University of Minnesota, Minneapolis, Minnesota, USA. Neurons obtain their stereotyped ..... the recov ely of lluorescence in axons indicates that neither is ...
Grokth Christopher University
cone motility
Cypher
and Paul C. Letourneau
of Minnesota,
Minneapolis,
Minnesota,
USA
Neurons obtain their stereotyped morphologies and connections as a result of growth cone migration. In the past year, studies on growth cone migration and pathfinding have helped to define certain properties of cytoskeletal filaments and cell membranes that may be important in growth cone function. Antisense mRNAs have proved to be particularly useful for examining the roles of specific neurite proteins.
Current
Opinion
in Cell
Introduction Nerve growth cones at the tips of elongating axons and dendrites perform several interconnected functions. They respond to extracellular stimuli, transduce these signals into directed motility that is driven by a constantly remodeled cytoskeleton, and assemble the elongating neurite. Two comprehensive monographs on growth cones have been published recently [ 1,2]. This review will focus on recent papers that address the mechanisms underlying neurite outgrowth. The signal transduction aspect of growth cone function will not be covered as this has been reviewed elsewhere recently [l-3]. Growth
cone
behavior
As growth cones advance, they apply rearward traction forces on the substratum. These forces can be observed when they are exerted on movable objects. Heidemann et al. [4**] have monitored the interactions of growth cones of cultured chick neurons as they contacted and displaced other neurites. The majority of these interactions were termed ‘frlopodial contractions’ in which the tip of a lilopodium adhered to the side of a neurite. Usually the neurite was drawn toward the growth cone as the lilopodia shortened and thickened. This behavior is similar to that described by O’Connor et a[. [5**] who observed the Iilopodia of grasshopper pioneer neurons contacting a guidepost neuron in situ. After contact, the filopodium expanded as it hIled with cytoplasm, thus becoming the preferred pathway for axon elongation. In this way, a single filopodial contact orients the growth cone towards the highly adhesive guidepost neuron. It is possible that these two groups [4**,5*-1 have observed the same phenomenon. In the former case, the filopodium contacted a deformable neurite that was pulled toward the growth cone [4=*], masking somewhat the filopodial engorgement, and the Iilopodium appeared to contract. In the latter case, contact was made with an immovable guidepost cell and the filopodial dilation
Biology
1992,
4:4-7
was seen more clearly [5**]. In this latter case, lilopodial contraction is not obvious. In both cases, contact was made by the tip of the filopodium, and this was followed by filopodial dilation and the exertion of a rearward tmction force on the substratum. The possibility that the forward flow of cytoplasm during filopodial dilation may be caused by shear-induced gel-sol transitions in the palmate regions of growth cones is discussed by Heidemann et al [4**]. It is also noteworthy that when a growth cone passed over and deflected an obstacle neurite toward the neurite shaft (retrogradely), the force on the obstacle neurite decreased after it had passed under the distal portion of the growth cone [4**]. After that time, the neurite moved in the direction of growth cone advance (anterogradely) until it was no longer deflected. This indicates that the traction force applied to the substratum is exerted in the distal most regions of the growth cone. As Heidemann et al. noted [ 4**], the chick sensory neurons they studied possessed many filopodia, but me morphologies of different types of growth cones are variable and complex. Although O’Connor et a[. [5**], using very adhesive substrata, did observe Iilopodial dilation behavior similar to that seen by Heidemann et al. [40*], they also reported two other types of growth cone behavior that appear to be substrata-dependent. As grasshopper pioneer growth cones migrated on a relatively homogeneous substratum, they extended filopodia in several directions. They did not advance along any particular lilopodia for a great distance. They extended lamellar veils, and preferentially elongated proximal axial branches. As these growth cones travelled proximally, they encountered a dorsal-ventral segment boundary at which there is a distal band of high-adhesivity cells and a proximal band of lower-adhesivity cells. Proximal growth ceased and branches were extended dorsally and ventraIly on the higher-adhesivity cells. Eventually the neurites always turned ventrally by withdrawing or pruning the dorsal branch. These three types of steering processes (lilopodial dilation, veil extension and microprun-
Abbreviations MAP-microtubule-associated
@ Current
protein;
Biology
N-CAM-neural
cell-adhesion
Ltd ISSN 0955674
molecule
Growth
ing) thus seem to be substratum-dependent and to involve different growth cone morphologies and steering strategies (see also [6*] >. Cytoskeletal
dynamics
Growth cone morphology is very dynamic as neurites elongate. Underlying this constant remodeling are changes in the assembly state, localization and stability of the filamentous actin and microtubules comprising the growth cone’s cytoskeleton. Additionally, these filaments are assembled and consolidated into the length of new neurite forming just proximal to the growth cone. Recently, techniques for imaging fluorescently labeled microtubules have been developed, which have allowed Sabry et al. [7**] to examine spatial and temporal changes in microtubule distribution during the steering events of the grasshopper growth cones mentioned above [5**]. A rich network of microtubules was observed throughout the growth cone ( [ 70~1;see also [8*] >. During orientation towards guidepost cells by frlopodial contact and dilation, microtubules entered selectively only the branch forming towards the guidepost cell [7=*]. During other steering events, however, microtubules were present in several branches but were retained or stabilized selectively only in the branch in the direction of future elongation. These observations indicate that, whereas microtubules are not necessary for branch formation, the generation of asymmetric microtubule arrays is important in growth cone orientation. The dynamism of microtubules and actin filaments in neurites has been examined by several groups using photobleaching or photoactivation techniques [ 90, lo*, 1 la*]. Okabe and Hirokawa [p], for example, followed the recovery of fluorescence after photobleaching either labeled actin or tubulin that had been injected into cultured dorsal root ganglion neurons. They observed that bleached areas of actin in neurites do not move or spread, suggesting that a substantial portion of the poly mer is not transported within axons. There was a small amount of rapid diffusional recovery, and a greater slow recovery, suggesting that the filaments are stationary but still dynamic. Similar results were obtained when labeled tubulin was photobleached in axons [Y]; bleached areas did not move, there was a small amount of ditfusional recovery, and a larger slow recovery. From these data it appears that neither actin nor tubulin is transported within axons as a polymer, that for both, the pools of freely diffusible monomers present are small, and that both filaments do turnover slowly throughout axons. Okabe and Hirokawa [lo=] have extended their analysis of actin dynamics to growth cones. They used inmunoelectron microscopy to localize biotin-labeled actin injected into fused PC12 cells. They found that the label appeared first in distal parts of filament bundles and lilopodia, and membrane-associated regions of the actin network. In cells fixed at later time points, actin filaments were labeled uniformly. In this same study [lo*], dorsal root ganglion neurons were injected with fluorescein-labeled actin and photobleached. When the leading edges
cone motility
Cypher
and Letourneau
of motile growth cones were bleached, fluorescence recovery was seen first at the distal margin. Over time, a minority of the bleached regions appeared to move centripetally at rates faster than the growth cones advanced. No regions that were photobleached proximal to the leading edge, however, appeared to be translocated. The fluorescence recovery of bleached filopodia did occur in a centripetal fashion. These results, and those of other studies [ 121, indicate that actin filaments are transported rearward in the distal regions of motile growth cones. The results on tubulin photobleaching of Okabe and Hirokawa [9*] are in agreement with those of other groups, notably Lim et al. [ 13,141, who demonstrated further that fluorescence recovery occurred more rapidly in growth cones than in neurite shahs [ 131. These results are not in agreement, however, with those of Reinsch el al [ 1 la-1 who used a more recently developed technique to create distinct areas of photoactivated-labeled tubulin within Xenopus neurons. These areas remained coherent and translocated distally at rates that were generally slower than growth cone advance. The photoactivated Ruorescent zones remained after the cells had been extracted with detergent to solubilize tubulin monomers. These results [ ll**] suggest that tubulin is assembled into polymers in or near the cell body and transported distally in neurites. They do not, however, exclude assembly throughout the axon or at the growth cone. Reinsch et al. [ ll**] discuss several possible explanations for the conflicting results obtained by the IWO methodologies used to label microtubules, including the different cell types used, the sensitivities of the two techniques, and the potential of both to perturb either the microtubules or associated motor proteins. At the leading
edge
Several recent studies have examined the dynamic properties of membranes at the leading edges of motile cells. Although most of this work has not been carried out with growth cones, it offers important insights into the mechanisms that might underlie growth cone motility. While considering such studies, however, it is important to remember that, whereas most moving cells transport themselves in toto, growth cones are cellular extensions within which materials are transported and assembled into the growing neurite. This synthetic aspect of growth cone function places demands on growth cone physiology that are distinct from those placed on other motile ceU types. There is, for example, substantial expansion of the plasma membrane of a migrating growth cone. Lockerbie et al. [15*] examined this membrane addition in a population of isolated growth cone particles. They observed a calcium-dependent externalization of wheat germ agglutinin-binding sites on these particles, that was independent of neurotransmitter release. This observation of regulated plasmalemmal expansion is consistent with other studies suggesting that calcium is important in neurite elongation [ 16,17*]. It has also been found recently that membrane glycoproteins in mouse macrophages are moved rearward from
5
6
Cytoplasm
and cell motility
.
the leading edge by an actin filament-dependent mechanism rather than by bulk flow of membrane lipid [ 181. This is likely to be true for growth cones. This finding has been verified by Kucik er al [19”1 who observed that Con-A-coated latex beads are much more likely to be actively transported centripetally on the dorsal surfaces of gold&h epidermal keratocytes if they have attached within 0.5 microns of the cell’s leading edge. Beads attached to the central region of the lamella diffuse randomly on the membrane. Using a laser optical trap to manipulate the beads, it was demonstrated further that, over time, the strength of bead binding to the transport system increases. This apparent specialization of the leading edge for loading glycoproteins onto a rearward, cytoskeletal-linked transport system has important implications for cell motility models [19-l. The specialized nature of growth cone margins is reflected in the non-uniform distributions of actin-binding and other proteins that have been shown to be concentrated at the leading edges of growth cones [ 20,211. How such distributions are obtained was the subject of a study carried out by Sheetz et al. [22-l on the localization and movement of two cell-surface molecules, neural celladhesion molecule (N-CAM) and the 2Al antigen, on the growth cones of cultured murine neurons. 40.~1 gold particles were coated with antibodies to these two proteins and allowed to bind to growth cone plasma membranes. The movements of these particles were followed by video-enhanced differential interference contrast microscopy. Compared with control beads coated with nonspedic rat IgG, the beads coated with antibodies to N-CAM and the 2Al antigen were concentrated at the edges of growth cones. Examination of the particle movements indicated that their forward movements to the growth cone margin were An-dependent. At the lamellipodial edges, the particles move rapidly parallel to the edge and rarely move back onto the central region of the growth cone; they are trapped at the edges. Taken together, these results indicate that there are selective transport mechanisms that move membraneassociated molecules to and from the leading edges of motile cells. This selectivity is based on the molecules transported and the direction of transport, thus allowing certain molecules to become concentrated at the leading edges of motile cells. Such concentrations of membrane molecules would be expected to facilitate greatly the navigational and motility functions of growth cones. The filopodial contractions and steering events discussed above may reflect traction exerted on adhesive sites, involving the linkage of concentrated adhesive ligands to the actin filament system of the subplasmalemmal cortex.
24 h, one neurite
per cell greatly
exceeds
the others
in
length and becomes an axon. Formation of an asymmetric axon, however, does not occur in cultures treated with antisense tau ~&WAS, even though the initial exploratory neurites are not affected. Whereas Caceres and Kosik [23**] presented cells with oligonucleotides in the cell culture medium, Dinsmore and Solomon [ 25.0 ] transfected undifferentiated cells of an embryonal mouse carcinoma cell line with sense and antisense MAP-2 mRNAs. Transfected cells expressed the introduced RNA constitutively. Normally, when the cells are induced with retinoic acid to differentiate into a neuronal phenotype, they synthesize the MAP-2 protein. In cells transfected with the antisense mRNA, MAP-2 protein and mRNA were reduced IO-fold and twofold, respectively. Such cells had shorter and many fewer neurites than controls. Both sets of results are intriguing and demonstrate the potential of antisense oligonucleotides as probes to help elucidate the roles of specific proteins in neurite outgrowth. Conclusion
Nerve growth cones have been and will continue to be studied extensively because of the importance and complexity of their functions. The papers discussed here reflect the variety of preparations and experimental techniques that are being used to examine growth cone motility and neurite assembly. In the hture, these and other novel approaches till be required to help us to understand growth cone behavior on as many levels as possible. Acknowledgements We thank manuscript.
Drs KA Support
References
Mesce and DM Snow for comments was from NM grant HD19950.
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1. 2. 3.
LETOURNEAU
PC,
on
the
reading within
the annual
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Antisense
probes
The functions of two microtubule-associated proteins (MAPS), tau and M-2, in neurite outgrowth have been examined recently using antisense RNAs to inhibit the synthesis of these proteins. In the case of tau, reduced expression blocked neurite polarization in cultures of rat cerebellar neurons [23-•,24]. In untreated cultures, or cultures treated with a control sense oligonucleotide, these cells initially extend several short neurites. After
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ZHENC J. L%MOLIRELIX P, DENNEW T. B~XBAUM RE, HEIDEMANN SR: Tensile Regulation of Axonal Elongation and Initiation. J Neurasci 1991, 11:1117-1125. Extending previous results, this study shows that the rate of neurite elongation is a linear function of the constant tension applied to them during ‘towed growth’. Tension applied to cell bodies without neurites can initiate neurltes, .some of which produce apparently normal motile growth cones. SABRY JH, O’CONNOR TP, EVANS I TOROW-RAYMOND A KIRSCHNER M, BEN~IIY D: Microtubule Behavior During Guidance of Pioneer Neuron Growth In Situ J Cell Biol 1991, 115:381-395. Individual microtubules were visualized during growth cone steering. They were observed to either selectively enter or be retained in elongating branches. TANAKA Growth
EM, KIRSCHNER M: Microtubule Behavior in the Cones of Living Neurons During Axon Elongation. J Cell Biol 1991, 115:34>363. Fluorescently labeled microtubules were observed to change between splayed, looped and bundled configurations. Individual microtubules also underwent growth. shrinkage and translocation.
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The externalization of wheat germ agglutinin-binding sites on isolated growth cone particles is shown to be regulated and calcium-dependent, but not linked to neurotransmitter release. 16.
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LANKFORD Kl, IET~~RNF.A~ PC: Roles of Actin Filaments and Three Second-messenger Systems in Short-term Regulation of Chick Dorsal Root Ganglion Neurite Outgrowth. Cell Mold Cyhkeleton 1991, 20~7-29. Growth cone behavior and actin filament stability are shown to be very sensitive to small changes in calcium levels. Although elevating CAMP levels or stimulating protein kinase C both inhibit neurite outgrowth, only the former may a&t calcium levels. 18.
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REIN~CH SS. MIT~HISON n, KIRSCHNER M: Microtubule Polymer Assembly and Transport During Axonal Elongation. J Cell Rio1 1991, 115:365-379. Areas of photoactlvated tuhulin move distally within neurites and behave as if the labeled tubulin were incorporated into polymers that are transported distauy 12.
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SHEEIZ MP, BAUMRIND NI WAYNE DB, Pm AL Concentration of Membrane Antigens by Forward Transport and Trapping in Neuronal Growth Cones. Cell 1990, 61:231-241. Video-enhanced microscopy was used to follow the movements on growth cones of gold particles coated with antibodies to two neuronal cell-surface antigens. The particles are transported to, and concentrated at, the edges of growth cones. CACERES A, KOSM KS: lnhibition of Neurite Polarity by tau Antisense OIigonucleotides in Primary Cerebellar Neurons. Nalure 1330, 343:46143. Antisense mRNA inhibition of tau expression in primary neuronal cultures prevents the formation of elongated asymmetric axons, but not of short exploratory neurites. 23. ..
24.
CACERE~ A POIREBIC S. KOSIK KS: The Effect of tau Antisense Oligonucleotides on Neurite Formation of Cultured Cerebellar Macroneurons. J Neurosci 1991, 11:151%1523.
DINSMORE JH, SOLOMON F: lnbibition of MAP2 Expression Affects both Morphological and Cell Division Phenotypes of Neuronal Differentiation. Cell 1991. 64:817-826. Embryonal carcinoma cells were transfected with MAP-2 antisense. or control sense, mRNA and induced to differentiate to a neuronal phenotype, The MAP-2 antisense, but not sense, construct lowered the number and length of neurites formed, but did not block ceU division. 25. ..
C Cypher and PC Letoumeau, Department of Cell Biology and Neuroanatomy, University of Minnesota, 4-135 Jackson Hall, 321 Church Street SE.. MiMapOliS, MiMesOta 55455, USA
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