Localization of ganglioside 9‐O‐acetyl GD3 in point contacts of neuronal growth cones

Gangliosides are a large group of sialylated glycosphingolipids widely expressed in mammalian tissues. We have shown previously that the expression of 9‐O‐acetyl GD3 is highly correlated with periods of neurite outgrowth in the developing nervous system, and that the advance of dorsal root ganglia growth cones on laminin was halted in presence of an antibody specific for 9‐O‐acetyl GD3. In this work, we examined by immunocytochemistry and confocal microscopy whether this ganglioside is localized in point contacts in neuronal growth cones. We identified point contacts by immunoreactions with proteins, such as vinculin and β1 integrin, known to be associated with these structures in growth cones. Our observations indicate that 9‐O‐acetyl GD3 is specifically associated with vinculin and β1 integrin in point contacts of growth cones, suggesting a possible role for this particular ganglioside in the modulation of these contacts during neurite outgrowth. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 31–37, 2003     

Mice Lacking GD3 Synthase Display Morphological Abnormalities in the Sciatic Nerve and Neuronal Disturbances during Peripheral Nerve Regeneration

The ganglioside 9-O-acetyl GD3 is overexpressed in peripheral nerves after lesioning, and its expression is correlated with axonal degeneration and regeneration in adult rodents. However, the biological roles of this ganglioside during the regenerative process are unclear. We used mice lacking GD3 synthase (Siat3a KO), an enzyme that converts GM3 to GD3, which can be further converted to 9-O-acetyl GD3. Morphological analyses of longitudinal and transverse sections of the sciatic nerve revealed significant differences in the transverse area and nerve thickness. The number of axons and the levels of myelin basic protein were significantly reduced in adult KO mice compared to wild-type (WT) mice. The G-ratio was increased in KO mice compared to WT mice based on quantification of thin transverse sections stained with toluidine blue. We found that neurite outgrowth was significantly reduced in the absence of GD3. However, addition of exogenous GD3 led to neurite growth after 3 days, similar to that in WT mice. To evaluate fiber regeneration after nerve lesioning, we compared the regenerated distance from the lesion site and found that this distance was one-fourth the length in KO mice compared to WT mice. KO mice in which GD3 was administered showed markedly improved regeneration compared to the control KO mice. In summary, we suggest that 9-O-acetyl GD3 plays biological roles in neuron-glia interactions, facilitating axonal growth and myelination induced by Schwann cells. Moreover, exogenous GD3 can be converted to 9-O-acetyl GD3 in mice lacking GD3 synthase, improving regeneration.     

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.     

Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin- deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.     

Developmental patterns of ganglioside sialosylation coincident with neuritogenesis in cultured embryonic chick brain neurons.

Chick brain precursor neurons were observed to introduce sialic acid biosynthetically into only three specific gangliosides: monosialosyl lactosyl ceramide (GM3), disialosyl lactosyl ceramide (GD3), and disialosyl gangliotrihexosyl ceramide (GD2), when sialic acid was labeled metabolically by its obligate precursor, [3H] ManNAc. Sialosyl donor CMP-[3H]NeuAc supplied in the culture medium gave rise uniquely to surface-labeled GD3. Thus sialosyl transferase/GD3 synthase activity is expressed both intraneuronally and in the neuronal exofacial surface. Upon epidermal growth factor-induced onset of neurite outgrowth, labeled complex sialosyl gangliotetrahexosyl ceramide species of gangliosides began to appear in the embryonic neuronal plasma membrane. However, intraneuronal and exofacial sialosyl transferase/GD3 synthase activities remained constant, with or without neurite outgrowth. Moreover, simpler species of gangliosides maintained a steady quantitative sialosyl level (1.6 +/- 0.2 micrograms of sialic acid/mg of protein), whereas more complex species completely absent before neurite outgrowth accrued and reached 4.8 +/- 0.9 micrograms of sialic acid/mg of protein with full neurite development. This analysis of developmental patterns of ganglioside sialosylation has provided evidence that stable neurite outgrowth depends upon generation by the neuron of special plasma membrane with a massive content of complex higher species of gangliosides.     

Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts

We have studied the function and distribution of the alpha 1 beta 1, alpha 5 beta 1 and alpha 6 beta 1 heterodimers on type-1 astrocytes with antibodies specific for integrin subunits (alpha 1, alpha 5, alpha 6, and beta 1). The alpha 1 beta 1 heterodimer mediates adhesion to laminin and collagen, the alpha 5 beta 1 to fibronectin in an RGD- dependent manner. The alpha 5 beta 1 integrin is found in focal contacts in long-term cultures of well-spread astrocytes colocalizing with vinculin and the termini of actin stress fibers. alpha 1 beta 1 heterodimers can occasionally be found as small aggregates within focal contacts but they do not accumulate there. Instead, alpha 1 beta 1 integrins are found in punctate deposits called point contacts which are distributed over the upper and the lower cell surfaces whether laminin, collagen, fibronectin or polylysine is used as a substratum. Unlike focal contacts, point contacts contain clathrin but rarely codistribute with actin or vinculin. Two observations indicate that these point contacts are functional. First, mAb 3A3, directed against the rat alpha 1 subunit, inhibits the attachment of astrocytes to laminin and collagen. Second, during the spreading of astrocytes, a band of point contacts forms around the cell perimeter at a time when no focal contacts are visible. While alpha 1 beta 1 integrins are found only in point contacts in astrocytes, the alpha 6 beta 1 integrin, another laminin receptor, is localized within focal contacts. Moreover, alpha 1 beta 1 heterodimers accumulate in focal contacts in fibroblasts. Thus, the alpha subunit contributes, independent of its ligand, to functional integrin heterodimer accumulation in focal contacts or in point contacts. This accumulation varies among different cell types with apparently identical heterodimers as well as with the motile state (spreading vs. flattened) of the same cells.     

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.     

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.     

Isolation and characterization of the major glycosphingolipids from the liver of the rainbow trout (Oncorhynchus mykiss): identification of an abundant source of 9-O-acetyl GD3

The carbohydrate structures of the major glycosphingolipids from the liver of the rainbow trout Oncorhynchus mykiss have been examined. We have isolated and identified four major neutral (glucosylceramide, galactosylceramide, lactosylceramide, and globoside) and five acidic (sulfatide, GM3, GM2, GD1a, and 9-O-Acetyl GD3) glycosphingolipids from trout liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, fast atom bombardment mass spectrometry, and specific monoclonal antibodies. Significantly, the relatively scarce ganglioside 9-O-acetyl GD3 was found to comprise approximately 23% of the total ganglioside content of normal rainbow trout liver. 9-O-Acetyl GD3 is, however, abundant in human melanoma and as such, trout liver may be a suitable source of this antigen.     

CD9-alpha6beta1 interactions in migratory parietal endoderm cells

Tetraspanins modulate the function of a variety of membrane proteins, including integrin receptors. We show here that the tetraspanin CD9 preferentially coimmunoprecipitates with the alpha6beta1 integrin heterodimer in F9-derived parietal endoderm cells in comparison to F9 stem cells. We also show that CD9 function-blocking antibody inhibits parietal endoderm migration in an embryoid body outgrowth assay. In addition, both CD9 and alpha6beta1 colocalize with vinculin to apparent focal adhesion sites in parietal endoderm cells. The data presented here suggests a role for CD9 in localizing the integrin to the focal adhesion. In addition, the data suggest a role for CD9 in alpha6beta1 mediated migration of parietal endoderm.     

Ganglioside modulation of neural cell adhesion molecule and N-cadherin- dependent neurite outgrowth

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.     

Rac1-dependent actin filament organization in growth cones is necessary for β1-integrin–mediated advance but not for growth on poly-D-lysine

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine–phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine–phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D -lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine–phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for β1 integrin–mediated growth cone advance, but not for growth on poly-D lysine. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 524–540, 1998     

Ganglioside GD3 monoclonal antibody-induced paxillin tyrosine phosphorylation and filamentous actin assembly in cerebellar growth cones

We have demonstrated that antibody to ganglioside GD3 (R24) immunoprecipitates src-family tyrosine kinase Lyn from primary cerebellar granule cells and R24 treatment of the intact cells induces Lyn activation and rapid tyrosine phosphorylation of several substrates, suggesting the functional association of ganglioside GD3 with Lyn. In this study, R24 treatment of primary cerebellar granule cells enhances phosphorylation of paxillin at tyrosine residue 118 and induces filamentous actin assembly and neurite outgrowth. R24 treatment of cerebellar growth cone membrane fraction induces prominent tyrosine phosphorylation of 68 kDa protein which comigrates with phosphopaxillin at tyrosine residue 118. Tyrosine phosphorylation of paxillin is known to regulate actin cytoskeleton-dependent changes in cell morphology. Signal transduction by ganglioside GD3 is involved in growth cone morphology via tyrosine phosphorylation of paxillin.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Growth cone interactions with a glial cell line from embryonic Xenopus retina

We have isolated a nonneuronal cell line from Xenopus retinal neuroepithelium (XR1 cell line). On the basis of immunocytochemical characterization using monoclonal antibodies generated in our laboratory as well as several other glial-specific antibodies, we have established that the XR1 cells are derived from embryonic astroglia. A monolayer of XR1 cells serves as an excellent substrate upon which embryonic retinal explants attach and elaborate neurites. This neurite outgrowth promoting activity appears not to be secreted into the medium, as medium conditioned by XR1 cells is ineffective in promoting outgrowth. Cell-free substrates were prepared to examine whether outgrowth promoting activity is also associated with the XR1 extracellular matrix (ECM). Substrates derived from XR1 cells grown on collagen are still capable of promoting outgrowth following osmotic shock and chemical extraction. This activity does not appear to be associated with laminin or fibronectin. Scanning electron microscopy was used to examine growth cones of retinal axons on XR1 cells and other substrates that supported neurite outgrowth. Growth cones and neurites growing on a monolayer of XR1 cells, or on collagen conditioned by XR1 cells, closely resemble the growth cones of retinal ganglion cells in vivo. A polyclonal antiserum (NOB1) generated against XR1 cells effectively and specifically inhibits neurite outgrowth on XR1-conditioned collagen. We therefore propose that neurite outgrowth promoting factors produced by these cells are associated with the extracellular matrix and may be glial specific.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

Beta 1 integrin-dependent and -independent polymerization of fibronectin

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.     

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013