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Chapter 8 Regulation of events within the growth cone by extracellular cues: tyrosine phosphorylation
F.J.Seil (Ed.)
Progress in Brain Research, Vol 103 Q 1994 Elsevier Science BV. All rights reserved.
CHAPTER 8
Regulation of events within the growth cone by extracellular cues: tyrosine phosphorylation Daniel J. Goldberg and Da-Yu Wu Department of Pharmacology and Centerfor Neurobiology and BehaLnor, Columbia Uniwrsiw, 630 W 168th St., New York W10032, U S A
Introduction
The growth cone is a critical site for the interactions with environmental cues that produce directed axonal growth during development and regeneration. The digitate filopodia that project tens of microns from the body of the growth cone to sample the environment are key sites for these interactions. Filopodia become more numerous when a growth cone reaches a region in which one of multiple potential pathways must be selected (Tosney and Landmesser, 1985; Bovolenta and Mason, 1987). In the developing grasshopper limb, contact of a filopodium of the first ingrowing axon with a critical landmark in such a region is responsible for turning of the axon onto the correct pathway (Caudy and Bentley, 1986; O’Connor et al., 1990). If the formation of filopodia is pharmacologically prevented in this ‘pioneer’ axon, its growth is wayward (Bentley and Toroian-Raymond, 1986). Intracellular mechanisms mediating the detection of cues by filopodia and their transduction into changes in growth cone behavior are not well understood. In this article, we will discuss evidence implicating protein-tyrosine phosphorylation in the functioning of filopodia. Several findings suggest an involvement of tyrosine phosphorylation in axon growth. The src protein-tyrosine kinase (PTK) is particularly highly expressed during nervous sys-
tem development and is enriched in the growth cone (Matten et al., 1990). Another PTK, abl, as well as certain protein-tyrosine phosphatases (PTPs), are transiently expressed in certain axonal tracts of the developing Drosophila nervous system (Elkins et al., 1990; Hariharan et al., 1991; Tian et al., 19911, and genetic deletion of the abl PTK contributes to wayward axonal growth (Elkins et al., 1990). Certain molecules capable of stimulating axonal growth cause rapid changes in protein-tyrosine phosphorylation in neurons or neuron-like cell lines (Maher, 1988; Atashi et al., 1992). While suggesting the involvement of proteintyrosine phosphorylation in axon growth, the foregoing results do not define specific roles. In fact, the only role that has so far been established for protein-tyrosine phosphorylation is at the beginning of the signaling pathway for nerve growth factor, whose receptor is a PTK which undergoes autophosphorylation (Loeb et al., 1991). A role in the filopodium would put protein-tyrosine phosphorylation in a key spot to mediate effects of environmental cues on axonal growth, particularly pathfinding. Concentration of phosphotyrosine at tip of filopodium
The bulk of our studies have involved the use of
76
Fig. 1. Concentrated phosphotyrosine at the tips of the filopodia of an AprySia growth cone. The neuron was cultured on a polylysine substrate in protein-free, defined medium and labeling with a monoclonal antibody to phosphotyrosine was visualized by indirect immunofluorescence. The growth cone was photographed with a 35-mm camera connected to the microscope. Arrowheads point to two of the many brightly stained filopodial tips. The filopodia are short in this growth cone. Bar, 10 pm.
isolated large neurons from the central nervous system (CNS) of the marine slug, Aplysia, displaying regenerative axon growth in culture. These neurons can produce growth cones which are especially suitable for the video microscopic techniques we employ. The axons grow quite slowly when cultured in protein-free medium on a substrate coated only with polylysine; growth is much faster when the substrate has also been pre-exposed to hemolymph from the animal, which contains a high molecular weight protein which promotes growth (Burmeister et al., 1991). Fluorescent staining of Apbsia growth cones on the polylysine substrate with an antibody specific for phosphorylated tyrosine residues yields a striking pattern: the tips of most of the filopodia are intensely bright (Fig. 1). The pattern is especially impressive when viewed with video intensified fluorescence microscopy, with the image magnification increased several-fold (Fig. 2A). The bright staining is often associated with a swelling of the tip, but it is clear that the intensity of the staining results not simply from increased volume but from a concentration of phosphotyrosine at the tip. This is evident when the pattern of staining of phosphotyrosine (Fig. 2A) is compared with
staining of the same growth cone with Texas Red (Fig. 2B), a fluorescent probe which indiscriminately stains cellular protein. The difference in intensity of staining between filopodial tip and shaft is much greater for phosphotyrosine than for Texas Red. We have also detected bright staining of filopodial tips of embryonic chick sympathetic and retinal ganglion neurons and neonatal rat hippocampal neurons cultured in serumfree medium on polylysine or polyornithine (Figs. 3 and 4A), though the fraction of filopodial tips that are brightly stained is substantially less than in the Aplysza cultures. The role of the protein-tyrosine phosphorylation described here must be different from the single defined role, noted above, of initiating a signaling cascade in response to nerve growth factor (or analogous growth factors). This is clear because the ApZysia neurons are cultured in a protein-free medium on a protein-free substrate in the absence of substantial numbers of other neurons or non-neuronal cells. Yet, there are indications that the phosphorylation is involved in mediating interactions with environmental cues, as its location at the tips of filopodia would suggest. When ApZysia neurons are cultured on a substrate pre-exposed to hemolymph as well as polylysine, far fewer filopodial tips display intense staining with the anti-phosphotyrosine antibody (Wu and Goldberg, 1993). Phosphotyrosine disappears from tips within a few minutes of addition of hemolymph to neurons cultured on a polylysine substrate (Wu and Goldberg, 1993). In addition, we have found integrin to coconcentrate with phosphotyrosine in filopodial tips of cultured chick sympathetic neurons (Fig. 4). Integrin is the membrane receptor for the extracellular matrix proteins, laminin and collagen, which promote neurite growth in these cells. Can the interaction of an individual filopodium with an environmental cue cause a change in the amount or distribution of phosphotyrosine in that filopodium? We do not yet know because, in the aforementioned experiments, the entire growth cone, not only the filopodium, is exposed to the growth promoting material of hemolymph. We are currently assessing the effects on phosphory-
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Fig. 2. Concentrated phosphotyrosine at the tips of the filopodia of an Aplysia growth cone, as recorded by video intensified fluorescence microscopy. Preparation was as described for Fig. 1,but the image was recorded with a SIT video camera connected to the microscope. This is a different growth cone from the one shown in Fig. 1. (B) This is the same growth cone as in panel A, showing the staining pattern obtained with Texas Red fluorophore, which indiscriminately stains proteins. Bar, 5 pm.
lation of exposure of filopodia alone to cues. It does appear that the machinery for dephosphorylation is present at the tip, since administration of an inhibitor of PTKs to a growth cone on polylysine results in dimming of the tip staining (Fig. 5). Role of tyrosine phosphorylationin the filopodium
What is the role of the tyrosine phosphorylation in the filopodium? This question should be considered in the context of evidence that the phos-
Fig. 3. Concentrated phosphotyrosine at the tips of filopodia of a neonatal rat hippocampal neuron. The neuron was cultured on a polylysine substrate in a defined, serum-free medium. Staining was as described in previous figures, and this and all subsequent fluorescent images were recorded with the SIT camera. Arrowheads point to the stained tips. The shafts of these short filopodia are so lightly stained that they are difficult to see in this view. Bar, 5 pm.
photyrosine associates or interacts with actin filaments. The core of the filopodiumis a bundle of actin filaments (Fig. 6A); at the distal end of this bundle is the concentrated phosphotyrosine (Fig. 6B), though we do not know if there is a physical connection. We have recently shown that filopodia-like protrusions that have a core bundle of microtubules form for a short time after transection of Aplysia axons in culture (Goldberg and Burmeister, 1992). These microtubule based protrusions do not develop concentrations of phosphotyrosine at their tips, while conventional actin-based filopodia forming at the same time do (see Fig. 8B). In addition, one can cause the network of actin filaments in the peripheral region of the growth cone to rapidly withdraw into the central region by administering the actinspecific drug, cytochalasin (Fig. 7A). Phosphotyrosine withdraws from the tip in clumps, sometimes fragmenting or spreading (Fig. 7B). Thus, the concentration of phosphotyrosine in the filopodial tips depends on the presence of actin filaments. With this in mind, we will consider three possible roles for the tyrosine phosphorylation.
Formation of jilopodia The first is in the formation of filopodia. Nonneuronal motile cells, such as fibroblasts, have peripheral actin-rich regions similar to that found in the growth cone. The actin filament bundles underlying their microspikes (short filopodia) form
78
Fig. 4. Concentrated phosphotyrosine at the tips of filopodia of an embryonic chick sympathetic neuron. The neuron was cultured on a polyomithine substrate in a defined, serum-free medium. (B) The same growth cone was also labeled with a polyclonal antibody to the PI subunit of integrin, and visualized with a different fluorophore from that used for panel A. Concentrated integrin is present at the tips, which displayed concentrated phosphotyrosine. Bar, 5 pm.
by an initial coalescence of the distal ends of filaments, followed by a disto-proximal zippering of the filaments into a bundle (Izzard, 1988). The localization of phosphotyrosine at the distal end of the actin bundle in the growth cone filopodia suggests it could be involved in this coalescence. However, we think this is not the case. As mentioned above, axotomy causes massive numbers of
Fig. 5. Dimming of staining for phosphotyrosine at the tips of filopodia of an APlYsia growth cme which had been exposed for 10 min to 100 p M genistein, a broad-specificity inhibitor of PTKs. The arrowheads point to filopodia which have relatively dim staining, while the arrows point to the positions of filopodia which cannot be seen because they have lost all their staining. Bar, 5 pm.
filopodia to form within a short time and so is a convenient means for studying factors underlying their formation. Contrary to the expectation if tyrosine phosphorylation were involved in the initial stages of filopdial formation, bright tip staining is not present when the filopodium first forms but only develops later. Figure 8, which shows an axon 25 min after axotomy (actin based filopodia start to form about 10 min after axotomy), demonstrates that many of the filopodia have no discernible concentration of phosphotyrosine at their tips. Even for those filopodia that display tip staining, the concentrations of phosphotyrosine are more modest than many seen later after axotomy (Fig. 9). Regulation of filopodial length A second possible role is in regulating filopodial length by regulating the polymerization of the core bundle of actin filaments. This is once again suggested by the location of the concentrated phosphotyrosine at the distal end of the core bundle. FiloDodia are dvnamic structures, movingin and out bemeen pauses. The distal end of the filaments is the preferred site for addition and loss of subunits; in fact, there is a addition of subunits at this end, regardless of
79
Fig. 6. Concentrated phosphotyrosine is adjacent to the distal ends of the bundles of actin filaments that comprise the cores of the filopodia.(A) This panel shows staining of an Aplysia growth cone with fluorescent phalloidin, which binds to filamentous actin. (B) The same growth cone is also stained with antibody to phosphotyrosine, visualized with a different fluorophore. Bar, 5 pm.
whether the axon is growing rapidly (Forscher and Smith, 1988; Okabe and Hirokawa, 1991). Thus, the distal tip of the filopodium should be a key site for the regulation of filopodial dynamics. In support of the idea that the tip phosphorylation is involved in regulating filopodial dynamics are our findings that filopodia exposed acutely to inhibitors of PTKs (such as genistein) or to hemolymph not only lose phosphotyrosine from their tips but lengthen considerably as a result of a change in their dynamics (Fig. 10). Underlying the
lengthening of the filopodium is a lengthening of the core bundle of actin filaments (Wu and Goldberg, 1993). While these results indicate an association between loss of tip phosphotyrosine and acute lengthening, we cannot assuredly say that the former causes the latter. Both the addition of PTK inhibitors and hemolymph have strong effects in the central region of the growth cone. These effects probably involve the release of large numbers of actin subunits, which could diffuse to the filopodial tips to drive actin polymerization
Fig. 7. Withdrawal of phosphotyrosine from the tips of filopodia when actin filaments are caused to withdraw by application of cytochalasin. (A) Video enhanced contrast-differential interference contrast (VEC-DIC) micrograph of an Aplysiu growth cone 4 min after the application of 1 p M cytochalasin D. The arrowheads point to the distal border of the network of actin filaments as it withdraws from the margin of the growth cone. The area distal to the arrowheads appears flatter because of the withdrawal of the actin network. (B) Phosphotyrosine is visualized by indirect immunofluorescence in a different growth cone, also 4 min after the addition of cytochalasin. Arrows point to filopodial tips which are not stained and arrowheads point to clumps of tyrosine phosphorylated protein apparently in the process of moving proximally along the filopodia. Bar, 5 pm.
80
Fig. 8. Concentrated phosphotyrosine is not common in filopodial tips soon after formation of filopodia. (A) This VEC-DIC micrograph shows one side of a single Aplysia axon in culture which was transected 25 min previously. Numerous filopodia have grown from the side of the axon. (B) Only three of these filopodia (arrows) have marked concentrations of phosphotyrosine at their tips, as detected by indirect immunofluorescence.Bar, 5 pm.
and, thus, filopodial lengthening. So, additional data are needed to conclude that tip phosphorylation is involved in regulating filopodial dynamics. It should be useful, as noted above, to determine whether exposure of individual filopodia to PTK inhibitors or hemolymph (to avoid primary effects elsewhere in the growth cone which could secondarily influence the filopodia) affects filopodial dynamics. In addition, not all filopodia on a polylysine substrate have large concentrations of phosphotyrosine at their tips, nor are all of the
Fig. 9. Large tip concentrations of phosphotyrosine develop in filopodia after axotomy. This axon was fked 45 min after transection. Several fdopodial tips (arrows) have large concentrations of phosphotyrosine. Some others which have lesser concentrations have concentrations of phosphotyrosine more proximally along the shaft (arrowheads). Bar, 5 pm.
filopodia on a polylysine/hemolymph substrate lacking bright tip staining (Wu and Goldberg, 1993). So, we should be able to determine whether naturally occurring differences in tip phosphorylation among filopodia of individual growth cones correlate with differences in dynamics. Attachment formation with cells or extracellular matrix A last possible role to consider for tyrosine phosphorylation in the filopodium is in forming attachments with cells or extracellular matrix along the path of growth. Specialization of the tip of some filopodia for adhesion has been described (Tsui et al., 1985), and interactions of only the distal part of the filopodium with environmental cues seem sufficient to alter growth cone behavior (Hammarback and Letourneau, 1986; Bandtlow et al., 1990; O’Connor et al., 1990). The one site where phosphotyrosine has been found to be heavily concentrated in non-neuronal cells is at the adherens junction, either between cells or between a cell and the substrate (focal contact). The fact that the phosphotyrosine of an adherens junction is at the ‘barbed‘ end of a bundle of polarized actin filaments, as in the filopodium, addition, we increases the appeal of this idea, have preliminary evidence that, on a polylysine/hemolymph substrate where the majority of filopodial tips do not have concentrated
81
Fig. 10. Application of an inhibitor of PTKs causes filopodia to lengthen. VEC-DIC micrographs show an Aplysia growth cone immediately before (A) and 7 min after (B) application of 100 pm genistein. Bar, 5 pm.
phosphotyrosine, some sites of contact between filopodia show highly elevated levels of phosphotyrosine (Fig. 11). However, it is clear that tyrosine phosphorylation in the filopodium need not be associated with attachments. The filopodial tips that display intense staining for phosphotyrosine on a polylysine substrate are quite mobile and can be seen by video microscopy to lift off the substrate frequently and move about (Wu and Goldberg, 1993).
Fig. 11. Concentrated phosphotyrosine at filopodium-file podium contacts. An Aplysia neuron was placed in culture on a substrate pre-exposed both to polylysine and to hemolymph. Arrows point to several filopodial tips which do not have marked concentrations of phosphotyrosine. Two sites of filopodium-filopodium contact display large concentrations of phosphotyrosine (arrowheads). Bar, 5 pm.
Assessment of the separation of these tips from the substrate by interference reflection microscopy confirms that these are not focal, or even close, contacts (Wu and Goldberg, 1993). Thus, the accumulation of phosphotyrosine occurs independently of the formation of contacts. It has been suggested that protein tyrosine phosphorylation facilitates the formation of protein assemblies at the plasma membrane in response to growth-factor binding (Koch et al., 1991). Aggregates of non-organellar material have been described moving rapidly forward and rearward in growth cone filopodia (Sheetz et al., 1990). We find that high concentrations of phosphotyrosine at filopodial tips are often associated with swellings (Fig. 2; Wu and Goldberg, 1993) and that phosphotyrosine withdrawing from the tips of filopodia after cytochalasin treatment often moves back as a clump (Fig. 7B). We have also looked at the development of phosphotyrosine in newly formed filopodia after axotomy. Filopodia with intense tip staining tend to display bright staining only at the tip, whereas ones with dimmer tip staining, presumably earlier in development, have bright clumps along their shafts (Fig. 9B), suggesting that tyrosine phosphorylated protein moves in clumps towards the tip of the filopodium. Thus, protein tyrosine phosphorylation may facilitate formation of assemblies of proteins, such as integrin, or facilitate their association with actin filaments (or both), to promote their movement to
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and concentration in distal parts of the filopodium, where they would participate in pathfinding activities. Conclusion
We have found tyrosine phosphorylated protein concentrated in the tips of some growth cone filopodia in culture. Its concentration there is acutely sensitive to the presence of growth promoting material on the substrate. These findings strongly suggest that one function of proteintyrosine phosphorylation during axonal growth is in mediating or facilitating certain functions of filopodia, structures which detect and respond to environmental cues that guide axonal growth. Two possible specific roles for tyrosine phosphorylation are in regulating the dynamics, and thus the length, of filopodia and in facilitating the accumulation in the filopodium of membrane receptors for environmental cues with which the filopodium interacts. It will be important to identify the tyrosine phosphorylated proteins. It should also be illuminating to define the effects of well-defined, important environmental cues, such as identified growth promoting or inhibiting factors or synaptic target and non-target neurons on the amount and distribution of phosphotyrosine in individual filopodia. Acknowledgements
This work was supported by NIH training grant MH15174, NIH fellowship NS09225, NIH research grant NS25161 and NIH program project GM32099. References Atashi, J.R., Klinz, S.G., Ingraham, C.A., Matten, W.T., Schachner, M. and Maness, P.F. (1992) Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes. Neuron, 8: 831-842. Bandtlow, C., Zachleder, T. and Schwab, M.E. (1990) Oligodendrocytes arrest neurite growth by contact inhibition. J. Neurosci., 10: 3837-3848.
Bentley, D. and Toroian-Raymond, A. (1986) Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment. Nature (Lorad.), 323: 712-715. Bovolenta, P. and Mason, C. (1987) Growth cone morphology varies with position in the developing mouse visual pathway from retina to first targets. J. Neurosci., 7 1447-1460. Burmeister, D.W., Rivas, R.J. and Goldberg, D.J. (1991) Substrate-bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation. Celf Motil. Cyfoslcel., 19: 255-268. Caudy, M. and Bentley, D. (1986) Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities. J. Neurosci., 6: 1781-1795. Elkins, T., Zinn, K., McAllister, L., Hoffmann, F.M. and Goodman, C.S. (1990) Genetic analysis of a Drosophila neural cell adhesion molecule: interaction of fasciclin I and Abelson tyrosine kinase mutations. Cell, 60: 565-575. Forscher, P. and Smith, S.J. (1988) Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J. Cell Biol., 107 1505-1516. Goldberg, D.J. and Burmeister, D.W. (1992) Microtubulebased filopodium-like protrusions form after axotomy. J. Neurosci., 1 2 4800-4807. Hammarback, J.A. and Letoumeau, P.C. (1986) Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding. Deu. Biol., 117: 655-662. Hariharan, I.K., Chuang, P.-T. and Rubin, G.M. (1991) Cloning and characterization of a receptor-class phosphotyrosine phosphatase gene expressed on central nervous system axons in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA, 88: 11266-11270. Izzard, C.S. (1988) A precursor of the focal contact in cultured fibroblasts. Cell Motil. Cytoskel., 10: 137-142. Koch, C.A., Anderson, D., Moran, M.F., Ellis, C. and Pawson, T. (1991) SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science, 252: 668-674. Loeb, D.M., Maragos, J., Martin-Zanca, D., Chao, M.V., Parada, L.F. and Greene, L.A. (1991) The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines. Cell, 66: 961-966. Maher, P.A. (1988) Nerve growth factor induces protein-tyrosine phosphorylation. Proc. Natl. Acad. Sci. USA, 85: 6788-6791. Matten, W.T., Aubry, M., West, J. and Maness, P.F. (1990) Tubulin is phosphorylated at tyrosine by pp60c.s'c in nerve growth cone membranes. J. Cell Biol., 111: 1959-1970. O'Connor, T.P., Duerr, J.S. and Bentley, D. (1990) Pioneer growth cone steering decisions mediated by single filopodial contacts in situ. J. Neurosci., 10: 3935-3946. Okabe, S. and Hirokawa, N. (1991) Actin dynamics in growth cones. J. Neurosci.. 11: 1918-1929.
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Sheetz, M.P., Baumrind, N.L., Wayne, D.B. and Pearlman, A.L. (1990) Concentration of membrane antigens by forward transport and trapping in neuronal growth cones. Cell, 61: 231-241. Tian, S.-S., Tsoulfas, P. and Zinn, K. (1991) Three receptorlinked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Cell, 61: 615-685. Tosney, K.W. and Landmesser, L.T. (1985) Growth cone rnor-
phology and trajectory in the lumbosacral region of the chick embryo. J. Neurosci., 5: 2345-2358. Tsui, H.-C.T., Lankford, K.L. and Klein, W.L. (1985) Differentiation of neuronal growth cones: specialization of filopodial tips for adhesive interactions. Proc. Natl. Acad. Sci. USA, 82: 8256-8260. Wu, D.-Y. and Goldberg, D.J. (1993) Regulated tyrosine phosphorylation at the tips of growth cone filopodia. J. Cell Biol., 123: 653-664.
Progress in Brain Research, Vol 103 Q 1994 Elsevier Science BV. All rights reserved.
CHAPTER 8
Regulation of events within the growth cone by extracellular cues: tyrosine phosphorylation Daniel J. Goldberg and Da-Yu Wu Department of Pharmacology and Centerfor Neurobiology and BehaLnor, Columbia Uniwrsiw, 630 W 168th St., New York W10032, U S A
Introduction
The growth cone is a critical site for the interactions with environmental cues that produce directed axonal growth during development and regeneration. The digitate filopodia that project tens of microns from the body of the growth cone to sample the environment are key sites for these interactions. Filopodia become more numerous when a growth cone reaches a region in which one of multiple potential pathways must be selected (Tosney and Landmesser, 1985; Bovolenta and Mason, 1987). In the developing grasshopper limb, contact of a filopodium of the first ingrowing axon with a critical landmark in such a region is responsible for turning of the axon onto the correct pathway (Caudy and Bentley, 1986; O’Connor et al., 1990). If the formation of filopodia is pharmacologically prevented in this ‘pioneer’ axon, its growth is wayward (Bentley and Toroian-Raymond, 1986). Intracellular mechanisms mediating the detection of cues by filopodia and their transduction into changes in growth cone behavior are not well understood. In this article, we will discuss evidence implicating protein-tyrosine phosphorylation in the functioning of filopodia. Several findings suggest an involvement of tyrosine phosphorylation in axon growth. The src protein-tyrosine kinase (PTK) is particularly highly expressed during nervous sys-
tem development and is enriched in the growth cone (Matten et al., 1990). Another PTK, abl, as well as certain protein-tyrosine phosphatases (PTPs), are transiently expressed in certain axonal tracts of the developing Drosophila nervous system (Elkins et al., 1990; Hariharan et al., 1991; Tian et al., 19911, and genetic deletion of the abl PTK contributes to wayward axonal growth (Elkins et al., 1990). Certain molecules capable of stimulating axonal growth cause rapid changes in protein-tyrosine phosphorylation in neurons or neuron-like cell lines (Maher, 1988; Atashi et al., 1992). While suggesting the involvement of proteintyrosine phosphorylation in axon growth, the foregoing results do not define specific roles. In fact, the only role that has so far been established for protein-tyrosine phosphorylation is at the beginning of the signaling pathway for nerve growth factor, whose receptor is a PTK which undergoes autophosphorylation (Loeb et al., 1991). A role in the filopodium would put protein-tyrosine phosphorylation in a key spot to mediate effects of environmental cues on axonal growth, particularly pathfinding. Concentration of phosphotyrosine at tip of filopodium
The bulk of our studies have involved the use of
76
Fig. 1. Concentrated phosphotyrosine at the tips of the filopodia of an AprySia growth cone. The neuron was cultured on a polylysine substrate in protein-free, defined medium and labeling with a monoclonal antibody to phosphotyrosine was visualized by indirect immunofluorescence. The growth cone was photographed with a 35-mm camera connected to the microscope. Arrowheads point to two of the many brightly stained filopodial tips. The filopodia are short in this growth cone. Bar, 10 pm.
isolated large neurons from the central nervous system (CNS) of the marine slug, Aplysia, displaying regenerative axon growth in culture. These neurons can produce growth cones which are especially suitable for the video microscopic techniques we employ. The axons grow quite slowly when cultured in protein-free medium on a substrate coated only with polylysine; growth is much faster when the substrate has also been pre-exposed to hemolymph from the animal, which contains a high molecular weight protein which promotes growth (Burmeister et al., 1991). Fluorescent staining of Apbsia growth cones on the polylysine substrate with an antibody specific for phosphorylated tyrosine residues yields a striking pattern: the tips of most of the filopodia are intensely bright (Fig. 1). The pattern is especially impressive when viewed with video intensified fluorescence microscopy, with the image magnification increased several-fold (Fig. 2A). The bright staining is often associated with a swelling of the tip, but it is clear that the intensity of the staining results not simply from increased volume but from a concentration of phosphotyrosine at the tip. This is evident when the pattern of staining of phosphotyrosine (Fig. 2A) is compared with
staining of the same growth cone with Texas Red (Fig. 2B), a fluorescent probe which indiscriminately stains cellular protein. The difference in intensity of staining between filopodial tip and shaft is much greater for phosphotyrosine than for Texas Red. We have also detected bright staining of filopodial tips of embryonic chick sympathetic and retinal ganglion neurons and neonatal rat hippocampal neurons cultured in serumfree medium on polylysine or polyornithine (Figs. 3 and 4A), though the fraction of filopodial tips that are brightly stained is substantially less than in the Aplysza cultures. The role of the protein-tyrosine phosphorylation described here must be different from the single defined role, noted above, of initiating a signaling cascade in response to nerve growth factor (or analogous growth factors). This is clear because the ApZysia neurons are cultured in a protein-free medium on a protein-free substrate in the absence of substantial numbers of other neurons or non-neuronal cells. Yet, there are indications that the phosphorylation is involved in mediating interactions with environmental cues, as its location at the tips of filopodia would suggest. When ApZysia neurons are cultured on a substrate pre-exposed to hemolymph as well as polylysine, far fewer filopodial tips display intense staining with the anti-phosphotyrosine antibody (Wu and Goldberg, 1993). Phosphotyrosine disappears from tips within a few minutes of addition of hemolymph to neurons cultured on a polylysine substrate (Wu and Goldberg, 1993). In addition, we have found integrin to coconcentrate with phosphotyrosine in filopodial tips of cultured chick sympathetic neurons (Fig. 4). Integrin is the membrane receptor for the extracellular matrix proteins, laminin and collagen, which promote neurite growth in these cells. Can the interaction of an individual filopodium with an environmental cue cause a change in the amount or distribution of phosphotyrosine in that filopodium? We do not yet know because, in the aforementioned experiments, the entire growth cone, not only the filopodium, is exposed to the growth promoting material of hemolymph. We are currently assessing the effects on phosphory-
77
Fig. 2. Concentrated phosphotyrosine at the tips of the filopodia of an Aplysia growth cone, as recorded by video intensified fluorescence microscopy. Preparation was as described for Fig. 1,but the image was recorded with a SIT video camera connected to the microscope. This is a different growth cone from the one shown in Fig. 1. (B) This is the same growth cone as in panel A, showing the staining pattern obtained with Texas Red fluorophore, which indiscriminately stains proteins. Bar, 5 pm.
lation of exposure of filopodia alone to cues. It does appear that the machinery for dephosphorylation is present at the tip, since administration of an inhibitor of PTKs to a growth cone on polylysine results in dimming of the tip staining (Fig. 5). Role of tyrosine phosphorylationin the filopodium
What is the role of the tyrosine phosphorylation in the filopodium? This question should be considered in the context of evidence that the phos-
Fig. 3. Concentrated phosphotyrosine at the tips of filopodia of a neonatal rat hippocampal neuron. The neuron was cultured on a polylysine substrate in a defined, serum-free medium. Staining was as described in previous figures, and this and all subsequent fluorescent images were recorded with the SIT camera. Arrowheads point to the stained tips. The shafts of these short filopodia are so lightly stained that they are difficult to see in this view. Bar, 5 pm.
photyrosine associates or interacts with actin filaments. The core of the filopodiumis a bundle of actin filaments (Fig. 6A); at the distal end of this bundle is the concentrated phosphotyrosine (Fig. 6B), though we do not know if there is a physical connection. We have recently shown that filopodia-like protrusions that have a core bundle of microtubules form for a short time after transection of Aplysia axons in culture (Goldberg and Burmeister, 1992). These microtubule based protrusions do not develop concentrations of phosphotyrosine at their tips, while conventional actin-based filopodia forming at the same time do (see Fig. 8B). In addition, one can cause the network of actin filaments in the peripheral region of the growth cone to rapidly withdraw into the central region by administering the actinspecific drug, cytochalasin (Fig. 7A). Phosphotyrosine withdraws from the tip in clumps, sometimes fragmenting or spreading (Fig. 7B). Thus, the concentration of phosphotyrosine in the filopodial tips depends on the presence of actin filaments. With this in mind, we will consider three possible roles for the tyrosine phosphorylation.
Formation of jilopodia The first is in the formation of filopodia. Nonneuronal motile cells, such as fibroblasts, have peripheral actin-rich regions similar to that found in the growth cone. The actin filament bundles underlying their microspikes (short filopodia) form
78
Fig. 4. Concentrated phosphotyrosine at the tips of filopodia of an embryonic chick sympathetic neuron. The neuron was cultured on a polyomithine substrate in a defined, serum-free medium. (B) The same growth cone was also labeled with a polyclonal antibody to the PI subunit of integrin, and visualized with a different fluorophore from that used for panel A. Concentrated integrin is present at the tips, which displayed concentrated phosphotyrosine. Bar, 5 pm.
by an initial coalescence of the distal ends of filaments, followed by a disto-proximal zippering of the filaments into a bundle (Izzard, 1988). The localization of phosphotyrosine at the distal end of the actin bundle in the growth cone filopodia suggests it could be involved in this coalescence. However, we think this is not the case. As mentioned above, axotomy causes massive numbers of
Fig. 5. Dimming of staining for phosphotyrosine at the tips of filopodia of an APlYsia growth cme which had been exposed for 10 min to 100 p M genistein, a broad-specificity inhibitor of PTKs. The arrowheads point to filopodia which have relatively dim staining, while the arrows point to the positions of filopodia which cannot be seen because they have lost all their staining. Bar, 5 pm.
filopodia to form within a short time and so is a convenient means for studying factors underlying their formation. Contrary to the expectation if tyrosine phosphorylation were involved in the initial stages of filopdial formation, bright tip staining is not present when the filopodium first forms but only develops later. Figure 8, which shows an axon 25 min after axotomy (actin based filopodia start to form about 10 min after axotomy), demonstrates that many of the filopodia have no discernible concentration of phosphotyrosine at their tips. Even for those filopodia that display tip staining, the concentrations of phosphotyrosine are more modest than many seen later after axotomy (Fig. 9). Regulation of filopodial length A second possible role is in regulating filopodial length by regulating the polymerization of the core bundle of actin filaments. This is once again suggested by the location of the concentrated phosphotyrosine at the distal end of the core bundle. FiloDodia are dvnamic structures, movingin and out bemeen pauses. The distal end of the filaments is the preferred site for addition and loss of subunits; in fact, there is a addition of subunits at this end, regardless of
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Fig. 6. Concentrated phosphotyrosine is adjacent to the distal ends of the bundles of actin filaments that comprise the cores of the filopodia.(A) This panel shows staining of an Aplysia growth cone with fluorescent phalloidin, which binds to filamentous actin. (B) The same growth cone is also stained with antibody to phosphotyrosine, visualized with a different fluorophore. Bar, 5 pm.
whether the axon is growing rapidly (Forscher and Smith, 1988; Okabe and Hirokawa, 1991). Thus, the distal tip of the filopodium should be a key site for the regulation of filopodial dynamics. In support of the idea that the tip phosphorylation is involved in regulating filopodial dynamics are our findings that filopodia exposed acutely to inhibitors of PTKs (such as genistein) or to hemolymph not only lose phosphotyrosine from their tips but lengthen considerably as a result of a change in their dynamics (Fig. 10). Underlying the
lengthening of the filopodium is a lengthening of the core bundle of actin filaments (Wu and Goldberg, 1993). While these results indicate an association between loss of tip phosphotyrosine and acute lengthening, we cannot assuredly say that the former causes the latter. Both the addition of PTK inhibitors and hemolymph have strong effects in the central region of the growth cone. These effects probably involve the release of large numbers of actin subunits, which could diffuse to the filopodial tips to drive actin polymerization
Fig. 7. Withdrawal of phosphotyrosine from the tips of filopodia when actin filaments are caused to withdraw by application of cytochalasin. (A) Video enhanced contrast-differential interference contrast (VEC-DIC) micrograph of an Aplysiu growth cone 4 min after the application of 1 p M cytochalasin D. The arrowheads point to the distal border of the network of actin filaments as it withdraws from the margin of the growth cone. The area distal to the arrowheads appears flatter because of the withdrawal of the actin network. (B) Phosphotyrosine is visualized by indirect immunofluorescence in a different growth cone, also 4 min after the addition of cytochalasin. Arrows point to filopodial tips which are not stained and arrowheads point to clumps of tyrosine phosphorylated protein apparently in the process of moving proximally along the filopodia. Bar, 5 pm.
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Fig. 8. Concentrated phosphotyrosine is not common in filopodial tips soon after formation of filopodia. (A) This VEC-DIC micrograph shows one side of a single Aplysia axon in culture which was transected 25 min previously. Numerous filopodia have grown from the side of the axon. (B) Only three of these filopodia (arrows) have marked concentrations of phosphotyrosine at their tips, as detected by indirect immunofluorescence.Bar, 5 pm.
and, thus, filopodial lengthening. So, additional data are needed to conclude that tip phosphorylation is involved in regulating filopodial dynamics. It should be useful, as noted above, to determine whether exposure of individual filopodia to PTK inhibitors or hemolymph (to avoid primary effects elsewhere in the growth cone which could secondarily influence the filopodia) affects filopodial dynamics. In addition, not all filopodia on a polylysine substrate have large concentrations of phosphotyrosine at their tips, nor are all of the
Fig. 9. Large tip concentrations of phosphotyrosine develop in filopodia after axotomy. This axon was fked 45 min after transection. Several fdopodial tips (arrows) have large concentrations of phosphotyrosine. Some others which have lesser concentrations have concentrations of phosphotyrosine more proximally along the shaft (arrowheads). Bar, 5 pm.
filopodia on a polylysine/hemolymph substrate lacking bright tip staining (Wu and Goldberg, 1993). So, we should be able to determine whether naturally occurring differences in tip phosphorylation among filopodia of individual growth cones correlate with differences in dynamics. Attachment formation with cells or extracellular matrix A last possible role to consider for tyrosine phosphorylation in the filopodium is in forming attachments with cells or extracellular matrix along the path of growth. Specialization of the tip of some filopodia for adhesion has been described (Tsui et al., 1985), and interactions of only the distal part of the filopodium with environmental cues seem sufficient to alter growth cone behavior (Hammarback and Letourneau, 1986; Bandtlow et al., 1990; O’Connor et al., 1990). The one site where phosphotyrosine has been found to be heavily concentrated in non-neuronal cells is at the adherens junction, either between cells or between a cell and the substrate (focal contact). The fact that the phosphotyrosine of an adherens junction is at the ‘barbed‘ end of a bundle of polarized actin filaments, as in the filopodium, addition, we increases the appeal of this idea, have preliminary evidence that, on a polylysine/hemolymph substrate where the majority of filopodial tips do not have concentrated
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Fig. 10. Application of an inhibitor of PTKs causes filopodia to lengthen. VEC-DIC micrographs show an Aplysia growth cone immediately before (A) and 7 min after (B) application of 100 pm genistein. Bar, 5 pm.
phosphotyrosine, some sites of contact between filopodia show highly elevated levels of phosphotyrosine (Fig. 11). However, it is clear that tyrosine phosphorylation in the filopodium need not be associated with attachments. The filopodial tips that display intense staining for phosphotyrosine on a polylysine substrate are quite mobile and can be seen by video microscopy to lift off the substrate frequently and move about (Wu and Goldberg, 1993).
Fig. 11. Concentrated phosphotyrosine at filopodium-file podium contacts. An Aplysia neuron was placed in culture on a substrate pre-exposed both to polylysine and to hemolymph. Arrows point to several filopodial tips which do not have marked concentrations of phosphotyrosine. Two sites of filopodium-filopodium contact display large concentrations of phosphotyrosine (arrowheads). Bar, 5 pm.
Assessment of the separation of these tips from the substrate by interference reflection microscopy confirms that these are not focal, or even close, contacts (Wu and Goldberg, 1993). Thus, the accumulation of phosphotyrosine occurs independently of the formation of contacts. It has been suggested that protein tyrosine phosphorylation facilitates the formation of protein assemblies at the plasma membrane in response to growth-factor binding (Koch et al., 1991). Aggregates of non-organellar material have been described moving rapidly forward and rearward in growth cone filopodia (Sheetz et al., 1990). We find that high concentrations of phosphotyrosine at filopodial tips are often associated with swellings (Fig. 2; Wu and Goldberg, 1993) and that phosphotyrosine withdrawing from the tips of filopodia after cytochalasin treatment often moves back as a clump (Fig. 7B). We have also looked at the development of phosphotyrosine in newly formed filopodia after axotomy. Filopodia with intense tip staining tend to display bright staining only at the tip, whereas ones with dimmer tip staining, presumably earlier in development, have bright clumps along their shafts (Fig. 9B), suggesting that tyrosine phosphorylated protein moves in clumps towards the tip of the filopodium. Thus, protein tyrosine phosphorylation may facilitate formation of assemblies of proteins, such as integrin, or facilitate their association with actin filaments (or both), to promote their movement to
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and concentration in distal parts of the filopodium, where they would participate in pathfinding activities. Conclusion
We have found tyrosine phosphorylated protein concentrated in the tips of some growth cone filopodia in culture. Its concentration there is acutely sensitive to the presence of growth promoting material on the substrate. These findings strongly suggest that one function of proteintyrosine phosphorylation during axonal growth is in mediating or facilitating certain functions of filopodia, structures which detect and respond to environmental cues that guide axonal growth. Two possible specific roles for tyrosine phosphorylation are in regulating the dynamics, and thus the length, of filopodia and in facilitating the accumulation in the filopodium of membrane receptors for environmental cues with which the filopodium interacts. It will be important to identify the tyrosine phosphorylated proteins. It should also be illuminating to define the effects of well-defined, important environmental cues, such as identified growth promoting or inhibiting factors or synaptic target and non-target neurons on the amount and distribution of phosphotyrosine in individual filopodia. Acknowledgements
This work was supported by NIH training grant MH15174, NIH fellowship NS09225, NIH research grant NS25161 and NIH program project GM32099. References Atashi, J.R., Klinz, S.G., Ingraham, C.A., Matten, W.T., Schachner, M. and Maness, P.F. (1992) Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes. Neuron, 8: 831-842. Bandtlow, C., Zachleder, T. and Schwab, M.E. (1990) Oligodendrocytes arrest neurite growth by contact inhibition. J. Neurosci., 10: 3837-3848.
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