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Growth cones are able to navigate over long distances to find their appropriate target by following guidance cues that are often presented to them in the form of an extracellular gradient. These external cues are converted into gradients of specific signaling molecules inside growth cones, while at the same time these internal signals are amplified. The amplified instruction is then used to generate asymmetric changes in the growth cone turning machinery so that one side of the growth cone migrates at a rate faster than the other side, and thus the growth cone turns toward or away from the external cue. This review examines how signal specification and amplification can be achieved inside the growth cone by multiple second messenger signaling pathways activated downstream of guidance cues. These include the calcium ion, cyclic nucleotide, and phosphatidylinositol signaling pathways., (© 2013 Wiley Periodicals, Inc.)

Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general., (Copyright © 2014 the authors 0270-6474/14/347165-14$15.00/0.)

Calcium imaging allows us to measure the spatial and temporal changes in intracellular calcium concentration in living cells. Localized calcium elevation often functions as the polarizing signal during guided migration including axon guidance. In this chapter, we describe a protocol to quantitatively monitor the spatiotemporal dynamics of calcium signals in neuronal growth cones in the presence of an extracellular concentration gradient of axon guidance cue.

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.

Neuronal network formation relies on the motile behavior of growth cones at the tip of navigating axons. Accumulating evidence indicates that growth cone motility requires spatially controlled endocytosis and exocytosis that can redistribute bulk membrane and functional cargos such as cell adhesion molecules. For axon elongation, the growth cone recycles cell adhesion molecules from its rear to its leading front through endosomes, thereby polarizing growth cone adhesiveness along the axis of migration direction. In response to extracellular guidance cues, the growth cone turns by retrieving membrane components from the retractive side or by supplying them to the side facing the new direction. We propose that polarized membrane trafficking creates adhesion gradients along and across the front-to-rear axis of growth cones that are essential for axon elongation and turning, respectively. This review will examine how growth cone adhesiveness can be patterned by spatially coordinated endocytosis and exocytosis of cell adhesion molecules. This article is part of a Special Issue entitled 'Neuronal Function'., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca(2+)) in an inositol 1,4,5-trisphosphate (IP(3))-dependent fashion as well as local activation of the Ca(2+) effector Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP(3)-Ca(2+)-CaMKK signaling in axon specification.

Semaphorin3A (Sema3A) is a repulsive guidance molecule for axons, which acts by inducing growth cone collapse through phosphorylation of CRMP2 (collapsin response mediator protein 2). Here, we show a role for CRMP2 oxidation and thioredoxin (TRX) in the regulation of CRMP2 phosphorylation and growth cone collapse. Sema3A stimulation generated hydrogen peroxide (H2O2) through MICAL (molecule interacting with CasL) and oxidized CRMP2, enabling it to form a disulfide-linked homodimer through cysteine-504. Oxidized CRMP2 then formed a transient disulfide-linked complex with TRX, which stimulated CRMP2 phosphorylation by glycogen synthase kinase-3, leading to growth cone collapse. We also reconstituted oxidation-dependent phosphorylation of CRMP2 in vitro, using a limited set of purified proteins. Our results not only clarify the importance of H2O2 and CRMP2 oxidation in Sema3A-induced growth cone collapse but also indicate an unappreciated role for TRX in linking CRMP2 oxidation to phosphorylation.

Graded distributions of extracellular cues guide developing axons toward their targets. A network of second messengers - Ca(2+) and cyclic nucleotides - shapes cue-derived information into either attractive or repulsive signals that steer growth cones bidirectionally. Emerging evidence suggests that such guidance signals create a localized imbalance between exocytosis and endocytosis, which in turn redirects membrane, adhesion and cytoskeletal components asymmetrically across the growth cone to bias the direction of axon extension. These recent advances allow us to propose a unifying model of how the growth cone translates shallow gradients of environmental information into polarized activity of the steering machinery for axon guidance.

The activity of PI3K is necessary for polarized cell motility. To guide extending axons, environmental cues polarize the growth cone via asymmetric generation of Ca(2+) signals and subsequent intracellular mechanical events, including membrane trafficking and cytoskeletal reorganization. However, it remains unclear how PI3K is involved in such events for axon guidance. Here, we demonstrate that PI3K plays a permissive role in growth cone turning by facilitating microtubule (MT)-dependent membrane transport. Using embryonic chick dorsal root ganglion neurons in culture, attractive axon turning was induced by Ca(2+) elevations on one side of the growth cone by photolyzing caged Ca(2+) or caged inositol 1,4,5-trisphosphate. We show that PI3K activity was required downstream of Ca(2+) signals for growth cone turning. Attractive Ca(2+) signals, generated with caged Ca(2+) or caged inositol 1,4,5-trisphosphate, triggered asymmetric transport of membrane vesicles from the center to the periphery of growth cones in a MT-dependent manner. This centrifugal vesicle transport was abolished by PI3K inhibitors, suggesting that PI3K is involved in growth cone attraction at the level of membrane trafficking. Consistent with this observation, immunocytochemistry showed that PI3K inhibitors reduced MTs in the growth cone peripheral domain. Time-lapse imaging of EB1 on the plus-end of MTs revealed that MT advance into the growth cone peripheral domain was dependent on PI3K activity: inhibition of the PI3K signaling pathway attenuated MT advance, whereas exogenous phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K-catalyzed reactions, promoted MT advance. This study demonstrates the importance of PI3K-dependent membrane trafficking in chemotactic cell migration.

Asymmetric Ca(2+) elevations across the axonal growth cone mediate its turning responses to attractive and repulsive guidance cues. Here we show that clathrin-mediated endocytosis acts downstream of Ca(2+) signals as driving machinery for growth cone turning. In dorsal root ganglion neurons, the formation of clathrin-coated pits is facilitated asymmetrically across the growth cone by a directionally applied chemorepellent, semaphorin 3A, or by Ca(2+) signals that mediate repulsive guidance. In contrast, coated pit formation remains symmetric in the presence of attractive Ca(2+) signals. Inhibition of clathrin-mediated endocytosis abolishes growth cone repulsion, but not attraction, induced by Ca(2+) or extracellular physiological cues. Furthermore, asymmetric perturbation of the balance of endocytosis and exocytosis in the growth cone is sufficient to initiate its turning toward the side with less endocytosis or more exocytosis. With our previous finding that growth cone attraction involves asymmetric exocytosis, we propose that the balance between membrane addition and removal dictates bidirectional axon guidance., (Copyright 2010 Elsevier Inc. All rights reserved.)

The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.

Inositol 1,4,5-trisphosphate (IP(3)) is generally viewed as a global messenger that increases cytosolic calcium ion (Ca(2+)) concentration. However, the spatiotemporal dynamics of IP(3) and the functional significance of localized IP(3) production in cell polarity remain largely unknown. Here, we demonstrate the critical role of spatially restricted IP(3) signals in axon guidance. We found that IP(3) and ensuing Ca(2+) signals were produced asymmetrically across growth cones exposed to an extracellular gradient of nerve growth factor (NGF) and mediated growth cone turning responses to NGF. Moreover, photolysis-induced production of IP(3) on one side of a growth cone was sufficient to initiate growth cone turning toward the side with the higher concentration of IP(3). Thus, locally produced IP(3) encodes spatial information that polarizes the growth cone for guided migration.

Asymmetric Ca(2+) signals across the growth cone mediate attractive or repulsive axon guidance depending on the occurrence of Ca(2+)-induced Ca(2+) release (CICR) through ryanodine receptors (RyRs). Although the neuronal isoform of nitric oxide (NO) synthase (nNOS) is highly expressed in developing dorsal root ganglion (DRG) neurons, the role of NO in axon guidance remains essentially unknown. Here we report that the NO-cGMP pathway negatively regulates CICR to control the directional polarity of DRG axon guidance. Intracellular levels of NO and cGMP depend on extracellular substrates: laminin activates the NO-cGMP pathway, whereas the adhesion molecule L1 does not. The activity of NO and cGMP determines the turning direction of growth cones with respect to asymmetric Ca(2+) signals that are produced by photolysing caged Ca(2+). The Ca(2+) signals cause growth cone repulsion on a laminin substrate, which is converted to attraction by pharmacological blockade of the NO-cGMP pathway or genetic deletion of nNOS. Conversely, Ca(2+)-induced growth cone attraction on an L1 substrate is converted to repulsion by increasing NO levels. Such NO-mediated switching of turning direction involves the regulation of CICR through RyRs. Furthermore, growth cone repulsion induced by an extracellular gradient of a physiological cue, neurotrophin-4, is dependent on Ca(2+) signals and converted to attraction by inhibiting the NO-cGMP pathway. These results suggest that, on contact with different adhesive environments, growth cones can change their turning responses to axon guidance cues by modulating CICR via endogenous NO and cGMP.

During development, asymmetric Ca(2+) signals across the growth cone mediate bidirectional axon guidance depending on intracellular levels of cyclic AMP: Ca(2+) signals trigger attractive or repulsive turning when cyclic AMP levels are high or low, respectively. Here, we report that the cell adhesion molecule L1 elevates cyclic AMP levels in neurons via ankyrin(B), a protein that links the L1 cytoplasmic tail with the spectrin network. We also show that the loss of ankyrin(B) expression converts Ca(2+)-triggered attraction to repulsion when the growth cone migrates via an L1-dependent mechanism. These results indicate that ankyrin(B) regulates axon guidance via cyclic AMP.

Asymmetric elevation of the Ca(2+) concentration in the growth cone can mediate both attractive and repulsive axon guidance. Ca(2+) signals that are accompanied by Ca(2+)-induced Ca(2+) release (CICR) trigger attraction, whereas Ca(2+) signals that are not accompanied by CICR trigger repulsion. The molecular machinery downstream of Ca(2+) signals, however, remains largely unknown. Here we report that asymmetric membrane trafficking mediates growth cone attraction. Local photolysis of caged Ca(2+), together with CICR, on one side of the growth cone of a chick dorsal root ganglion neuron facilitated the microtubule-dependent centrifugal transport of vesicles towards the leading edge and their subsequent vesicle-associated membrane-protein 2 (VAMP2)-mediated exocytosis on the side with an elevated Ca(2+) concentration. In contrast, Ca(2+) signals without CICR had no effect on the vesicle transport. Furthermore, pharmacological inhibition of VAMP2-mediated exocytosis prevented growth cone attraction, but not repulsion. These results strongly suggest that growth cone attraction and repulsion are driven by distinct mechanisms, rather than using the same molecular machinery with opposing polarities.

Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric Ca2+ elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89-93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909-916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates Ca2+-induced Ca2+ release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when Ca2+ signals are not accompanied by RyR3-mediated CICR. We also propose that the source of Ca2+ influx, rather than its amplitude or the baseline Ca2+ level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.

Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with alpha-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.