Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II functions

Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension.     

Alpha-actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones

We have used biochemical and immunocytochemical techniques to investigate the possible involvement of membrane cytoskeletal elements such as alpha-actinin, calspectin (brain spectrin or fodrin), and actin in growth cone activities. During NGF-induced differentiation of PC12 cells, alpha-actinin increased in association with neurite outgrowth and was predominantly distributed throughout the entire growth cone and the distal portion of neurites. Filopodial movements were sensitive to Ca2+ flux. Two types of alpha-actinin, with Ca2(+)-sensitive and -insensitive actin binding abilities, were identified in the differentiated cells. Ca2(+)-sensitive alpha-actinin and actin filaments were concentrated in filopodia. The Ca2(+)-insensitive protein was distributed from the body of the growth cone to the distal portion of neurites, corresponding to the substratum-adhesive sites. The location of calspectin in growth cones was similar to that of the Ca2(+)-insensitive alpha-actinin. These results are consistent with the hypothesis that Ca2(+)-sensitive alpha-actinin and actin filaments are involved in Ca2(+)-dependent filopodial movement and Ca2(+)-insensitive alpha-actinin and calspectin are associated with adhesion of growth cones.     

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.     

Cell-substratum adhesion of neurite growth cones, and its role in neurite elongation.

The adhesion of chick embryo sensory neurons to glass coverslips was examined with interference reflection optics. On untreated glass, adhesive contacts are common only beneath growth cones and are small. On polylysine-treated glass growth cones are highly spread, microspikes reach treat lengths and extensive adhesive contacts underlie growth cones, microspikes and nerve fibers. Veils, expanded from the growth cone, are adherent to the substratum either centrally or laterally, while the extending edge of the cell margin is non-adherent. Linear adhesions are frequent beneath microspikes and pass centrally beneath the growth cone margin. The distribution of linear adhesions resembles that of microfilament bundles seen within whole mounts of growth cones. Adhesive contacts stabilize extensions of the growth cone margin and may influence the organization of the microfilamentous network within the growth cone. Regulation of microfilament organization by adhesion may influence microfilament functions in growth cone mobility and the assembly of neurite structures.     

Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Localization of pp60c-src in growth cone of PC12 cell

By immunocytochemical and biochemical techniques, we observed the localization and expression of pp60c-src in nerve growth factor (NGF)-treated PC12 cells. Immunostaining of pp60c-src is detected in the neuronal soma and the tips of neurites (growth cones). Immunofluorescence in the neurites is less significant. High-resolution microscopy reveals that the location of pp60c-src in growth cone is in good agreement with the adhesive site of growth cone to the substratum. The pp60c-src kinase activity and the pp60c-src protein level increase 3.1- to 3.5-fold and 2.0-fold during differentiation of PC12 cells, respectively. The pp60c-src levels in the neurite fraction are also higher than those in the neuronal soma fraction. These results support the immunocytochemical finding that pp60c-src is localized in growth cones of differentiated PC12 cells. Furthermore, we discuss the possible role of pp60c-src in growth cone.     

Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization.

We have generated a polyclonal antibody against myosin II from a neuronally derived cell line in order to assess potential roles for myosin II in growth cone movement and synaptic transmission. The distribution of neuronal myosin II, in isolated cells as well as in tissues of the adult rat brain and spinal cord, was examined at the light microscopic and ultrastructural levels. In isolated neuroblastoma cells and dorsal root ganglion neurons, myosin II was found at the leading edge of growth cones, within neuritic processes and cell soma, and adjacent to the plasma membrane. The subcellular distribution of myosin II overlapped significantly with that of both actin and single-headed myosin I. These results implicate both myosin I and myosin II as molecular motors required for neurite elongation and growth cone motility. An exclusive postsynaptic distribution of myosin II in neurons of the mature central nervous system suggests that myosin II cannot play a role in the mobilization of synaptic vesicles, but could participate in synaptic plasticity.     

Growth cone behavior and production of traction force

The growth cone must push its substrate rearward via some traction force in order to propel itself forward. To determine which growth cone behaviors produce traction force, we observed chick sensory growth cones under conditions in which force production was accommodated by movement of obstacles in the environment, namely, neurites of other sensory neurons or glass fibers. The movements of these obstacles occurred via three, different, stereotyped growth cone behaviors: (a) filopodial contractions, (b) smooth rearward movement on the dorsal surface of the growth cone, and (c) interactions with ruffling lamellipodia. More than 70% of the obstacle movements were caused by filopodial contractions in which the obstacle attached at the extreme distal end of a filopodium and moved only as the filopodium changed its extension. Filopodial contractions were characterized by frequent changes of obstacle velocity and direction. Contraction of a single filopodium is estimated to exert 50-90 microdyn of force, which can account for the pull exerted by chick sensory growth cones. Importantly, all five cases of growth cones growing over the top of obstacle neurites (i.e., geometry that mimics the usual growth cone/substrate interaction), were of the filopodial contraction type. Some 25% of obstacle movements occurred by a smooth backward movement along the top surface of growth cones. Both the appearance and rate of movements were similar to that reported for retrograde flow of cortical actin near the dorsal growth cone surface. Although these retrograde flow movements also exerted enough force to account for growth cone pulling, we did not observe such movements on ventral growth cone surfaces. Occasionally obstacles were moved by interaction with ruffling lamellipodia. However, we obtained no evidence for attachment of the obstacles to ruffling lamellipodia or for directed obstacle movements by this mechanism. These data suggest that chick sensory growth cones move forward by contractile activity of filopodia, i.e., isometric contraction on a rigid substrate. Our data argue against retrograde flow of actin producing traction force.     

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.     

Myosin VI stabilizes an actin network during Drosophila spermatid individualization

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Growth cones contain myosin II bipolar filament arrays

Nonmuscle myosin II is among the most abundant forms of myosin in nerve growth cones. At least two isoforms of myosin II (A and B) that have overlapping but distinct distributions are found in growth cones. It appears that both myosin IIA and IIB may be necessary for normal nerve outgrowth and motility, but the molecular interactions responsible for their activity remain unclear. For instance, it is unknown if these myosin II isoforms produce bipolar "minifilaments" in growth cones similar to those observed in other nonmuscle cells. To determine if minifilaments are present in growth cones, we modified the electron microscopy preparative procedures used to detect minifilaments in other cell types. We found structures that appeared very similar to bipolar minifilaments found in noneuronal cells. They also labeled with antibodies to either myosin IIA or IIB. Thus, the activity of myosin II in growth cones is likely to be similar to that in other nonmuscle cells. Bipolar filaments interacting with oppositely oriented actin filaments will produce localized contractions or exert tension on actin networks. This activity will be responsible for the myosin II dependent motility in growth cones.     

Myosin II functions in actin-bundle turnover in neuronal growth cones

Retrograde actin flow works in concert with cell adhesion to generate traction forces that are involved in axon guidance in neuronal growth cones. Myosins have been implicated in retrograde flow, but identification of the specific myosin subtype(s) involved has been controversial. Using fluorescent speckle microscopy (FSM) to assess actin dynamics, we report that inhibition of myosin II alone decreases retrograde flow by 51% and the remaining flow can be almost fully accounted for by the 'push' of plus-end actin assembly at the leading edge of the growth cone. Interestingly, actin bundles that are associated with filopodium roots elongated by approximately 83% after inhibition of myosin II. This unexpected result was due to decreased rates of actin-bundle severing near their proximal (minus or pointed) ends which are located in the transition zone of the growth cone. Our study reveals a mechanism for the regulation of actin-bundle length by myosin II that is dependent on actin-bundle severing, and demonstrate that retrograde flow is a steady state that depends on both myosin II contractility and actin-network treadmilling.     

Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X- 100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.     

Cytochemical Localization of Actin and Myosin Aggregates in Interphase Nuclei in Situ

Biochemical and ultrastructural studies on isolated nuclear compartments have previously shown actin and myosin to be constituents of interphase nuclei. In the present work, immunocytochemistry, in conjunction with confocal microscopy and ultrastructural immunogold techniques, shows that interphase nuclei of intact dorsal root ganglion neurons and of PC12 cells contain actin and myosin. Nuclear actin was observed to be distributed throughout the nucleoplasm occurring as distinct aggregates. Frequently, prominent actin aggregates were associated with the nucleolar periphery, often near nucleolar satellites. Ultrastructurally, actin was observed to be associated with linear, electrondense structures, putatively identified as chromatin fibers, extending from nucleoli. Use of three antibodies against subclasses of α-actin isoforms revealed that nuclear actin is more closely related to α-sarcomeric actin than to α-smooth muscle actin. Those aggregates associated with the nucleolus were found to be in the polymerized F-actin form, in a small fraction of neurons, as assessed by FITC-phalloidin. A myosin-like antigen was also observed to occur as intranuclear aggregates. Quantitative assays of the distribution of actin and myosin aggregates by nearest neighbour analysis indicated a distribution characterized as uniform and failed to reveal statistically significant associations between any set of aggregates, The evidence presented herein indicates that actin and myosin are constituent proteins of interphase nuclei in situ of both normal mammalian and transformed mammalian cells.     

Geometry of isolated sensory neurons in culture. Effects of embryonic age and culture substratum

Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.     

Tension and compression in the cytoskeleton of PC 12 neurites

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.     

Growth cone-growth cone interactions in cultures of rat sympathetic neurons

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.     

RhoA‐kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA‐kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP‐actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F‐actin patches, the latter being an effect attributable to ROCK‐mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F‐actin polymerization underlying protrusive activity in the distal axon. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006