Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction.

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.     

Different modes of growth cone collapse in NG 108-15 cells

In the fundamental process of neuronal path-finding, a growth cone at the tip of every neurite detects and follows multiple guidance cues regulating outgrowth and initiating directional changes. While the main focus of research lies on the cytoskeletal dynamics underlying growth cone advancement, we investigated collapse and retraction mechanisms in NG108-15 growth cones transiently transfected with mCherry-LifeAct and pCS2+/EMTB-3XGFP for filamentous actin and microtubules, respectively. Using fluorescence time lapse microscopy we could identify two distinct modes of growth cone collapse leading either to neurite retraction or to a controlled halt of neurite extension. In the latter case, lateral movement and folding of actin bundles (filopodia) confine microtubule extension and limit microtubule-based expansion processes without the necessity of a constantly engaged actin turnover machinery. We term this previously unreported second type fold collapse and suggest that it marks an intermediate-term mode of growth regulation closing the gap between full retraction and small scale fluctuations.     

Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone

Filopodial motility is critical for many biological processes, particularly for axon guidance. This motility is based on altering the F-actin-based cytoskeleton, but the mechanisms of how this occurs and the actin-associated proteins that function in this process remain unclear. We investigated two of these proteins found in filopodia, talin and vinculin, by inactivating them in subregions of chick dorsal root ganglia neuronal growth cones and by observing subsequent behavior by video-enhanced microscopy and quantitative morphometry. Microscale chromophore-assisted laser inactivation of talin resulted in the temporary cessation of filopodial extension and retraction. Inactivation of vinculin caused an increased incidence of filopodial bending and buckling within the laser spot but had no effect on extension or retraction. These findings show that talin acts in filopodial motility and may couple both extension and retraction to actin dynamics. They also suggest that vinculin is not required for filopodial extension and retraction but plays a role in the structural integrity of filopodia.     

Spinal neurite reabsorption and regrowth in vitro depend on the polarity of an applied electric field

Retraction and regrowth of frog neural tube neurites have been studied in vitro in control cultures and in the presence of a small, continuously applied electrical field. In control cultures, some degree of retraction was seen in 39% of neurites while 7% were reabsorbed completely. Reabsorption of anodal-facing neurites was at least twice as common, with 67% showing some retraction and 17% almost totally reabsorbed. Cathodal-facing neurites were spared from retraction. Following extreme reabsorption of anodal-facing neurites, reversal of the electric field promoted regeneration in 47% (9/19) of cases studied. growth cone morphology also was determined by the polarity of the applied field. Anodal-facing growth cones had fewer filopodia than cathodal-facing growth cones sharing the same cell body. Field reversal induced a polarity-specific change in filopodia number on individual growth cones: a shift from anodal to cathodal increased filopodia numbers and vice versa. Some possible mechanisms involved and the significance of these results are discussed.     

Distinct calcium signaling within neuronal growth cones and filopodia

Previous findings indicate that spatial restriction of intracellular calcium levels within growth cones can regulate growth cone behavior at many levels, ranging from filopodial disposition to neurite extension. By combining techniques for focal stimulation of growth cones with those for measurement of filopodia and for capturing low intensity calcium signals, we demonstrate that filopodia on individual growth cones can respond to imposed stimuli independently from one another. Moreover, filopodia and their parent growth cones appear to represent functionally and morphologically distinct domains of calcium regulation, possessing distinct calcium sources and sinks. Both are sensitive to calcium influx; however, application of the calcium ionophore A23187 to cells in calcium-free medium demonstrated the presence of potential intracellular calcium pools in the growth cone proper, but not in isolated filopodia. Thapsigargin significantly reduced the rise in growth cone calcium levels associated with excitatory neurotransmitters, further implicating release from calcium pools as one component of growth cone calcium regulation. The relative contributions of these pools were examined in response to excitatory neurotransmitters by quantitative calcium measurements made in both growth cones and isolated filopodia. Striking differences were observed; filopodia were sensitive to a low concentration of dopamine and serotonin, while growth cones displayed an amplified rise at a higher concentration. The spatial distribution of organelles that could serve as morphological correlates to such calcium amplification was examined using confocal microscopy. While the majority of organelles were located in the central core of the growth cone proper, peripheral organelles were detected at the base of a subset of filopodia. The distinctive distribution of calcium regulation within motile growth cones suggests one mechanism by which growth cones may regulate their complex behavior. © 1996 John Wiley & Sons, Inc.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis

Interactions between dynamic microtubules and actin filaments (F-actin) underlie a range of cellular processes including cell polarity and motility. In growth cones, dynamic microtubules are continually extending into selected filopodia, aligning alongside the proximal ends of the F-actin bundles. This interaction is essential for neuritogenesis and growth-cone pathfinding. However, the molecular components mediating the interaction between microtubules and filopodial F-actin have yet to be determined. Here we show that drebrin, an F-actin-associated protein, binds directly to the microtubule-binding protein EB3. In growth cones, this interaction occurs specifically when drebrin is located on F-actin in the proximal region of filopodia and when EB3 is located at the tips of microtubules invading filopodia. When this interaction is disrupted, the formation of growth cones and the extension of neurites are impaired. We conclude that drebrin targets EB3 to coordinate F-actin-microtubule interactions that underlie neuritogenesis.     

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.     

Acetylcholine influences growth cone motility and morphology of developing thalamic axons

The neurotransmitter acetylcholine (ACh) is expressed in the developing telencephalon at the time when thalamic axons project to the cortex, long before synapses are being formed. Since previous studies demonstrated an influence of ACh on neurite extension we used different in vitro assays to examine possible effects of ACh on the growth of thalamic axons. In explant cultures, application of ACh reduced the length of thalamic axons in a dose dependent manner, an effect that could also be evoked by selective muscarinic and nicotinic agonists. Time-lapse imaging of thalamic axons exposed to microscopic gradients of ACh revealed that growth cones no longer advanced, but maintained high filopodial activity. This growth cone pausing was not accompanied by axon retraction or growth cone collapse. It could at least partially be blocked by muscarinic and nicotinic antagonists, indicating that both types of ACh receptors contribute to mediate these effects on thalamic axons. Finally, we also found that ACh changed the morphology of growth cones; they became larger and extended more filopodia. Since such changes in the structure and motility of growth cones are observed at decision regions along the path of many fiber populations including thalamic axons, we suggest that ACh plays a role during the elaboration of thalamocortical projections.Key words: cortical development, thalamocortical projections, neurotransmitter, acetylcholine, growth cone, axonal guidance, wiring molecules     

Regulation of neuronal growth cone filopodia by nitric oxide

Nitric oxide (NO) has been proposed to play an important role during neuronal development. Since many of its effects occur during the time of growth cone pathfinding and target interaction, we here test the hypothesis that part of NO's effects might be exerted at the growth cone. We found that low concentrations of the NO-donors DEA/NO, SIN-1, and SNP caused a rapid and transient elongation of filopodia as well as a reduction in filopodial number. These effects resulted from distinct changes in filopodial extension and retraction rates. Our novel findings suggest that NO could play a physiological role by temporarily changing a growth cone's morphology and switching its behavior from a close-range to a long-range exploratory mode. We subsequently dissected the pathway by which NO acted on growth cones. The effect of NO donors on filopodial length could be blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase (sGC), indicating that NO acted via sGC. Supporting this idea, injection of cyclic GMP (cGMP) mimicked the effect of NO donors on growth cone filopodia. Moreover, application of NO-donors as well as injection of cGMP elicited a rapid and transient rise in intracellular calcium in growth cones, indicating that NO acted via cGMP to elevate calcium. This calcium rise, as well as the morphological effects of SIN-1 on filopodia, were blocked by preventing calcium entry. Given the role of filopodia in axonal guidance, our new data suggest that NO could function at the neuronal growth cone as an intracellular and/or intercellular signaling molecule by affecting steering decisions during neuronal pathfinding.     

Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.     

Induction of filopodia by direct local elevation of intracellular calcium ion concentration.

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from approximately 60 nM to approximately 1 microM within the illuminated zone, and then returned to resting level in approximately 10-15 s. New filopodia arose in this area within 1-5 min, and persisted for approximately 15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia.     

Rapid growth cone translocation on laminin is supported by lamellipodial not filopodial structures

To determine the relationship between growth cone structure and motility, we compared the neurite extension rate, the form of individual growth cones, and the organization of f-actin in embryonic (E21) and postnatal (P30) sympathetic neurons in culture. Neurites extended faster on laminin than on collagen, but the P30 nerites were less than half as long as E21 neurites on both substrata. Growth cone shape was classified into one of five categories, ranging from fully lamellipodial to blunt endings. The leading margins of lamellipodia advanced smoothly across the substratum ahead of any filopodial activity and contained meshworks of actin filaments with no linear f-actin bundles, indicating that filopodia need not underlie lamellipodia. Rapid translocation (averaging 0.9-1.4 microns/min) was correlated with the presence of lamellipodia; translocation associated with filopodia averaged only 0.3-0.5 microns/min. This relationship extended to growth cones on a branched neurite where the translocation of each growth cone was dependent on its shape. Growth cones with both filopodial and lamellipodial components moved at intermediate rates. The prevalence of lamellipodial growth cones depended on age of the neurites; early in culture, 70% of E21 growth cones were primarily lamellipodial compared to 38% of P30 growth cones. A high percentage of E21 lamellipodial growth cones were associated with rapid neurite elongation (1.2 mm/day), whereas a week later, only 16% were lamellipodial, and neurites extended at 0.5 mm/day. Age-related differences in neurite extension thus reflected the proportion of lamellipodial growth cones present rather than disparities in basic structure or in the rates at which growth cones of a given type moved at different ages. Filopodia and lamellipodia are each sufficient to advance the neurite margin; however, rapid extension of superior cervical ganglion neurites was supported by lamellipodia independent of filopodial activity.     

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

Background

During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.

Results

To investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.

Conclusions

This study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.     

Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid.

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.     

Antagonistic forces generated by cytoplasmic dynein and myosin-II during growth cone turning and axonal retraction

Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dynein-driven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.     

Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells

Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology, including neurite extension and retraction. Rho activation leads to neurite retraction and cell rounding, whereas Rac and Cdc42 are implicated in the promotion of filopodia and lamellipodia formation in growth cones and, therefore, in neurite extension. In this study, we examined the morphological role of Rnd1, a new member of Rho family GTPases, in PC12 cells, and found that expression of Rnd1 by itself caused the formation of many neuritic processes from the cell body with disruption of the cortical actin filaments, the processes having microtubules but few filamentous actin and neurofilaments. Treatment with cytochalasin D, an inhibitor of actin polymerization, could mimic the effects of expression of Rnd1, in that this inhibitor disrupted the cortical actin filaments and induced the formation of many thin processes containing microtubules. The process formation induced by Rnd1 was inhibited by dominant negative Rac1. These results suggest that Rnd1 induces the Rac-dependent neuritic process formation in part by disruption of the cortical actin filaments.     

Ena/VASP: proteins at the tip of the nervous system

The emergence of neurites from a symmetrical cell body is an essential feature of nervous system development. Neurites are the precursors of axons and dendrites and are tipped by growth cones, motile structures that guide elongating axons in the developing nervous system. Growth cones steer the axon along a defined path to its appropriate target in response to guidance cues. This navigation involves the dynamic extension and withdrawal of actin-filled finger-like protrusions called filopodia that continuously sample their environment. Ena/VASP proteins, a conserved family of actin-regulatory proteins, are crucial for filopodia formation and function downstream of several guidance cues. Here we review recent findings into Ena/VASP function in neurite initiation, axon outgrowth and guidance.     

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.     

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.