LIM kinase and slingshot are critical for neurite extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.     

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.     

EphA signaling promotes actin-based dendritic spine remodeling through slingshot phosphatase

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.     

The bound nucleotide of actin

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G-actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F-actin and the interaction of F-actin · AMPPNP with myosin was studied. F-actin · AMPPNP activated the ATPase of myosin to the same extent as did F-actin · ADP. However, the rate of superprecipitation was slower in the case of F-actin · AMPPNP than in the control.     

The reverse turn of actin

A model of actin sliding is proposed, which is based on the occurrence of actin-myosin interactions and the generation in actin filaments of elastic deformations of torsion, compression, and tension. Previously in experiments in vitro, an unusually stable right-handed axial rotation of actin filaments sliding relative to myosin with a rotation pitch of 1 microm was observed. However, the reported value of the rotation pitch is significantly greater (14 times) than the pitch of actin right-handed double helix (72 nm), which supposedly determines the rotation pitch of actin. This result is explained in terms of the model by the presence of the reverse turn of actin at each elementary step.     

The place of inactivated actin and its kinetic predecessor in actin folding-unfolding

The kinetics of actin unfolding induced by guanidine hydrochloride of different concentrations was studied. The parametric representation of the kinetic dependencies of tryptophan fluorescence intensity changes recorded at two wavelengths allowed us to detect and characterize a new essentially unfolded kinetic intermediate. Its characteristics suggested that this intermediate state is a premolten globule. It was shown that the equilibrium transition between inactivated and completely unfolded states is also a two-step process and proceeds via an essentially unfolded kinetic intermediate. The new kinetic pathway of actin unfolding--refolding was proposed. According to it, the founded essentially unfolded kinetic state is the on-pathway intermediate, while inactivated actin is the off-pathway misfolded state stabilized by aggregation of partially folded macromolecules of protein.     

The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin

Glucocorticoid induced tumor necrosis factor receptor (GITR) is a new member of the tumor necrosis factor-nerve growth factor receptor superfamily of which the function has not been well studied. The extracellular domain of GITR was produced in Escherichia coli and purified as a single band of predicted M(r) of 18.0 kDa. GITR and GITR ligand were expressed constitutively on the surface of Raw 264.7 macrophage cell line and murine peritoneal macrophages. An extracellular domain of GITR can activate murine macrophages to express inducible nitric oxide synthase and to generate nitric oxide in a dose- and time-dependent manner.     

The viscoelastic properties of actin solutions

The viscoelastic properties in actin solutions were investigated by measuring their elastic modulus and viscous modulus using a rheometer. The polymerization/gelation process of actin solutions was accompanied by an increase of both parameters, indicating the formation of a protein network. High shear rotational motion destroyed this network which, however, would reanneal if left undisturbed. At 25 °C under low ionic strength conditions, the viscoelastic moduli of a Spudich-Watt globular (G) actin preparation increased with time, while G-actin, purified by gel filtration maintained low viscoelastic moduli. The rigidity of the filamentous (F) actin network in a solution of Spudich-Watt actin, measured by the elastic modulus, was somewhat lower than that of gel-filtration-purified actin at the same protein concentration. The crosslink density of these F-actin networks was estimated, using models from rubber elasticity theory. The calculated density was 1 crosslink/50 actin monomers for the purified actin and 1 crosslink/120 actin monomers for Spudich-Watt actin. The results are consistent with the idea that a small amount of regulatory factor(s), which could be removed by the gel filtration step, modulates the structure of an actin network.     

The actin cytoskeleton in presynaptic assembly

Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly.     

The formation of actin rods composed of actin tubules in Dictyostelium discoideum spores

A new type of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.     

The interaction of actin with dystrophin

Proton NMR spectroscopy of synthetic peptides corresponding to defined regions of human dystrophin has been employed to study the interaction with F-actin. No evidence of interaction with a C-terminal region corresponding to amino acid residues 3429-3440 was obtained. F-actin restricted the mobility of residues 19-27 in a synthetic peptide corresponding to residues 10-32. This suggests that this is a site of F-actin interaction in the intact dystrophin molecule. Identical sequences to that of residues 19-22 in dystrophin, namely Lys-Thr-Phe-Thr are also present in the N-terminal regions of the alpha-actinins implying this is also a site of F-actin interaction with alpha-actinin.