Nitric oxide regulates growth cone filopodial dynamics via ryanodine receptor-mediated calcium release

Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca(2+)](i)). Moreover, these NO-induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca(2+)](i) that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO-induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process.     

Pioneer growth cone steering decisions mediated by single filopodial contacts in situ

In grasshopper embryo limb buds, the sibling Ti1 pioneers are the first neurons to initiate axonogenesis. The pioneer growth cones migrate from the limb tip to the CNS along a in direction comprising discrete steering events. Filopodial exploration of the cellular terrain in the vicinity of the advancing growth cone appears to be important for steering. Some information is available on the identity of cells and cell types, on cell-surface characteristics, and on the involvement of basal lamina in these steering decisions. In the work reported here, we have used computer-enhanced fluorescence video microscopy to examine filopodial behavior and the process of growth cone migration and reorientation resulting from interactions with the normal guidance cues on the in situ substrate. We observed several different kinds of migration and steering events, which appear to be related to the absolute and relative affinities of the contacted substrates. On a relatively homogeneous substrate of intrasegmental epithelium, growth cones advance by extending veils between filopodia, as is commonly observed on uniform substrates in vitro. Where growth cones confront an orthogonal border between substrates of dissimilar affinity, they remain on the higher-affinity substrate by extending new branches along it. Subsequently, reorientation in the preferred direction on the higher-affinity substrate is accomplished by regression of branches extended in the nonselected direction. By contrast, a single filopodial contact with a very high-affinity substrate, such as a guidepost neuron, can reorient a growth cone, even when it is migrating on a favorable substrate. In this situation, the filopodium that contacts the high-affinity substrate expands in diameter until it becomes the nascent axon.     

The selective inhibition of growth cone extension by specific neurites in culture

We have found that neurites from embryonic chick retinal and sympathetic explants do not mix in culture, while retinal neurites mix with retinal neurites, and sympathetic neurites mix with sympathetic neurites. These results confirm those obtained with embryonic rat tissues by Bray et al. (1980). We have also used video time-lapse techniques to examine the behavior of individual retinal and sympathetic growth cones as they attempt to cross retinal and sympathetic neurites. We have found that retinal growth cones cross retinal neurites without delay, retinal growth cones usually retract from sympathetic neurites but often cross them on a second advance, sympathetic growth cones repeatedly retract from retinal neurites, rarely managing to cross them at all, and sympathetic growth cones cross sympathetic neurites but are sometimes delayed. Our results indicate that retinal and sympathetic growth cones can distinguish between specific labels associated with retinal and sympathetic neurites. They also suggest that active avoidance could play an important role in growth cone navigation.     

A quantitative analysis of branching,growth cone turning,and directed growth in zebrafish retinotectal axon guidance

The topographic projection from the eye to the tectum (amphibians and fish)/superior colliculus (birds and mammals) is a paradigm model system for studying mechanisms of neural wiring development. It has previously been proposed that retinal ganglion cell axons use distinct guidance strategies in fish vs. mammals, with direct guidance to the tectal target zone in the former and overshoot followed by biased branching toward the target zone in the latter. Here we visualized individual retinal ganglion cell axons as they grew over the tectum in zebrafish for periods of 10‐21 hours and analyzed these results using an array of quantitative measures. We found that, although axons were generally guided directly toward their targets, this occurred without growth cone turning. Instead, axons branched dynamically and profusely throughout pathfinding, and successive branches oriented growth cone extension toward a target zone in a stepwise manner. These data suggest that the guidance strategies used between fish and mammals may be less distinct than previously thought. J. Comp. Neurol., 521:1409–1429, 2013. © 2012 Wiley Periodicals, Inc.     

In vivo imaging of growth cone and filopodial dynamics: evidence for contact-mediated retraction of filopodia leading to the tiling of sibling processes

In the leech embryo, the peripheral comb cell (CC) sends out many nonoverlapping, growth cone-tipped processes that grow in parallel and serve as a scaffold for the migrating myocytes of the later-developing oblique muscle layer. To explore how the parallel arrangement is generated we first examined the arrangement of CC cytoskeletal components by expressing a tubulin-binding protein and actin, both tagged with fluorescent reporters. This revealed that the growth cones were compartmentalized into F-actin-rich filopodia and a microtubule-rich central region. Time-lapse analysis with a 2-photon laser scanning microscope revealed that the growth cones of the CC are highly dynamic, undergoing rapid filopodial extension and retraction. Measurements of filopodial lifespan and length revealed that most filopodia at the leading edge of the growth cone achieved significantly longer lifespans and length than lateral filopodia. Furthermore, for the short-lived lateral filopodia, apparent interaction with a neighboring process was found to be a significant predictor of their nearly immediate (within 2-4 minutes) retraction. When contact was experimentally prevented by ablating individual CCs, the filopodia from the growth cones of adjacent segmental neighbors were found to be significantly lengthened in the direction of the removed homolog. Treatment with low doses of cytochalasin D to disrupt F-actin assembly led to filopodial retraction and growth cone collapse and resulted in the bifurcation of many CC processes, numerous crossover errors, and the loss of parallelism. These findings indicate the existence of a contact-mediated repulsive interaction between processes of the CC.     

Multiple signaling pathways regulate FGF-2-induced retinal ganglion cell neurite extension and growth cone guidance

Growth cones use cues in their environment in order to grow in a directed fashion to their targets. In Xenopus laevis, fibroblast growth factors (FGFs) participate in retinal ganglion cell (RGC) axon guidance in vivo and in vitro. The main intracellular signaling cascades known to act downstream of the FGF receptor include the mitogen-activated protein kinase (MAPK), phospholipase Cgamma (PLCgamma) and phosphotidylinositol 3-kinase (PI3K) pathways. We used pharmacological inhibitors to identify the signaling cascade(s) responsible for FGF-2-stimulated RGC axon extension and chemorepulsion. The MAPK, PI3K and PLCgamma pathways were blocked by U0126, LY249002 and U73122, respectively. D609 was used to test a role for the phosphotidylcholine-PLC (PC-PLC) pathway. We determined that the MAPK and two PLC pathways are required for FGF-2 to stimulate RGC neurite extension in vitro, but the response of axons to FGF-2 applied asymmetrically to the growth cone depended only on the PLC pathways.     

Semi-automated quantification of filopodial dynamics

Cellular motility underlies critical physiological processes including embryogenesis, metastasis and wound healing. Nerve cells undergo cellular migration during development and also extend neuronal processes for long distances through a complex microenvironment to appropriately wire the nervous system. The growth cone is a highly dynamic structure that responds to extracellular cues by extending and retracting filopodia and lamellipodia to explore the microenvironment and to dictate the path and speed of process extension. Neuronal responses to a myriad of guidance cues have been studied biochemically, however, these approaches fail to capture critical spatio-temporal elements of growth cone dynamics. Live imaging of growth cones in culture has emerged as a powerful tool to study growth cone responses to guidance cues but the dynamic nature of the growth cone requires careful quantitative analysis. Space time kymographs have been developed as a tool to quantify lamellipodia dynamics in a semi-automated fashion but no such tools exist to analyze filopodial dynamics. In this work we present an algorithm to quantify filopodial dynamics from cultured neurons imaged by time-lapse fluorescence microscopy. The method is based on locating the end tips of filopodia and tracking their locations as if they were free-moving particles. The algorithm is a useful tool and should be broadly applicable to filopodial tracking from multiple cell types.     

Evidence for microtubule capture by filopodial actin filaments in growth cones.

We have studied, by immunofluorescence, the organization of microtubules in growth cones in dissociated neuronal cell cultures. Growth cones have a large pool of soluble tubulin and in conventionally fixed cultures the entire growth cone, including the filopodia, is labelled with tubulin antibodies, thus obscuring the microtubules. In contrast, in cultures fixed with fixatives containing detergent, the soluble pool of tubulin is removed allowing clear views of the microtubules. On entering the growth cone from the neurite, microtubules splay out and may extend as far as the base of filopodia. In most growth cones, one or more individual microtubules extend into filopodia, aligning with the parallel bundle of microfilaments. This observation suggests one way growth cone motility and neurite advance may be coupled.     

A common denominator of growth cone guidance and collapse?

Axonal guidance in the retinotectal system and in spinal nerve segmentation is based on repulsion or inhibition. In both systems the membrane glycoprotein responsible for the guiding activity is capable of inducing growth cone collapse. We discuss two models of axonal guidance that correlate axonal guidance and growth cone collapse. The models are applicable to axon guidance by membrane-associated or diffusible stimuli, and are not based on preferential adhesion of axons to certain substrata.     

ERM proteins regulate growth cone responses to Sema3A

Axonal growth cones initiate and sustain directed growth in response to cues in their environment. A variety of events such as receptor internalization, kinase activation, and actin rearrangement can be stimulated by guidance cues and are essential for mediating targeted growth cone behavior. Surprisingly little is known about how such disparate actions are coordinated. Our data suggest that ezrin, radixin, and moesin (ERMs), a family of highly homologous, multifunctional proteins may be able to coordinate growth cone responses to the guidance cue Semaphorin 3A (Sema3A). We show that active ERMs concentrate asymmetrically in neocortical growth cones, are rapidly and transiently inactivated by Sema3A, and are required for Sema3A-mediated growth cone collapse and guidance. The FERM domain of active ERMs regulates internalization of the Sema3A receptor, Npn1, and its coreceptor, L1CAM, while the ERM C-terminal domain binds and caps F-actin. Our data support a model in which ERMs can coordinate membrane and actin dynamics in response to Sema3A.     

Regulation of growth cone morphology by nerve growth factor: a comparative study by scanning electron microscopy

The object of this study was to document and analyze local regulation by nerve growth factor (NGF) of neuronal growth cone properties and to explore the possible diversity of this effect in various NGF-responsive preparations. In particular, scanning electron microscopy was used to characterize the morphology of neuronal growth cones in cultures of dissociated chick embryo dorsal root ganglia (DRG) under conditions of continuous NGF exposure, withdrawal of NGF for 5-6 hr, and restoration of NGF for various times. Comparison was made with similarly manipulated cultures of dissociated newborn rat sympathetic ganglia and neurite-bearing PC12 pheochromocytoma cells. The growth cones of most of the continuously NGF-treated DRG neurons (cultured on poly-L-lysine or collagen-coated glass coverslips) had relatively compact central flattened areas and numerous prominent filopodia. Withdrawal of NGF resulted in a marked spreading of the central growth cone area so that the average maximum width of this structure increased by about threefold as compared to nondeprived cultures. The mean number and lengths of filopodia were unaffected. Restoration of NGF brought about, over a time course of tens of minutes, a return of the original type of growth cone morphology. Rather different responses were observed for the sympathetic neuron and PC12 cultures. Here, surface ruffles, only rarely seen in the chick cultures, were a major feature of the growth cones, whereas filopodia, though present, were less prominent. Removal of NGF led to loss of ruffles and to rounding up of the growth cones; NGF readdition elicited a rapid (less than 30 sec) reinitiation of ruffling and a more gradual (over tens of minutes) respreading of growth cones. These findings illustrate not only that NGF can regulate growth cone properties, but also that there is a diversity as to how this is manifested. Possible mechanisms and biological roles for this regulation are discussed.     

Morphogens as growth cone signalling molecules

Morphogen signalling among cells is one of the most important mechanisms underlying the progressive patterning of embryos. Members of the hedgehog (Hh), wingless (Wnt), transforming growth factor-β (TGFβ), and fibroblast growth factor (Fgf) families of extracellular signalling molecules act as morphogens. Recent studies have demonstrated that members of these four families of proteins, secreted by well-characterised organiser centres in the central nervous system (CNS) as floor plate or midbrain–hindbrain boundary, are reused at later developmental stages to control axon growth. Here, we have summarised the evidence for this novel idea with a particular emphasis on those related to Shh and Wnt signalling—the object of some works in our laboratory.     

Actin-based growth cone motility and guidance

Nerve growth cones, the dilated tip of developing axons, are equipped with exquisite abilities to sense environmental cues and to move rapidly through complex terrains of developing brain, leading the axons to their specific targets for precise neuronal wiring. The actin cytoskeleton is the major component of the growth cone that powers its directional motility. Past research has provided significant insights into the mechanisms by which growth cones translate extracellular signals into directional migration. In this review, we summarize the actin-based mechanisms underlying directional growth cone motility, examine novel findings, and discuss the outstanding questions concerning the actin-based growth cone behaviors.     

Cortactin colocalizes with filopodial actin and accumulates at IgCAM adhesion sites in Aplysia growth cones

Both IgCAMs and the actin cytoskeleton play critical roles in neuronal growth cone motility and guidance. However, it is unclear how IgCAM receptors transduce signals from the plasma membrane to induce actin remodeling. Previous studies have shown that local clustering and immobilization of apCAM, the Aplysia homolog of NCAM, induces Src kinase activity and F‐actin polymerization in the peripheral domain of cultured Aplysia bag cell growth cones. Therefore, we wanted to test whether the Src kinase substrate and actin regulator cortactin could be a molecular link between Src activity and actin assembly during apCAM‐mediated growth cone guidance. Here, we cloned Aplysia cortactin and showed that it is abundant in the nervous system. Immunostaining of growth cones revealed a strong colocalization of cortactin with F‐actin in filopodial bundles and at the leading edge of lamellipodia. Perturbation of the cytoskeleton indicated that cortactin distribution largely depends on actin filaments. Furthermore, active Src colocalized with cortactin in regions of actin assembly, including leading edge and filopodia tips. Finally, we observed that cortactin, like F‐actin, localizes to apCAM adhesion sites mediating growth cone guidance. Altogether, these data suggest that cortactin is a mediator of IgCAM‐triggered actin assembly involved in growth cone motility and guidance. © 2008 Wiley‐Liss, Inc.     

Semaphorin 3A: from growth cone repellent to promoter of neuronal regeneration

正Highlight Semaphorin 3A is a classically known axonal guidance cue that mediates axonal growth cone repulsion and collapse.Recent works,however,suggest that it may have the apparently diametrically opposite activity of promoting neuronal regeneration.During embryonic development,the axonal guidance cues facilitate the navigation of axonal growth cones towards the targets     

Activation of ezrin/radixin/moesin mediates attractive growth cone guidance through regulation of growth cone actin and adhesion receptors

The development of a functioning neural network relies on responses of axonal growth cones to molecular guidance cues that are encountered en route to their target tissue. Nerve growth factor (NGF) and neurotrophin-3 serve as attractive cues for chick embryo sensory growth cones in vitro and in vivo, but little is known about the actin-binding proteins necessary to mediate this response. The evolutionarily conserved ezrin/radixin/moesin (ERM) family of proteins can tether actin filaments to the cell membrane when phosphorylated at a conserved threonine residue. Here we show that acute neurotrophin stimulation rapidly increases active phospho-ERM levels in chick sensory neuron growth cone filopodia, coincident with an increase in filopodial L1 and β-integrin. Disrupting ERM function with a dominant-negative construct (DN-ERM) results in smaller and less motile growth cones with disorganized actin filaments. Previously, we found that NGF treatment increases actin-depolymerizing factor (ADF)/cofilin activity and growth cone F-actin (Marsick et al., 2010). Here, we show this F-actin increase, as well as attractive turning to NGF, is blocked when ERM function is disrupted despite normal activation of ADF/cofilin. We further show that DN-ERM expression disrupts leading edge localization of active ADF/cofilin and free F-actin barbed ends. Moreover, filopodial phospho-ERM levels are increased by incorporation of active ADF/cofilin and reduced by knockdown of L1CAM.Together, these data suggest that ERM proteins organize actin filaments in sensory neuron growth cones and are crucial for neurotrophin-induced remodeling of F-actin and redistribution of adhesion receptors.