GAP-43, which is identical to several previously described proteins (B-50, F1, pp46 and p57) found in growth cones and nerve terminals (for a review see Skene, ...
Nerve growth cone motility K. Lankford, C. Cypher and P. Letourneau Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, Minnesota, USA Current Opinion in Cell Biology 1990, 2:80-85
Introduction As suggested by its name (given in 1890 by Santiago Ramon y Cajal), the nerve growth cone is the enlarged tip of a nerve fiber and is important in the elongation of axons and dendrites. Growth cones are locomotory structures, whose movements control the locations of axonal pathways and synaptogenesis. A growth cone is also a sensory apparatus that recognizes local variations in its environment and leads nerve fiber elongation in particular directions. The temporal and spatial distribution of cell adhesion molecules, as well as target-derived soluble molecules and their receptors on growth cones are major determinants of these sensory functions. These ideas have been discussed in several places (Bray and Hollenbeck, A n n u Rev CelIBiol 1988, 4:43-62; Dodd and Jessell, Science 1988, 242:692-699; Harrelson and Goodman, Science 1988, 242:700-708; Kater and Letoumeau, Biology of the Nerve Growth Cone, Alan R. Liss, 1985). The locomotory organization of growth cones generally resembles that of fibroblastic cells, especially the leading lameUa at the front of a migrating fibroblast [1]. Recent reviews of the regulation of the organization and functions of microtubules and actin filaments, as well as of the use of mechanochemical forces provide much information for understanding growth cone motility (Smith, Science 1988, 242:708-715) [2]. Our review focuses on recent papers that examine growth cone motility, the relationship of growth cone motility to neurite elongation, and intracellular regulatory mechanisms.
Neurite elongation and growth cone activity Microtubules and microftlaments are the dominant cytoskeletal components of neurites and growth cones. How do these organdies interact and contribute to neurite elongation and growth cone activity? Neuritic cytoplasm and the central mass of a growth cone resemble the previously described C-domains, rich in microtubules and organelles, while the front of a growth cone is a dynamic P-type region (Bridgman et al., J Cell
Biol 1986, 102:1510-1521) [3-5]. An elegant study of the large nerve growth cones of the sea slug Ap/ysia used Allen video-enhanced-contrast, differential interference contrast (AVEC-DIC) microscopy to show that disruption of actin filaments by cytochalasin B results in a rapid advance of microtubules and membranous organelles to the front edge of a growth cone [3]. When cytochalasin B is removed, the micrombules and membranous organelles are pushed back by the reorganization of the actin matrix. This retreat may involve rearward flow of the cortical actin matrix (Bray and White, Science 1988, 239:883--887). It is as yet unclear whether these changes involve microtubule translocation and assembly/disassembly or both. Com'ersely, the effects of the microtubule polymerizing drug taxol on neurite motility suggest that dense microtubules or a depleted mbulin pool restrict protrusive activity [6]. Since these results are poorly understood, the influence of microtubules on actin filament activity is not clear. However, the beautiful AVEC-DIC video pictures [3] suggest that the advance of microtubules and organelles is controlled by the actinrich peripheral front of the growth cone.
A lively debate has argued whether neurite elongation is 'pushed' forward by microtubule-based a.xonal transport or whether growth cones 'pull' the neurite forward via actin-based motility (Bray, Trends Neurosci 1987, 10:431--434; Goldberg and Burmeister, J Cell Biol 1986, 103:1921-1931; Letoumeau et al., Cell Motil Cytoskeleton 1987, 8:193-209). In considering this, Dennerll et al. [7] and Lamoureux et al. [8] have measured the mechanical forces within neurites. The mechanical properties of neurites and the effects of cytoskeletal drugs show that neurites are under tension produced by the cortical actin network, and that neurite microtubules resist this tension and experience compression [7]. Adhesion of the neurite to the substratum relieves some tension, but when adhesions are released, the additional compression on neuritic microtubules stimulates them to depolymerize. These workers have also measured the 'pull' or axial tensions that advancing growth cones exert on neurites [8]. Myosin, which may participate in this 'pulling' has been localized ultrastmcmrally to areas of density in the actin network at the front of a growth cone [9]. These find-
Abbreviations AVEC-DIC--AIIenvideo-enhanced-contrast,differentialinterferencecontrast; GAP~growth-associatedprotein; IP2--inositol4,5-bisphosphate;IP3--inositol 1,4,5-triphosphate; NCAM~neural cell adhesionmolecule;PMA--phorbol 12-myristate13-acetate. 80
(~) Current Science Ltd ISSN 0955-0674
Nerve growlh cone motility Lankford, Cypher and Letourneau ings have provided the first quantitative support for the notion that protrusion, spreading and 'pull' of the growth cone promotes neurite elongation through relief of compression on neuritic microtubules, thereby stimulating net microtubule polymerization. This model can be related to the reported faster elongation of neurites when thegrowth cone protrusions are lamellipodial rather than filopodial [10]. However, no one has compared the axial tensions generated by lamellipodial and filopodial growth cones. These discussions about 'push' and 'pull' are enlightened by three papers [11-13] that address how microtubules are advanced or polymerized in a growing neurite. Two papers investigated the incorporation of tubulin analogues into neuritic microtubules of nerve growth factor treated PCl2 cells [11,12]. Ultrastructural analysis showed that neuritic microtubules do grow at their plus ends, which are orientated towards the neurite tip [11], and photobteaching cells injected with fluorescent tubulin shows that microtubule turnover or replacement is unusually slow in the proximal neurite and is fast near the growth cone [12]. In addition, microtubules in proximal neurites seem to be relatively stable and non-moving [12]. On the other hand, these results indicate that microtubules in growth cones may be dynamic, growing and shrinking in equilibrium with a pool of distally transported tubulin. Eventually, these short-lived microtubules may be altered by capping, associations with microtubule associated proteins, or other post-translational modifications [2,12,13] that precede their organization into the stable microtubule bundles of neurites [4]. While there are recent reports that the more stable microtubules of neurites have undergone post-translational modifications (e.g. detyrosination or acetylation, [12,13]) it is unclear whether such modifications are the cause or consequence of stabilization. Taken together these studies suggest that microtubules and actin filaments have diverse interactions during neurite elongation; the dense cytoskeletal matrix and rearward cortical flow at the leading edge may restrict the physical advance of microtubules and associated organdies, while the forward pull generated by the leading edge may promote microtubule polymerization and/or stabilization. Stabilized microtubule bundles may then restrict the dynamic actin filament matrix, as a broadened growth cone is converted to a cylindrical neurite. Nearly all studies of neurite growth involve already extended neurites. An important exception is a detailed examination of the positions of several intracelluIar components shortly before the stereotypically located emergence of the first growth cone from an identified neuron in grasshopper embryos [14]. In the few hours before neurite initiation, the Golgi apparatus and dense aggregates of tubulin and actin filaments all accumulate in the region where the growth cone will soon emerge. Although these findings suggest that these intrinsic features determine the site of growth cone formation, improved temporal resolution and more information about possible interactions of these organelles is needed to understand how a growth cone is first organized.
GAP-43 may play a critical role in growth cone motility Growth-associated proteins (GAPs) are developmentally regulated polypeptides that are synthesized at elevated levels during neurite outgrowth and regeneration. The best characterized of these is the neuron-specific protein GAP-43, which is identical to several previously described proteins (B-50, F1, pp46 and p57) found in growth cones and nerve terminals (for a review see Skene, A n n u Rev Neurosci 1989, 12:127-156). While GAP-43 is most abundant during neurite outgrowth, it is also present during synaptogenesis and in some areas of mature brain. This suggests that GAP-43 may be involved not only in growth cone motility, but synaptic plasticity as well. While the exact role of GAP-43 in any of these processes is not fully elucidated, the recent data ofZuber et at [15] suggest that its functions are intimately associated with neuronal cytoarchitecture. Zuber et al. used a GAP-43 expression plasmid to transfect non-neuronal Cos and NIH 3T3 cells. Transient transfection of these cells resulted in the appearance of membrane-associated GAP-43 protein, and in the cells extending long, thin processes. Similar results were obtained in several CliO cell lines. This extension of filopodial-like processes by non-neuronal cells suggests that GAP-43 interacts in some way with the cytoskeleton, perhaps at the plasma membrane, to influence cell shape and motility. GAP-43 has several properties that may modulate its actions. First, it is a substrate for calcium and phospholipidd.ependent protein kinase C. In addition, GAP-43 binds calmodulin in the absence of calcium and releases calmodulin at elevated calcium concentrations (Andreasen et al., Biochemistry 1983, 22:4615--4618). This calmodulin binding is also regulated by the phosphorylation state of GAP-43 (Alexander et at, J Biol C,hem 1987, 262:6108-6118). Thus, GAP-43 may influence the amount of free calmodulin present within neurons. Another way in which GAP-43 may mediate calcium's effects in growth cones is by its phosphorytation-dependent regulation of phosphatidylinositol metabolism (Van Dongen et al., Biochem Biophys Res Commtm 1985, 128:1219-1227). When phosphorylated, GAP-43 may inhibit inositol 1,4,5triphosphate (IP3)-dependent calcium release. In addition to phosphorylation, GAP-43 can also be modified by reversible linkage to long chain fatty acids. Skene and Virag [16] have shown that isolated, intact growth cones will covatently link palmitic acid to membrane proteins, predominantly GAP-43. Their results suggest further that GAP-43 is dynamically acylated and deacylated in growth cones and thus may be redistributed between soluble and membrane- bound forms. A further aspect of GAP-4Ys ability to undergo translocation was recently shown by Van Hooff et al. [17]. They demonstrate that the intracellular localization of GAP-43 changes when PC12 cells are stimulated by nerve growth factor to extend neurites. Prior to nerve grovah factor treatment, GAP-43 is found primarily in lysosomal structures and the Golgi apparatus. After treatment, however, GAP-43 is
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Cytoplasmand cell motility found on the cytoplasmic face of the plasma membrane, especially on lameUipodia and filopodia. The phosphorylation of GAP-43 appears to occur hours after its synthesis, later than its acylation. Nelson et al. [18] have shown that GAP-43 is one of the major substrates for protein kinase C in a preparation of broken, isolated growth cone particles from fetal rat brain. Using various criteria, Nelson et al. identified the phosphoprotein pp46, found in adult rat hippocampal formation, as GAP-43. The phosphorylation of this protein is directly correlated with the persistence of long-term potentiation in adult rats. Thus, neurite outgrowth and synaptic plasticity may share some common mechanisms invoMng GAP-43. As GAP-43 and protein kinase C are found in the presynaptic nerve membrane, GAP-43 may mediate structural remodeling of the nerve terminal.
Intracellular second messengers that regulate growth cone motility Three major second messenger systems, calcium, c3vlic AblP, and protein kinase C have been implicated in control of growth cone motility. In a recent paper, all three have also been shown to act locally at the growth cone to control neurite outgrowth in rat hippocampal pyramidal neurons [19]. Focal application of the calcium ionophore A23187 or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) to pyramidal cell growth cones caused dendritic retraction, while the cTclic AMP activator forskolin promoted dendritic outgrowth. Neither PMA nor forskolin altered growth cone calcium levels or glutamate-induced increases in growth cone calcium, suggesting that the effects of PMA and forskolin were not mediated through calcium. Since the calcium channel blocker Co 2+, the protein kinase C inhibitor trifluoperizine, and the cyclic AMP activator forskolin, could all block glutamate-induced dendrite retraction, these observations suggest that several second messengers may be involved in the effects of glutamate on neurite outgrowth. Although other second messengers have begun to receive greater attention, the data continue to support a central role for calcium in mediating the growth promoting or inhibiting actions of a number of stimuli. Calcium influx into growth cones is implicated in the growth inhibiting actions of several neurotransmitters [19-21]. In identified B19 neurons of the snail Helisoma, the growth inhibiting actions of serotonin correlate with depolarization of the cell membrane and increases in growth cone calcium, presumably through activation of voltage-dependent calcium channels [20]. Acetytcholine has no effect on neurite outgrowth itself, but it antagonizes the effects of serotonin, hyperpolarizes B19 cells and blocks the serotonininduced increases in growth cone calcium [20]. Glutamate inhibition of pyramidal cell dendritic outgrowth is also correlated with high growth cone calcium levels, and the calcium channel blocker La3+ blocks the growthinhibiting action of glutamate [19].
Additional regulatory roles for calcium are indicated by the ability of La3+ and Co 2+ to block the galvanotropic reorientation of growth cones in an electric field [22], and there is suggestive evidence that the effects of adhesive guidance cues may also be mediated in part by intracellular calcium [23-25]. Exposure of PCl2 cells to antibodies to L1 or neural cell adhesion molecule (NCAbl), or to contact with other PC12 cells increases intracellular calcium and lowers inositol 4,5-bisphosphate (IP 2) and IP3 levels [24]. In platelet cells, binding of the fibrinogergfibronectin surface receptor to either ligands or antireceptor antibodies inhibits the calcium elevation and cytoskeletal stabilization that are induced by thrombin or ADP [25]. Finally, ta3 +, which can lower intracellular calcium levels, promotes Helisoma neurite outgrowth on fibronectin, which is normally a poor substrate for neurite outgrowth by these cells [23]. If calcium does play a key role in controlling motility, it is unclear how the local control of calcium levels in the growth cone is precisely regulated. In a recent paper, movement of the endoplasmic reticulum, a potential Ca2+ source, into the advancing growth cone was observed to precede movement of other membranous organelles, but the localization of the endoplasmic reticulum did not correlate precisely with areas of microtubule or growth cone advance [5].
Calcium may act primarily by controlling the cytoskeleton Although calcium ions are involved in many intmcellular processes, recent results suggest that calcium may control neurite outgrowth primarily by regulating the stability of the cytoskeleton, and particularly the stability of actin filaments [26]. An apparent loss of actin filaments occurred in the periphery, but not filopodia, of chick dorsal root ganglion growth cones subjected to calciumelevating conditions that induced growth cone collapse and neurite retraction. Micrombule numbers were also reduced in growth cones subjected to calcium elevating treatment. Exposure to calcium depletion inhibited neurite outgrowth, without causing changes in growth cone shape or neurite retraction, and both actin and microtubules appeared more abundant after this treatment. The actin-stabilizing drug phalloidin mimicked the behavioral effects of calcium depletion and could block or re~'erse the effects of treatment with calcium ionophore, suggesting that changes in actin stability are both necessary and sufficient for the behavioral effects of elevated intraeellular calcium. Taken together the observations suggest a model in which both assembly and disassembly of cytoskeletal structures are necessary for neurite extension, and the balance between assembly and disassembly is strongly influenced by intracellular calcium. This model of regulation of cS,toskeletal organization and turnover by calcium is consistent with the observed inhibition of veil expansion in Aplysia growth cones that were exposed to lowered extraeellular calcium or calcium channel blockers, and with the induction of new veils by focal application of calcium or calcium
Nerve growth cone motility Lankford, Cypher and Letourneau ionophores [27]. The very local stimulation of veil formation by focal calcium or calcium ionophore treatment is also consistent with the idea that calcium acts locally on peripheral growth cone structures. The apparent greater resistance to elevated calcium of chick dorsal root ganglion filopodial actin bundles, compax:ed with peripheral actin networks, implies that the composition of filopodial and lamellipodial actin filament systems may be different. Antibody labeling confirms that different actin-associated proteins can be preferentially located in different parts of the growth cone [1,9], implying that the composition of actin filament networks may differ. The observation that Helisoma growth cones in control medium have more filopodia than in the presence of the calcium channel blocker La3+ implies that filopodial structures are favored in higher calcium concentrations [23]. The observation that the rate of sympathetic neurite outgrowth is dependent on the amount of time spent in the filopodial as opposed to lamellipodial configuration [10] further argues that actin organization in the growth cone is an important influence on neurite outgrowth. However, it has been shown previously that
intact actin networks are not necessary for neurite extension (Letourneau et al., Cell Mota Cytoskeleton 1987, 8:193-209) and more recently it has been reported that intact actin filament networks are not necessary for some types of steering events, since disruption of actin filament networks with cytochalasin B does not abolish growth cone turning in response to applied electric fields [28].
Summary Although many issues remain unresoNed, the past )'ear has wimessed a number of advances in our understanding of the inter-relationships between extracellular influences, cell phenotype, growth associated proteins, second messengers, and cytoskeletal components in the control of neurite outgrowth and growth cone behavior. Some of the early events associated with process initiation have been tentatively identified, and more is known about the assembly and stabilization of the microtubular framework of growing neurites. The mechanical forces involved in neurite extension have begun to be quanti-
Neurotransmitter
Protein kinaseC ..-.~
Microtubules
Tubulin
?
i.° ., - ,.. . .* .. . .° . .° . .- • .° . •. .• -.• -., . -.. .- . - .
°.-•
-,-,-
..°.-•
-. •
CAM/SAM Fig. 1. Schematic diagram of a growth cone showing the relationships between the calcium second messenger system, extracellular growth regulating signals, and cytoskeletal structures. To simplify the diagram, other second messenger systems are not shown. Open arrows indicate established causal relationships between calcium, extracellular growth signals and cytoskeletal assembly. In this model, calcium levels are controlled by calcium influx and efflux from the extracellular space and storage and release from intracellular pools. Changes in calcium levels affect the assembly/disassembly of actin and microtubules, and the assembly/disassembly states of tubulin and actin affect one another. Pairs of tinted arrows facing opposite directions indicate the reversible processes of assembly and disassembly of cytoskeletal structures. Paired open arrows facing opposite directions indicate the reversible movement of calcium ions between a membrane-bound compartment, the cytosol, and the extracellular space• Arrows with question marks indicate possible relationships that can be inferred from indirect observations or studies in other cell types, such as a possible link between receptor binding to cell adhesion molecules (CAMs) or substrate adhesion molecules (SAMs) and intracellular calcium or cytoskeletal stability• Note that calcium channels and neurotransmitter receptors are thought to be mobile in the plane of the plasma membrane and their distribution may be affected by electric fields or other factors. Growth associated protein (GAP)-43 can also be present in a soluble compartment or a membrane-associated compartment.
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Cytoplasmand cell motility fled, and interactions between the actin and microtubule systems are being further characterized. The current data more strongly support a functional role for GAP-43 in control of motility. The data also tend to support a central role for cytoplasmic calcium in mediating the actions of many growth-regulating influences, and strongly implicate changes in actin filament stability as mediating the behavioral effects of calcium (Fig. 1).
Acknowledgements Preparation of this review and work in this laboratory are supported by National Institute of ttealth grants HD 19950 and NS24403. Thanks are given to Hung Russell for the original drawing of the growth cone model (Fig. 1).
sis in the growth cone and neurite shaft. Brain Res 1988, 450:60-68. Taxol treatment causes a loss of actin-rich lamellipodia and ill•podia and a reduction in ferritin or horse radish peroxidase endocytosis. 7.
DEh,'NERLLTJ, JosH1 ttC, STEEl. VL, BUXBAIJM RE, HEIDE,~L~,,'N SR: Tension and compression in the cytoskeleton o f PC12 Neurites If: quantitath'e measurements. J Cell Biol 1988, 107:665-674. Quantitative study of the reLa~e contributions of actin and microtubules to neurite tension. (1) Disrupting actin filaments decreases tension, and greatly reduces the spring constant. (2) Disruption of microtubules increases tension without affecting the spring constant. (3) Releasing tension by detachment, causes depol)Tnerization o f microtubules or at least reduced pobrnerization. ee
8.
LAMOUREUXP, BUXBAUMRE, HEIDE.~L~N,'NSR: Direct evidence that growth cones pull. Nature 1989, 340:159-162. First direct evidence showing that growth cone ackmace correlates with axial tension in the neurite. Axial tension increases as the growth cone ack'ances, up to a point where the tension stabilizes. ee
BRIDG~L~NPC, D.tU.EY ME: The organization of myosin and actin in rapid frozen nerve growth cones. J Cell Biol 1989, 108:95-109. Antimyosin staining is shown to be punctate and found primarily at the border of the central and peripheral regions of the growxh cone, in actively ruffling lameUipodia, and in spots along the filopodial shaft (especially at the base). 9.
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Annotated references and recommended reading • ••
Of interest Of outstanding interest
1. •
LETOURNEAUPC, SHATrUCK TA: Distribution and possible
interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones. Development 1989, 105:505-519. The leading margins of gro~-th cones contain several actin-binding proreins, myosin, tropomyosin, 0t-actinin and filamin, as well as vinculin and talin, proteins invoh'ed in ~loskeleta]-membmne interactions. These cytoskeletal components may interact with cell adhesion molecules o f growth cones such as NCAM, L1, N-cadherin and integrin. 2.
MrrcHtsoN T, KmSaL'~'ERM: Cytoskeletal dynamics and nerve growth. Neuron 1988, 1:761-772. A ~eview o f neuronal growth ~kich ira-•Ires 3 processes: (1) distal transport o f cytoskeletal precursors; (2) growth cone motility, and (3) distal assembly of the cytoskeleton. oe
3. ••
FORSCHERP, S.~UTH SJ..Actions o f cytochalasins on the organization o f actin filaments and microtubules in a neuronal growth cone. J Cell Biol 1988, 107:1505-1516. C)1ochalasin-induced loss of actin fiLaments proceeds in a ~m'e from the periphery to the central region. Recovery follows the same pattern. When actin filaments are disassembled, microtubules and organelles move into the growth cone periphery, when actin pob~nerizes microtubules and organelles are pushed back.
4. •
CHENG TPO, REESE "IS: Compartmentalization o f ant•r•gradely and retrogradely transported organelles in axons and growth cones from chick optic tectum. J Neurosci 1988, 8:3190--3199. Anterogradely and retrogradely transported organ•lies are closely ass•dated with microtubules in neurites and in the base of a growth cone. However, membranous organelles are not associated with the microtubules that project to the front of the growth cone. 5. •
DAit_vYhiE, BRtI~M&q PC: Dynamics o f the endoplasmic reticulum and other membranous organelles in the growtll cones o f cultured neurons. J Neurosci 1989, 9:1897-1909. DiOC6 ~-as used to monitor endopLasmic reticulum and other membranous organdies in living cells. The endopLasmic reticulum precedes other organelles in extending into adx~mcing growth cones. The positions o f the endoplasmic reticulum and other membranous organeUes correLate only approximately with the positions of rater•tubules. 6. •
S~cIAm GI, BAAS PW, HEIDEg~tA.'~,'NSR: Role of microtubules in cytoplasmic compartmentation o f neurons. II. End•oTto-
t0. •
KLE1TMANN, JOtlNSON MI: Rapid growth cone translocati6n on laminin is supported by lamellipodial not filopodial structures. Cell Motil Cytoskeleton 1989, 13:288-300. Rate of neurite outgrowth in embryonic and postnatal rat wmpathetic neurons correhtes with the reLa~'e amount of time spent in the lamellipodial as opposed to filopodial configuration, ,~ith lameUipodial growth cones moving faster. 11.
OKABES, |tlROKAWAN: Microtubule dynamics in n e n ' e cells: analysis using microinjection of biotinylated tubuUn in PC12 cells. J Cell Biol 1988, I07:651-6(~. Labeled tubulin is added primarily to the distal ends of microtubules along the length of neurites. The rate of addition is roughtly 0.3 lam/min. The evidence suggests that tubulin is transported as a monomer. t e
12. • •
LL~tS-S, S,*.MMAKPJ, BORIS'/GG: Progressive and spatially differentiated stability of microtubules in de~-eloping neuronal cells. J Cell Biol 1989, 109:253-263. Fluorescence photobleaching study of rater•injected rhodamine tubulin. Shows that (1) recovery of bleached areas is slower in more mature cells; (2) recovery is faster in grovah cones and the cell body than in neurites; (3) bleached areas do not move, and (4) regions with more stabile microtubules also show more acetylated tubulin. 13. •
ROBSONSJ, BURGOYNE RD: Differential localization of tyrosinat•d, detyrosinated, and acetylated alpha-tubulins in neurites and growth cones o f dorsal root ganglion neurons. Cell Motil Cytoskeleton 1989, 12:273-282. Tyrosinated ct-tubulin is found throughout the neurite and growth cone. Detyrosinated and acetylated ct-tubulins, however, are restricted to the neurite shaft. Post-transLation modification of (x-tubulln may, therefore, stabilize microtubules in neurite shafts. 14. • •
LEFCORTF, BENTLEY D: Organization o f cytoskeletal elements and organelles preceding growth cone emergence from an identified neuron in situ. J Cell Biol 1989, 108:1737-1749. This is the first study showing the structural changes that precede neurite outgro~:,-h in a neuron in sittt A microtubule cap, the Golgi apparatus and an actin filament cap accumuLate and predict the site of axon initiation.
ZUBERl~,[X, GOOD~I.~N DW, KAP~'~SI.R, FlStLSt&'~MC: The neuronal growth-associated protein GAP-43 induces ill•podia in non-neuronal ceils. Science 1989, 244:1193-1195. First demonstration of a function for GAP-43. Filopodial-like processes are produced in normally spherical cells after transfection with, and expression of, the gene for GAP-43. 15. o•
Nerve growth cone motility Lankford, Cypher and Letourneau 16. •
SKENEJHP, VIRAG I: Posttranslational m e m b r a n e a t t a c h m e n t and d y n a m i c fatty acylation of neuronal grov,a h c o n e protein GAP43. J Cell Biol 1989, 108:613-624. This paper describes the reversible attachment of f a w acids to GAP-43 and other aspects of GAP43 association with membranes. 17. ••
VAN IIOOFF COM, HOL'IMUISJCM, OESTREICHERAB, BOONSTRA J, DE GRA&q PNE, GISPEN V¢]t: Nerve g r o w t h factor-induced c h a n g e s in t h e intracellular localization of t h e protein kinase C substrate B-50 in p h e o c h r o m o c y t o m a PCI2 cells. J Cell Biol 1989, 108:1115-1125. The intracellular distribution of GAP-43 changes wanen PCI2 cells are treated with nerve growth factor. GAP-43 immunoreac~'ity shifts from br,osomal stmctures and the Golgi apparatus to the plasma membrane, especially to protruding micro~illi, lamellipodia and filopodia. 18. •
NELSON RB, LINDEN DJ, ttY~t~ C, PFEN,'NLNGER K}|, RotrITENBERGA: T h e two major p h o s p h o p r o t e i n s in g r o w t h c o n e s are probably identical to two protein kinase C substrates correlated with the persistence of long term potentiation. J Neurosci 1989, 9:381-389. E~idence is given that 2 protein kinase C substrates in growth cones, o n e of which is GAP-43, are identical to 2 proteins phosphorylated in adult rat hippocampal formation following induced long-term potentiation. These data suggest that a c o m m o n mechanism may underlie neurite outgrowth in development and neural plasticity in adult rats. 19. • •
M A ~ N MP, GtmlRm PB, KATER SB: Intracellular m e s s e n gets in t h e generation and degeneration o f hippocampal neuroarchitecture. J Neurosci Res 1988, 21:447-464. Focal application of A23187 or PMA to pyramidal cell growth cones is shown to induce dendritic retraction, "afiile forskolin promotes dendritic outgrowth. These results indicate that all 3 second messenger systems can act locally at the growth cone to control neurite outgrowth, and that all 3 can be operable in the same cell type. 20. • •
MCCOBBDP, COHAN CS, CON,~ORJA, KATERSB: interactive effecrs o f serotonin and acetylcholine on neurite elongation. Neuron 1988, 1:377-385. Acetylcholine and serotonin affect membrane potential and calcium influx of identified IIelisoma neurons in opposite w'a}~, and acetylchollne blocks the inhibitory effects of serotonin on neurite elongation. 21. •
McCOBB DP, KATERSB: Membrane voltage and neurotransmitter regulation of neuronal g r o w t h c o n e motility. Dev Biol 1988, 130:599-609. tt)perpolarization of cell blocks the motility inhibiting actions of dopamine and serotonin. 22. ••
McCPaaG CD: Studies on t h e m e c h a n i s m o f embryonic frog nerve orientation in a small applied electric field. J Cell Sci 1989, 93:723-730. This paper s h o ~ that inorganic calcium channel blockers prevent growth cone steering in an electric field, and presents evidence from
other treatment conditions that filopodial reorientation and growth cone turning responses m~" be separately controlled. The authors present a model for electric field induced steering in which gro~.h cone mining is caused by as)rnmetric calcium influx. 23. •
MATTSONMP, GUTHRIE PB, KATER SB: C o m p o n e n t s o f neurite o u t g r o w t h that d e t e r m i n e neuronal cytoarchitecture: influence o f calcium and t h e g r o w t h substrate. J Neurosci Res 1988, 20:331-345. Lowering extracellular calcium makes a previously poor substrate a good one for neurite grow~.h. 24. o•
ScHtmOt U, LOHSE MJ, SCHACI-LNERK{: Neural cell adhesion molecules influence s e c o n d m e s s e n g e r systems. Neuron 1989, 3:13-20. Antibodies to L1 or NCAM, or cell-cell contacts between PC12 cells, increase intracellular Ca2+ while reducing levels of IP3 and IP 2. These observations provide sugges~'e evidence for calcium and/or G-protein inx~ob.ement in mediating the effects of cell-cell adhesion on neurite outgro~',.h. 25. •
SINIGAGLL~F, BtSlO A, TORn M, BALDUL\'I CL, BERTOL~O G, BALDUtX'IC: Effects of G P l l b - l l l a c o m p l e x ligands o n calcium ion m o v e m e n t and q,'toskeleton organization in activated platelets. Bic~bem Bioplo~ Res Commun 1988, 154:258--264. Activation o f the fibronectin/fibrinogen receptor with the s3~mhetic peptide GRGDS, or an antibody inhibits the intracellular calcium elevation and c~loskeletal reorganization seen when platelets are stimulated ~ith thrombin or ADP. 26. • •
LANKFORDKL, Iz'rouR.\'EAtJ PC: Evidence that calcium m a y control neurite o u t g r o w t h by regulating t h e stability of actin filaments. J Cell Biol 1989, 109:1229-1243. This light and whole m o u n t electron microscopic study shows that a loss of peripheral actin filaments is associated with treatments that increase growth cone calcium and cause neurite retraction. Phalloidin blocks or reverses the effects of calcium-elerating conditions and mimics the effects of calciumlowering conditions, suggesting that changes in actin filaments are both necessary and sufficient to explain the beha~ioral response to calcium manipulations. 27. GOLDBERGDJ: Local role o f Ca 2+ in formation of veils in • • g r o w t h cones. J Neurosci 1988, 8:2596-2605. VEC-DIC study showing that: (1) low calcium inhibits lameUipodial advance, and (2) local calcium application can induce new lamelllpodia. 28. •
MCCRtdGCD: Nerve g r o w t h in t h e absence of g r o w t h cone filopodia and t h e effects o f a small applied electric field. J Cell Sci 1989, 93:715-721. Neurons treated with cytochalasin B continue to exhibit growth cone turning towards the cathode in an applied electric field, indicating that intact actin filament networks are not necessary for s o m e t~pes of steering events.
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