Filopodia and lamellipodia are withdrawn, and some- times neurites retract a short distance. Obviously, the motile apparatus can be disrupted in many ways.
Cell Motility and the Cytoskeleton 20:267-271 (1991)
Views and Reviews Regulation of Growth Cone Motility Paul C. Letourneau and Christopher Cypher Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis
Nerve growth cones are the motile tips of elongating axons and dendrites. The migration and behavior of growth cones are responsible for axonal pathfinding to synaptic targets, and for the branching patterns of dendritic trees. Growth cones are studied at all levels of organization, molecular, cellular, and within intact embryos. Preparations of growth cone fragments from neural tissues or neural cell lines are being used to identify the molecular components of growth cones. Sophisticated videomicroscopy and image processing are revealing the structure, intracellular motility, and metabolism of living growth cones at high resolution. Similar methods permit recording of growth cone activity within intact neural tissues, even complex vertebrate neural structures. These techniques and their findings were the focus of a recent meeting, honoring the discovery of growth cones in 1890 by the Spanish neurobiologist Santiago Ramon y Cajal. The speakers at this meeting wrote chapters for The Nerve Growth Cone, a thorough presentation of growth cone biology [Letourneau et al., 19911. Information in this monograph, as well as recently published papers, has provided several current issues for discussion. Rather than focus on the cytoskeleton and the assembly, disassembly, and organization of actin and tubulin polymers in growth cones, we will emphasize recent studies pertaining to how growth cones interact with their environment. The topics include a new aspect of growth cone behavior, the protein GAP-43 as a specialized component of growth cone motility, second messengers that may regulate growth cone motility, and signal transduction systems that link external cues to the machinery of growth cone migration. For another perspective on growth cone behavior see the recent Views and Reviews by Heidemann and Buxbaum [ 19901. BEING POSITIVE ABOUT NEGATIVE BEHAVIOR!
Studies of growth cone migration have emphasized positive interactions such as adhesive affinities, pathway 0 1991 Wiley-Liss, Inc.
selection, and chemotaxis. Lately, several papers have shown that the inhibition of growth cone migration may be an equally important navigational cue [Bandtlow et al., 1990; Cox et al., 1990; Davies et al., 1990; Raper and Kapfhammer, 1990; Walter et al., 19901. Like highway dividers and “Do Not Enter” signs, certain molecules may cause growth cones to avoid particular regions, thereby providing guidance. These inhibitory substances have different sources (myelin, neurotransmitters, cell membranes) and regional origins, but a common feature of their effects seems to be that the motile peripheral margins of growth cones collapse within several minutes of exposure to each substance. Filopodia and lamellipodia are withdrawn, and sometimes neurites retract a short distance. Obviously, the motile apparatus can be disrupted in many ways. However, these phenomena have physiological relevance in the tissue sources of the inhibitory activities, and specificity in the neuronal types that show growth cone collapse. The mechanisms and similarities of these responses are unknown, since no group has described the status of, or changes in, the organization of actin filaments or microtubules in growth cones after exposure to a collapsing factor. It remains unknown whether collapse is initiated by contraction, depolymerization, or other disruption of the growth cone cytoskeleton. Eventually, it will be necessary to dissect the action of each putative inhibitor, and show whether growth cone collapse is regulated by the same signaling systems and second messengers that control the advance of growth cones.
Received August 14, 1991; accepted August 28, 1991. Address reprint requests to Paul C. Letourneau, Department of Cell Biology and Neuroanatomy, 4-135 Jackson Hall, University of Minnesota, Minneapolis, MN 55455.
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HOW SPECIALIZED IS THE MOTILE ACTIVITY OF GROWTH CONES?
functions of GAP-43, the data suggest that GAP-43 may have an essential role in neurite elongation. On the other hand, a PC 12 cell clone that synthesizes only very small amounts of a truncated form of GAP-43 exhibits normal neurite formation, although altered motility at the neurite tips is mentioned briefly in the paper [Baetge and Hammang, 19911. It has also been reported that GAP-43 immunoreactivity is present in axons, but not dendrites after hippocampal neurons establish their polarity in vitro [Goslin et al., 19901. It is unclear how dendritic and axonal growth cones differ, although rates of axonal elongation are several fold greater than dendritic elongation. In addition, an abstract reports that GAP-43 immunoreactivity is not present during neurite formation by certain neuroblastoma cells [Megerian and Klein, 19901. The meaning of these studies for growth cone motility is further clouded, because none of them has sufficiently probed the effects of these perturbations on the underlying cytoskeletal organization or the filopodial and lamellipodial protrusion that is typical of growth cones. Furthermore, other than its presence in a detergent-resistant membrane cytoskeleton, there is no detailed evidence on the association of GAP-43 with actin or other specific cytoskeletal components. At this point, it appears that GAP-43 may not be essential for the fundamental protrusive and migratory activities of growth cones, but it may mediate regulatory influences that link motility to neurite assembly or transduce signals generated by intrinsic or extrinsic factors.
Filopodial and lamellipodial protrusion and the cytoskeletal structure of the leading margins of growth cones generally resemble the leading edges of locomotory fibroblastic cells [Bridgman, 19911. Likewise, analyses of growth cone particles from developing neural tissues reveal a commonly observed collection of actinbinding and other cytoskeletal proteins that are believed to participate in locomotory functions of other cells [Cypher and Letourneau, 19911. A recent interesting example is that two groups report that growth cones contain myosin I, which is implicated in locomotory processes that involve movement of the plasmalemma and other membranous structures [Bridgman, 1991; Phelan et al., 19911. For example, myosin I may be involved in the extension of filopodia or their exertion of tension on contact points with the substratum in order to promote forward migration and neurite elongation. Myosin I1 is also present in growth cones, and it is intriguing to speculate whether these two myosins have separate locations and functions in the events of neurite elongation and growth cone migration. There is one protein, GAP-43, that may have a special role in growth cone motility. GAP-43 may be present only in neurons, and its expression is greatest during periods of neurite elongation. Growth cone particles are significantly enriched for GAP-43, and immunoelectron microscopy localizes GAP-43 to the cytoplasmic side of the plasmalemma of intact growth cones [Van Lookeren Campagne et al., 19911. Some tempting evi- SECOND MESSENGERS REGULATE THE dence for a GAP-43 function is the demonstration that GROWTH CONE CYTOSKELETON numerous filopodial-like protrusions are extended by An explanation of the regulation of growth cone COS cells transfected with a GAP-43 DNA construct motility must clarify how motility is turned on and off, as [Zuber et al., 19891. Other properties of GAP-43 that well as the more subtle, but critical features of growth may be significant include its association with detergent- cone turning and branching. Two cytoplasmic second insoluble growth cone membrane cytoskeletons [Meiri messengers, Ca+ and protein kinases, are thought to and Gordon-Weeks, 19901, its binding to calmodulin and be important regulators of growth cone motility. Calcium G,, [Strittmatter et al., 19901, its phosphorylation by pro- ion has been investigated most intensively, and much tein kinase C(PkC), and its inhibition of phosphatidyli- evidence indicates that fluctuations in [Ca' '1 may regnositol phosphate kinase. Thus, GAP-43 could be in- ulate several components of growth cone activity [Kater volved in the influences of several second messenger and Mills, 19911. In a model of plasmalemmal expansion systems on cytoskeletal activities. using growth cone particles, elevation of internal The current evidence from cell and molecular bio- [Ca'+] stimulates fusion of internal vesicles with the logical attempts to reveal the role of GAP-43 in growth surface membrane [Lockerbie et al., 19911. Studies that cone motility is unclear. The response of GAP-43-trans- indicate a relationship between [Ca' '1 and actin filafected COS cells is already mentioned. Incorporation of ment organization emphasize the importance of dynamic GAP-43 anti-sense oligonucleotides [Fidel et al., 1990; changes in actin filament organization during growth Schotman et al., 19901 or anti-GAP-43 antibodies into cone motility. Manipulations that lower growth cone primary neurons and two neural cell lines is reported to [Ca' '1 produce stabilization of actin filaments and halt block neurite formation [Shea et al., 19911. Although growth cone migration, while manipulations that raise these perturbations may interfere with intracellular me- [Ca' '1 destabilize actin filaments-lamellipodia are tabolism in ways that are not specifically related to the withdrawn, as growth cones collapse, and growth cone +
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migration stops [Lankford and Letourneau, 19911. These effects of any one extrinsic factor or drug may depend o n data suggest that filopodial and lemellipodial motility other past and present features of the external and interinvolves fluctuations in actin filament severing, poly- nal environment of a growth cone. Bixby and colleagues have investigated the role of merization, and depolymerization that are at least partially controlled by Ca+ '-sensitive proteins and regula- PkC in neurite outgrowth on a variety of purified adhetory molecules. Localized control of filopodial and sive molecules [Bixby and Jhabvala, 19901. Stimulation lamellipodial protrusion during growth cone navigation of PkC by phorbol ester enhances neurite outgrowth on could involve localization of the Ca' +-sensitive com- suboptimal concentrations of laminin, while inhibitors of ponents or local changes in cytoplasmic [Ca' +I, either PkC block neurite outgrowth on laminin. Activation of involving Ca+ release from internal Cat stores and/ protein kinase A by elevation of cAMP levels reduces the or Ca' ' channels in the plasma membrane. Although it extent of neurite outgrowth on laminin. On the other is unknown how [Ca' '1 fluctuations regulate actin fil- hand, inhibitors of PkC actually enhance neurite elongaament stability, some Ca+ +-sensitive regulatory pro- tion on substrata coated with the homophilic neural adteins have been demonstrated in growth cones, including hesion molecules L1 or N-cadherin. These results sugcalmodulin, which binds to GAP-43 at the plasma mem- gest that the pathway for stimulating neurite outgrowth brane. Evidence to support a view that growth cone mo- on laminin involves binding of neural surface laminin tility is controlled by local transmembrane Cat ' cur- receptors and activation of PkC, while ligand binding of rents includes observations of local protrusion of the homophilic adhesion molecules L 1 and N-cadherin lamellipodia in response to local Ca+ ' application via has different effects on second messenger systems of micropipettes [Goldberg, 19881, and the correlation of growth cones, perhaps dephosphorylation of proteins. It protrusive activity at the margins of growth cones with is interesting that specific adhesive interactions may do hot spots of high internal [Ca' '1, which may corre- more than just promote adhesion and traction for growth channels [Silver et al., cone migration, but they may activate particular metaspond with clusters of Caf bolic systems that regulate cytoskeletal organization and 19901. There is ample evidence for the presence of protein function in cell motility. Elucidation of these regulatory pathways requires kinases (Ca' +/calmodulin-dependent, CAMP-dependent, protein kinase C , and c-src) and phosphorylated identification of the molecules whose activity is reguproteins in growth cones. Major proteins that are known lated by [Ca' + ] , kinases, or other second messengers, to be phosphorylated in growth cones include GAP-43, and eventually demonstration that particular pathways tubulin, and vinculin, plus growth cone components that are responsible for the specific behaviors or changes in are known to be phosphorylated in other cells, such as growth cone motility that are characteristic of growth talin and integrins. The case for functional significance cone migration on different substrata. It will be a formiof the phosphorylation in any one of these proteins to dable task to determine how these different regulatory growth cone motility is unclear. Although pharmacolog- pathways are integrated in growth cones in vivo, as they ical studies that have manipulated cAMP levels or PkC produce characteristic neuronal morphologies and patactivity have produced conflicting results, two recent terns of neural circuits. This is particularly true in light of the redundancy of the cell adhesion molecules and other studies are notable. Growth cones of chick sensory neurons responded navigational cues to which growth cones are often simulto elevation of cAMP by an initial cessation of motility, taneously exposed. How does a growth cone know followed by recovery after 20-30 min [Lankford and whom to listen to and when? Letourneau, 19911. Interestingly, in growth cones with cytoplasmic Ca' ' levels >=500 nM, elevation of WHAT IS THE TRANSMEMBRANE LINKAGE OF cAMP caused a significant reduction in [Ca' 'I. If forGUIDANCE CUES TO THE MOTILE APPARATUS? skolin (which activates adenyl cyclase) is added simulThe results of Bixby et al. indicate that adhesive taneously with the calcium ionophore A23 187, the expected rise in intracellular [Ca' '1 that follows iono- interactions do influence second messenger systems of phore treatment is almost completely blocked. Further- neurons. Another interesting study by Schachner and more, the loss of cortical actin filaments that occurs colleagues found that binding of antibodies against the when [Ca''] is elevated by treatment with A23187 adhesive molecules NCAM and L1 to PC12 cells reduces alone is also blocked by the presence of forskolin. These intracellular levels of the inositol phosphates IP, and IP,, results show that regulation of growth cone motility can and increases intracellular [Ca' '1 [Schachner, 19911. involve interactions between second messenger systems, The antibody-induced increase in [Ca' '1 was inhibited in this case perhaps a CAMP-dependent kinase and the by pertussis toxin, suggesting involvement of a G protein components that control intracellular [Ca' '1. Thus, the in the transduction process. In general, G proteins are +
+
+
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activated by interactions with transmembrane proteins, such as hormone receptors, and they in turn regulate second messenger systems. The results of Schachner and colleagues are significant, because Fishman and colleagues report that the G protein, Go, is a major component of the growth cone membrane [Strittmatter et al., 19901. Go is known to regulate C a + + channels, K + channels, phospholipase C, and phospholipase A, [Strittmatter and Fishman, 19911. Furthermore, this group found that GAP-43 regulates the binding to Go of GTP, which controls the activity of Go. Although these studies involved isolated proteins they suggest important interactions that may regulate growth cone behaviors. It is conceivable that such interactions, the regulation of Go by an intracellular protein, GAP-43, might modulate not only Go,but also the proteins with which it interacts, ion channels, phospholipases, and even transmembrane substratum receptors. Thus, regulatory signals may be directed not only from the environment, across the plasmalemma to the growth cone interior, but internal events may regulate how the growth cone perceives its environment. In this case, the regulation of Go and perhaps other G proteins by GAP-43 may allow a growth cone to finetune its sensitivities to particular features of a spatially and temporally heterogeneous environment. These recent papers emphasize the complexity of the regulatory systems that control growth cone migration. Growth cones are now known to contain cell surface receptors, adhesive ligands, and other components that may interact with several second messenger systems to control the organization and activities of the membranes, actin filament arrays, and microtubules of growth cones. In addition, developmentally regulated changes in gene expression may alter the intrinsic machinery of specific populations of growth cones, so that changes in receptors and/or their regulatory systems allow participation in new behaviors, such as synaptogenesis. The work to elucidate these phenomena must continue at molecular, cellular, and higher levels of organization, in order to identify precisely what are the critical points in the regulation of growth cone motility, and uncover how specific extrinsic cues produce their effects. For example, Ivins et al. [ 19911 report that the rapid growth cone collapse induced by contact with unlike neurites is not associated with changes in growth cone cytoplasmic [Ca’ ‘I. However, there are several other regulatory systems to investigate as potential mediators of just this one growth cone behavior. The complexity of growth cone motility is just beginning to be revealed. ACKNOWLEDGMENTS
The preparation of this review was supported by NIH grant HD19950.
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