Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.     

High-resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.     

High‐resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three‐dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force‐distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F‐actin bundles are stiffer than surrounding F‐actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Growth cone guidance in the zebrafish central nervous system.

The accessibility and simplicity of the zebrafish embryo have allowed researchers to make a detailed characterization of pathfinding by identifiable growth cones. The growth cones follow precise cell-specific pathways to their targets. Analyses of pathfinding in mutant and experimentally manipulated wild type embryos have shown that growth cones accomplish this by interacting with specific cellular cues in their environment, many of which are likely to be redundant.     

Ultrastructure of an identified array of growth cones and possible substrates for guidance in the embryonic medicinal leech,Hirudo medicinalis

The oblique muscle organizer (Comb- or C-cell) in the embryonic medicinal leech, Hirudo medicinalis, provides an amenable situation to examine growth cone navigation in vivo. Each of the segmentally iterated C-cells extends an array of growth cones through the body wall along oblique trajectories. C-cell growth cones undergo an early, relatively slow period of extension followed by later, protracted and rapid directed outgrowth. During such transitions in extension, guidance might be mediated by a number of factors, including intrinsic constraints on polarity, spatially and temporally regulated cell and matrix interactions, physical constraints imposed by the environment, or guidance along particular cells in advance of the growth cones. Growth cones and their environment were examined by transmission electron microscopy to define those factors that might play a significant role in migration and guidance in this system. The ultrastructural examination has made the possibility very unlikely that simple, physical constraints play a prominent role in guiding C-cell growth cones. No anatomically defined paths or obliquely aligned channels were found in advance of these growth cones, and there were no identifiable physical boundaries, which might constrain young growth cones to a particular location in the body wall before rapid extension. There were diverse associations with many matrices and basement membranes located above, below, and within the layer in which growth cones appear to extend at the light level. Additionally, a preliminary examination of myocyte assembly upon processes proximal to the growth cones further implicates a role for matrix-associated interactions in muscle histogenesis as well as process outgrowth during embryonic development.     

Multiple strategies for directed growth cone extension and navigation of peripheral neurons

Leeches have a diverse constellation of peripheral neural elements that are challenged to extend growth cones in highly specific ways in a constantly changing embryonic environment. Two major systems are reviewed here. In one, peripheral afferents extend growth cones toward the central nervous system (CNS), forming common pathways, and then segregate into particular tracts within the CNS. A majority of these afferents depend on CNS-derived guidance cues and projections from the CNS to guide their way. However, not all of the nerves are established this way and at least one of the peripheral nerves is likely to be pioneered by sensillar sensory afferents. The distribution of particular antigens (such as the lan3–2 antigen) suggests the identity of molecules involved in homophilic adhesion along common pathways, whereas others (such as the lan4–2 and 3–6 antigens) are candidates for mediating specific pathway choices. In the second system, the myo-organizing Comb cell (C cell) projects multiple growth cones simultaneously along oblique trajectories not influenced by segmental or midline boundaries. Its parallel growth cones exhibit space-filling as well as directional growth and are guided by local cues to extend in discrete phases that are coordinated with the development of the environment. Both systems exhibit highly directed outgrowth orchestrated by a hierarchy of cues, establish patterns of neurites used to direct later migrating cells, and seem to be regulated temporally and spatially by interactions with the embryonic environment. These systems illustrate the strengths of examining neural development in vivo across several levels of analysis. © 1995 John Wiley & Sons, Inc.     

The role of repulsive guidance molecules in the embryonic and adult vertebrate central nervous system

During the development of the nervous system, outgrowing axons often have to travel long distances to reach their target neurons. In this process, outgrowing neurites tipped with motile growth cones rely on guidance cues present in their local environment. These cues are detected by specific receptors expressed on growth cones and neurites and influence the trajectory of the growing fibres. Neurite growth, guidance, target innervation and synapse formation and maturation are the processes that occur predominantly but not exclusively during embryonic or early post-natal development in vertebrates. As a result, a functional neural network is established, which is usually remarkably stable. However, the stability of the neural network in higher vertebrates comes at an expensive price, i.e. the loss of any significant ability to regenerate injured or damaged neuronal connections in their central nervous system (CNS). Most importantly, neurite growth inhibitors prevent any regenerative growth of injured nerve fibres. Some of these inhibitors are associated with CNS myelin, others are found at the lesion site and in the scar tissue. Traumatic injuries in brain and spinal cord of mammals induce upregulation of embryonic inhibitory or repulsive guidance cues and their receptors on the neurites. An example for embryonic repulsive directional cues re-expressed at lesion sites in both the rat and human CNS is provided with repulsive guidance molecules, a new family of directional guidance cues.     

Mechanisms of growth cone guidance and motility in the developing grasshopper embryo

During neuronal pathfinding in vivo, growth cones must reorient their direction of migration in response to extracellular guidance cues. The developing grasshopper limb bud has proved to be a model system in which to examine mechanisms of growth cone guidance and motility in vivo. In this review we examine the contributions of adhesion and multiple guidance cues (semaphorins 1 and 2) in directing a growth cone steering event. Recent observations have suggested that the tibial pioneer growth cones are not directed via mechanisms of differential adhesivity. We present a model of growth cone steering that suggests a combination of adhesive and guidance receptors are important for a correct steering event and that guidance molecules may be important regulators of adhesive interactions with the actin cytoskeleton.     

Regulation of growth cone actin dynamics by ADF/cofilin.

Nervous system development is reliant on neuronal pathfinding, the process in which axons are guided to their target cells by specific extracellular cues. The ability of neurons to extend over long distances in response to environmental guidance signals is made possible by the growth cone, a highly motile structure found at the end of neuronal processes. Growth cones detect directional cues and respond with either attractive or repulsive movements. The motility of growth cones is dependent on rapid reorganization of the actin cytoskeleton, presumably mediated by actin-associated proteins under the control of incoming guidance signals. This article reviews how one such family of proteins, the ADF/cofilins, are emerging as key regulators of growth cone actin dynamics. These proteins are essential for rapid actin turnover in a variety of different cell types. ADF/cofilins are heavily co-localized with actin in growth cones and are necessary for neurite outgrowth. ADF/cofilin activities are regulated through reversible phosphorylation by LIM kinases and slingshot phosphatases. LIM kinases are downstream effectors of the Rho GTPases Rho, Rac, and Cdc42. Growing evidence suggests that extracellular guidance cues may locally alter actin dynamics by regulating the activity of LIM kinase and ADF/cofilin phosphatases via the Rho GTPases. In this way, ADF/cofilins and their upstream effectors may be pivotal to our understanding of how guidance information is translated into physical alterations of the growth cone actin cytoskeleton.     

HuC–eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones

The field of axon guidance is taking advantage of the powerful genetic and imaging tools that are now available to visualise growth behaviour in living cells, both in vivo and in real time. We have developed a method to visualise individual neurons within the living zebrafish embryo which provides exceptional cellular resolution of growth cones and their filopodia. We generated a DNA construct in which the HuC promoter drives expression of eGFP. Injection of the plasmid into single cell fertilised zebrafish egg resulted in mosaic expression of eGFP in neurons throughout the developing embryo. By manipulating the concentration of injected plasmid, it was possible to optimise the numbers of neurons that expressed the construct so that individual growth cones could be easily visualised. We then used time-lapse high magnification widefield epifluorescence microscopy to visualise the growth cones as they were exploring their environment. Growth cones both near the surface of the embryo as well as deep within the developing brain of embryos at 20?h post fertilisation were clearly imaged. With time-lapse sequence imaging with intervals between frames as frequent as 1?s there was minimal loss of fluorescence intensity and the dynamic nature of the growth cones became evident. This method therefore provides high magnification, high resolution time-lapse imaging of living neurons in vivo and by use of widefield epifluorescence rather than confocal it is a relatively inexpensive microscopy method.     

Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3-7 and 7-23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10-40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.     

Guidance of pioneer growth cones: Filopodial contacts and coupling revealed with an antibody to Lucifer Yellow

We are interested in the factors that guide individual neuronal growth cones during embryonic development. We have developed an antibody to the fluorescent dye Lucifer Yellow. We use the antibody here to examine the specific filopodial contacts and dye coupling by the first growth cones in the grasshopper embryo that navigate in an axonless environment. We have studied the distribution and apparent selective adhesion of the filopodia from these pioneering growth cones in the central nervous system and periphery. Our results suggest that selective filopodial adhesion to specific “landmark” cells may play an important role in the guidance of pioneer growth cones.     

Suppression of Radixin and Moesin Alters Growth Cone Morphology,Motility, and Process Formation In Primary Cultured Neurons

In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin–radixin or ezrin–moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin–moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8–10 times slower than the one of growth cones from control neurons. Radixin–moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.     

Modeling the role of myosin 1c in neuronal growth cone turning

We addressed the mechanical basis for how embryonic chick dorsal root ganglion growth cones turn on a uniform substrate of laminin-1. Turning is significantly correlated with lamellipodial area but not with filopodial length. We assessed the lamellipodial contribution to turning by asymmetric micro-CALI of myosin isoforms that causes localized lamellipodial expansion (myosin 1c) or filopodial retraction (myosin V). Episodes of asymmetric micro-CALI of myosin 1c (or myosin 1c and V together) caused significant turning of the growth cone. In contrast, repeated micro-CALI of myosin V or irradiation without added antibody did not turn growth cones. These findings argue that lamellipodia and not filopodia are necessary for growth cone turning. To model the role of myosin 1c on growth cone turning, we fitted the measured trajectories from asymmetric micro-CALI of myosin 1c-treated and untreated growth cones to the persistent random walk model. The first parameter in this equation, root-mean-square speed, is indistinguishable between the two data sets whereas the second parameter, the persistence of motion, is significantly increased (2.5-fold) as a result of asymmetric inactivation of myosin 1c by micro-CALI. This analysis demonstrates that growth cone turning results from an increase in the persistence of directional motion rather than a change in speed. Taken together, our results suggest that myosin 1c is a molecular correlate for directional persistence underlying growth cone motility.     

Herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons

The mechanism of anterograde transport of alphaherpesviruses in axons remains controversial. This study examined the transport, assembly, and egress of herpes simplex virus type 1 (HSV-1) in mid- and distal axons of infected explanted human fetal dorsal root ganglia using confocal microscopy and transmission electron microscopy (TEM) at 19, 24, and 48 h postinfection (p.i.). Confocal-microscopy studies showed that although capsid (VP5) and tegument (UL37) proteins were not uniformly present in axons until 24 h p.i., they colocalized with envelope (gG) proteins in axonal varicosities and in growth cones at 24 and 48 h p.i. TEM of longitudinal sections of axons in situ showed enveloped and unenveloped capsids in the axonal varicosities and growth cones, whereas in the midregion of the axons, predominantly unenveloped capsids were observed. Partially enveloped capsids, apparently budding into vesicles, were observed in axonal varicosities and growth cones, but not during viral attachment and entry into axons. Tegument proteins (VP22) were found associated with vesicles in growth cones, either alone or together with envelope (gD) proteins, by transmission immunoelectron microscopy. Extracellular virions were observed adjacent to axonal varicosities and growth cones, with some virions observed in crescent-shaped invaginations of the axonal plasma membrane, suggesting exit at these sites. These findings suggest that varicosities and growth cones are probable sites of HSV-1 envelopment of at least a proportion of virions in the mid- to distal axon. Envelopment probably occurs by budding of capsids into vesicles with associated tegument and envelope proteins. Virions appear to exit from these sites by exocytosis.     

Responses of temporal retinal growth cones to ephrinA5-coated beads

The topographic positioning of retinal axons in the optic tectum is regulated, at least in part, by ephrinA/EphA repulsive interactions. Temporal axons, expressing high levels of EphA receptors, project to the ephrinA5-poor anterior tectum and avoid the ephrinA5-rich posterior tectum. To examine the dynamic behavior of temporal growth cones when they first encounter ephrinA, we manipulated ephrinA-coated beads with a laser tweezer into desired positions around the growth cones of chick retinal axons in culture. At high concentrations of ephrinA5 on the beads, growth cones typically collapsed on contacting the bead. At low concentrations, however, growth cones showed heterogeneous responses with some growth cones showing repulsive turning and others showing attractive turning after contacting the bead. Experiments with two beads indicate that retinal axons integrate guidance information that is provided simultaneously at two discrete locations. When a time-delay was introduced between exposure to the first and the second bead, individual axons exhibited a stereotyped response to the repeated stimuli, either responding with attraction followed by attraction, or showing repulsion followed by repulsion or collapse. Our results suggest the existence of at least two retinal subpopulations from the temporal retina, one being attracted, another being repelled by low levels of ephrinA5. These findings demonstrate that temporal retinal axons are not universally repelled by ephrinA5 and suggest that their ability to respond differentially to low concentrations may help them to map in a continuous manner over the surface of the anterior tectum.     

A comparison of calcium homeostasis in isolated and attached growth cones of the snail Helisoma

This study examines the capability of growth cones from identified neurons of the snail Helisoma trivolvis to perform calcium homeostasis. Calcium influx into the cytoplasm was eliminated or increased experimentally to alter [Ca]i, and the compensatory response of the growth cone was measured with the fluorescent calcium indicator Fura-2. Growth cones compensated for both increases and decreases in calcium influx by restoring [Ca]i towards basal levels under both types of challenges. The intrinsic ability of growth cones to control [Ca]i was examined in physically isolated growth cones. Isolated growth cones demonstrated essentially identical calcium homeostatic properties to their intact counterparts, indicating that mechanisms governing calcium homeostasis exist intrinsically in the growth cone. Such independence may add significantly to the growth cone's potential to locally interpret and respond to stimuli encountered en route to its appropriate target.     

Making connections in the fly visual system

Understanding the molecular mechanisms that regulate formation of precise patterns of neuronal connections within the central nervous system remains a challenging problem in neurobiology. Genetic studies in worms and flies and molecular studies in vertebrate systems have led to an increasingly sophisticated understanding of how growth cones navigate toward their targets and form topographic maps. Considerably less is known about how growth cones recognize their cellular targets and form synapses with them. Here, we review connection formation in the fly visual system, the methodological approaches used to study it, and recent progress in uncovering the molecular basis of connection specificity.     

Nerve growth cone migration onto Schwann cells involves the calcium-dependent adhesion molecule, N-cadherin

The role of calcium-dependent adhesion molecules in the migration of nerve growth cones onto the top of Schwann cells was probed by examination of sensory growth cone-Schwann cell interactions in medium containing either 1.0 mM Ca2+ or 0.1 mM Ca2+. In the presence of 1.0 mM Ca2+ growth cones rapidly migrated onto Schwann cells, spread, and remained for extended periods. However, in 0.1 mM Ca2+ growth cones still made frequent contacts with Schwann cells, but migration onto the upper cell surface was much reduced. This contrast in growth cone-Schwann cell interactions could be switched rapidly by changing the Ca2+ concentration of the culture medium. Growth cones of retinal neurons showed similar calcium-dependence in their migration onto Schwann cells. Antibodies to the calcium-dependent adhesion molecule, N-cadherin, also blocked growth cone migration onto Schwann cells, but antibodies to another neuronal adhesion molecule, L1, had no effect on growth cone-Schwann cell interactions. Immunocytochemical staining for N-cadherin and L1 indicated that growth cones and Schwann cells have N-cadherin on their surfaces, while L1 is present only on axons and growth cones. These results provide two kinds of evidence that N-cadherin is important in the initial interactions of growth cones and Schwann cells.     

Proper cellular reorganization during Drosophila spermatid individualization depends on actin structures composed of two domains, bundles and meshwork, that are differentially regulated and have different functions

During spermatid individualization in Drosophila, actin structures (cones) mediate cellular remodeling that separates the syncytial spermatids into individual cells. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. We show here that the two domains form separately in time, are regulated by different sets of actin-associated proteins, can be formed independently, and have different roles. Newly forming cones were composed only of bundles, whereas the meshwork formed later, coincident with the onset of cone movement. Polarized distributions of myosin VI, Arp2/3 complex, and the actin-bundling proteins, singed (fascin) and quail (villin), occurred when movement initiated. When the Arp2/3 complex was absent, meshwork formation was compromised, but surprisingly, the cones still moved. Despite the fact that the cones moved, membrane reorganization and cytoplasmic exclusion were abnormal and individualization failed. In contrast, when profilin, a regulator of actin assembly, was absent, bundle formation was greatly reduced. The meshwork still formed, but no movement occurred. Analysis of this actin structure's formation and participation in cellular reorganization provides insight into how the mechanisms used in cell motility are modified to mediate motile processes within specialized cells.