cgamma signaling pathway. Collectively, these data demonstrate that villin acts as a regulator of HGF-induced actin dynamics.     

Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase beta1 to the actin cytoskeleton

The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kbeta1 to the cytoskeleton. Recruitment of AtPI4Kbeta1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane.     

Novel mechanistic link between focal adhesion remodeling and glucose-stimulated insulin secretion

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion.     

alpha5 integrin signaling regulates the formation of spines and synapses in hippocampal neurons

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.     

Combinatorial morphogenesis of dendritic spines and filopodia by SPAR and α-actinin2

Rap small GTPases regulate excitatory synaptic strength and morphological plasticity of dendritic spines. Changes in spine structure are mediated by the F-actin cytoskeleton, but the link between Rap activity and actin dynamics is unclear. Here, we report a novel interaction between SPAR, a postsynaptic inhibitor of Rap, and α-actinin, a family of actin-cross-linking proteins. SPAR and α-actinin engage in bidirectional structural plasticity of dendritic spines: SPAR promotes spine head enlargement, whereas increased α-actinin2 expression favors dendritic spine elongation and thinning. Surprisingly, SPAR and α-actinin2 can function in an additive rather than antagonistic fashion at the same dendritic spine, generating combination spine/filopodia hybrids. These data identify a molecular pathway bridging the actin cytoskeleton and Rap at synapses, and suggest that formation of spines and filopodia are not necessarily opposing forms of structural plasticity.     

Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics.     

Protein kinase D regulates cofilin activity through p21-activated kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.     

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.     

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.     

Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentration

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1μM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.     

The flare gene, which encodes the AIP1 protein of Drosophila, functions to regulate F-actin disassembly in pupal epidermal cells

Adult Drosophila are decorated with several types of polarized cuticular structures, such as hairs and bristles. The morphogenesis of these takes place in pupal cells and is mediated by the actin and microtubule cytoskeletons. Mutations in flare (flr) result in grossly abnormal epidermal hairs. We report here that flr encodes the Drosophila actin interacting protein 1 (AIP1). In other systems this protein has been found to promote cofilin-mediated F-actin disassembly. In Drosophila cofilin is encoded by twinstar (tsr). We show that flr mutations result in increased levels of F-actin accumulation and increased F-actin stability in vivo. Further, flr is essential for cell proliferation and viability and for the function of the frizzled planar cell polarity system. All of these phenotypes are similar to those seen for tsr mutations. This differs from the situation in yeast where cofilin is essential while aip1 mutations result in only subtle defects in the actin cytoskeleton. Surprisingly, we found that mutations in flr and tsr also result in greatly increased tubulin staining, suggesting a tight linkage between the actin and microtubule cytoskeleton in these cells.     

Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control

Cortactin is an important molecular scaffold for actin assembly and organization. Novel mechanistic functions of cortactin have emerged with more interacting partners identified, revealing its multifaceted roles in regulating actin cytoskeletal networks that are necessary for endocytosis, cell migration and invasion, adhesion, synaptic organization and cell morphogenesis. These processes are mediated by its multi-domains binding to F-actin and Arp2/3 complex and various SH3 targets. Furthermore, its role in actin remodeling is subjected to regulation by tyrosine and serine/threonine kinases. Elucidating the mechanisms underlying cortactin phosphorylation and its functional consequences would provide new insights to various aspects of cell dynamics control.     

Fluid Shear Stress Upregulates E-Tmod41 via miR-23b-3p and Contributes to F-Actin Cytoskeleton Remodeling during Erythropoiesis

The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm²) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3’UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, P_E0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and P_E0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling.     

The epithelial cell cytoskeleton and intracellular trafficking. III. How is villin involved in the actin cytoskeleton dynamics in intestinal cells?

Villin plays a key role in the maintenance of the brush border organization by bundling F-actin into a network of parallel filaments. Our previous in vivo data on villin knockout mice showed that, although this protein is not necessary for the bundling of F-actin, it is important for the reorganization of the actin cytoskeleton elicited by stress conditions. We further investigated villin property to initiate actin remodeling in cellular processes such as hepatocyte growth factor-induced motility, morphogenesis, and bacterial infection. Our data suggest that villin is involved in actin remodeling necessary for many cellular processes requiring the actin cytoskeleton plasticity.     

Cofilin/Twinstar phosphorylation levels increase in response to impaired coenzyme a metabolism

Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture - a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics.