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Making the connection: Cytoskeletal rearrangements during growth cone guidance
Cell, Vol. 83, 171-176,
October
20, 1995, Copyright
0 1995 by Cell
Press
Making the Connection: Cytoskeletal Rearrangements during Growth Cone Guidance Elly Tanaka’ and James Sabryt *Ludwig Institute of Cancer Research London WlP 8BT England tDepartment of Biochemistry Stanford University Medical School Stanford, California 94305
One of the fundamental questions of neurobiology is how neurons acquire the intricate yet stereotyped pattern of connections characteristic of the adult nervous system. A century ago, Ramon y Cajal hypothesized that neurons grow by extending axons and dendrites through embryonic tissues guided by the expanded terminal structure that he named the growth cone. Two decades later, Granville Harrison directly observed the dynamic nature of the growth cone in vitro, thus confirming Ramon y Cajal’s hypothesis. Since then, it has become clear that neurites grow toward their targets and that this growth is regulated by the interaction of this growth cone with environmental cues. However, the path of the neuron is not asimple one. The growth cone is likely to be receiving signals simultaneously from molecules on the surface of other neuronal and nonneuronal cells, molecules in the extracellular matrix, and diffusible chemoattractants and chemorepellents (Grenningloh and Goodman, 1992; Keynes and Cook, 1995; Dodd and Schuchardt, 1995). How does the growth cone reliably interpret these signals to generate a change in its shape and motility that results in neurite extension in the correct direction? It has become clear that the cytoskeleton plays a central role during axonal guidance. The internal organization of actin filaments and microtubules changes rapidly within the growth cone before large-scale changes in growth cone shape can be seen. These cytoskeletal changes predict the direction of future growth, indicating that environmental cues steer neurites by stabilizing local changes of cytoskeletal polymers in the growth cone. Here we will review the changes in actin and microtubule organization that occur when growth cones turn toward a favorable cue and the mechanism by which these changes occur. Based on observations from diverse systems, we have subdivided turning into three stages-exploration, site selection, and the final stage, site stabilization and axon formation. It is unlikely that these steps form an obligatory sequence of events. Growth cones at turning decisions explore many options and assume many shapes before making a choice. Therefore, the process of growth cone steering is a highly flexible one, and the multitude of extracellular guidance cues probably exert their effects on different cytoskeletal elements at different stages of the turning decision. Stage One: The Growth Cone Explores Its Environment Growth Cone Shape Is Highly Dynamic Turning growth cones are characterized by the constant protrusion and retraction of several morphologically dis-
Review
tinct features from its cell surface. Filopodia are thin (0.20.5 urn) spike-like projections up to 40 frrn long that grow out at rates of up to 12 pmlmin and retract at similar rates. Lamella are web-like veils of cytoplasm that spread and retract, often between filopodia, as depicted by the arrow in Figure 1 (Bray and Chapman, 1985; Goldberg and Burmeister, 1986). So, while a growth cone maintains a fairly constant volume over time, it is continuously changing its shape. Filopodia and lamella are likely to serve several different functions: to detect the extracellular environment and to mediate motility and adhesion. Growth cones function as cellular antennae to sample a large volume of the environment and to increase the sensitivity to subtle gradients across the growth cone. In a typical cultured neurite, the axonal diameter measures intherangeof2-lOurn, butthewidthspanned byagrowth cone (including filopodia) can range from 5 to 50 km (Bray and Chapman, 1985; Goldberg and Burmeister, 1986). Contact of the filopodia with a cue is sufficient to cause neurons to turn (O’Connor et al., 1990). Furthermore, it has been shown that temporal retinal ganglion cells decrease their rate of outgrowth in the presence of an increasing gradient of a growth inhibitory factor (posterior tectal membranes) of as little as 1% over 25 urn (Baier and Bonhoeffer, 1992). Filopodia and lamella often mediate attachment of the growth cone to the substrate or cell surfaces and in some instances can generate the force necessary to pull the growth cone forward (Lamoureux et al., 1989). In addition, the protrusion of lamella provides new cytoplasmicvolume that can be invaded by organelles and expand to form the new growth cone (Goldberg and Burmeister, 1986). Therefore, these structures provide the basic components for the growth cone to move and sense its environment, and they are intimately linked to new axon formation. Actin Dynamics Control Growth Cone Shape The shape of filopodia and lamella is largely determined by the organization of the actin cytoskeleton. At the core of each filopodium is a dense, cross-linked bundle of actin filaments that extend into the lamella, while in lamella long actin filaments criss cross, appearing like woven fabric (Figure 1). The actin filaments in both filopodia and lamella are predominantly oriented with their faster growing ends at the periphery and their slower growing ends toward the center of the cell (Lewis and Bridgman, 1992). Drugs that disrupt F-actin cause lamellipodial and filopodial collapse and block the ability of neurons to pathfind (Chien et al., 1993). Although precisely how these structures are produced and what powers their movement are still controversial, their dynamic properties are generally thought to be determined by three main processes: the assembly of actin at the membrane, the disassembly of actin at sites in the growth cone center, and the translocation of the actin network from the leading edge of the cell toward the center in a process called retrograde flow (Forscher and Smith, 1988) (Figures 1 and 2). The size of the lamella appears to be determined by the balance of these three parameters. it
Cell 172
Figure
1. Growth
Cone
Exploration
Lamellipodia and filopodia protrude and retract, leading to rapid changes in growth cone shape. Lamellipodia often form between existing filopodia (arrow). Actin (red barbed filaments) fill the lamellipodia and filopodia and are mostly oriented with their faster growing ends toward the membrane. Assembly of actin occurs at the membrane (green subunits in right growth cone). Most microtubules (yellow lines) are found in the growth cone center, but some extend deep into the lamellipodia. They also change their distribution rapidly. This redistribution occurs by polymerization (black segments in right growth cone), movement, and bending.
has been suggested that when cell surface receptors bind extracellular matrix ligands, they recruit a multiprotein complex that links the receptor to the actin meshwork. This retards the retrograde flow of actin relative to the substrate, allowing the continued assembly of actin at the leading edge, or the action of actin-based motors to cause a
retrograde
Figure
2. Protrusion
of Lamella
fbw
by Inhibiting
Retrograde
Actin
Flow
(a) Retrograde flow is likely driven by myosin-type motors, which are hypothesized to bind to the membrane. Actin assembly (green subunits) occurs at the membrane, and actin disassembly occurs toward the center of the growth cone. (b) When ligands engage extracellular matrix receptors, it is proposed that a protein complex can then bind to actin (black boxes), retarding retrograde flow relative to the substrate. Continued assembly of actin and the continued action of myosin lead to forward protrusion of membrane.
protrusion of the lamella and cell movement in that direction (Figure 2) (Lin and Forscher, 1995). This mechanism clearly links extracellular cues with cell motility. But what powers retrograde flow in the first place? It has been proposed that this flux can be powered simply by the assembly of actin at the edge of the cell (Hill and Kirschner, 1982). However, if this assembly is blocked in growth cones by cytochalasin, flux is not inhibited (Forscher and Smith, 1988). Therefore, it is likely that an actin-based motor, probably a myosin, is responsible for retrograde flux. In nonneuronal cells, the unconventional type I myosins have been localized to lammella (Fukui et al., 1989). Interestingly, many of these myosins have recently been found in growth cones (Ruppert et al., 1993). Rho Family GTPases LCnk Extracellular Signals to the Actin Cytoskeleton While growth cones may have some intrinsic ability to form lamella and filopodia, their formation seems to be regulated by extracellularcues (Holt, 1989; Myers and Bastiani, 1993). These extracellular signals are likely to be transduced via the Rho subfamily of Ras-related GTPases, which includes CDC42, Rat, and Rho. In fibroblasts, growth factors signal through these GTPases to elicit changes in the actin cytoskeleton. Microinjection of activated Rat into fibroblasts rapidly stimulated the formation of lamellipodial ruffles and stress fibers while microinjection of a dominant negative mutant of Rat inhibited the ability of growth factors to stimulate ruffle formation (Ridley et al., 1992). Interestingly, the three GTPasesseem to be responsible for generating distinct actin-containing structures. Microinjection of activated Rho stimulates the formation of stress fibers while microinjection of Rat stimulates both lamella and stress fibers, and CDC42 stimulates filopodia, lamella, and stress fibers. The coordinated formation of all three actin structures by CDC42 occurs by activating endogenous Rat, which in turn stimulates Rho since injection of activated CDC42 in the presence of Rat and Rho inhibitors results in the formation only of filopodia. Hence, each GTPase induces a different structure: CDC42 stimulates the formation of filopodia, Rat stimulates the formation of stress fibers, and Rho stimulates the formation of lamella, but since CDC42, Rat, and Rho function in a signaling hierarchy, multiple actin structures can be formed from a single signal (Nobes and Hall, 1995). Work in Drosophila points to a potential role of these proteins in neuronal pathfinding. Rat and CDC42 homologs are abundantly expressed in the nervous system. Furthermore, if dominant negative or constituitively active mutants of Rat are expressed in certain peripheral neurons, those axons fail to elongate normally and actin organization in the axons is abnormal (Luo et al., 1994). It has not yet been shown how these GTPases elicit changes in the actin cytoskeleton-whether they modulate actin polymerization, translocation, or bundling. Their activation is associated with the accumulation of a complex of proteins, including paxillin, vinculin, and focal adhesion kinase (Nobes and Hall, 1995). However, it is not yet clear whether these focal adhesion complexes act solely as sites of attachment or whether they also constitute sites where new actin is assembled.
Review: 173
Growth
Cone
Guidance
et al., 1993) or by modulation thaler et al., 1988).
Figure
3. Site Selection
and Site Stabilization
in Growth
ConeTurning
During site selection, actin accumulates at the point where the growth cone contacts the guidance cue and is depleted in the zone adjacent to it. The growth cone remains spread in other directions. Microtubules extend toward the contact site. At this stage, microtubules may occupy other regions of the growth cone as well. During site stabilization, microtubules have coalesced toward the contact site and form a bundle in the growth cone. The growth cone membrane has begun to collapse around it.
Microtubules Probe the Intracellular Environment At first there appeared to be a clear separation between the function of actin in driving growth cone motility and of microtubules for providing structural support and for acting as tracks for vesicular transport in the axon (Yamada et al., 1970). Yet recent visualizations of microtubules in growing neurons indicate they are intimately associated with the dynamic actin-based protrusions in the growth cone and play a critical role in turning decisions. Microtubules in the axon form highly stable cross-linked bundles (Schnapp and Reese, 1982), but as they emerge from the axon into the growth cone, they spread into single filaments that continuously extend into and retract from the peripheral areas of the lamella and the bases of filopodia (see Figure 1) (Tanaka and Kirschner, 1991). Thisconstant exploration is attributable to a property of microtubules called dynamic instability, by which individual microtubules randomly transit between phases of polymerization (in growth cones, at 11 Bmlmin) and depolymerization (10 pm/min)(Mitchison and Kirschner, 1984). In growth cones, the more dynamic (positive) ends of microtubules are pointed toward the periphery of the growth cone (Heidemann et al., 1981). It has been proposed that dynamic instability enables microtubules to explore cytoplasmic space more efficiently than other forms of polymer kinetics (Holy and Leibler, 1994). The precise role of microtubules in growth cone motility is not yet clear. Disrupting microtubule assembly and inhibiting microtubule motors in moving cells reduce lamellipodial protrusions and persistent migration (Rodionov et al., 1993; Vasiliev et al., 1970). This suggests that the penetration of microtubules into the lamella promotes their formation either by local membrane insertion (Martenson
of actin organization
(Rinner-
Stage Two: Doing the Right or Left ThingSite Selection In the exploration stage, actin-driven protrusions are extending and retracting rapidly in multiple directions while inside the growth cone, the microtubules are exploring the intracellular environment created by those protrusions. How does this dynamic growth cone orient its growth and motility in response to extracellular cues? Observations in several systems indicate that during the early stages of turning, while the growth cone shape may still appear spread and uncommitted to a new direction, the internal actin and microtubule cytoskeletons are undergoing dramatic rearrangements resulting in a branch or region of the growth cone being chosen for the site of future growth. Two major changes occur: the accumulation of actin polymer at the site of future growth and the invasion of microtubules toward that site (Figure 3). Actin Marks the Spot Actin distribution was found to be highly dynamic within growth cones of grasshopper Til pioneer neurons as they turned toward natural cues (O’Connor and Bentley, 1993). When filopodia contacted certain guidepost cells, those filopodia showed a specific accumulation of F-actin. The spots of F-actin were observed to move down and accumulate at the base of the filopodia. Cultured Aplysia growth cones growing on polylysine assume very different morphologies from grasshopper neurons, elaborating a large fan-like lamella rather than long filopodia, but similar cytoskeletal changes occur during contact mediated turning (Lin and Forscher, 1993). When two Aplysia growth cones touch, they reorient their growth toward each other and their growth rate increases lo-fold. At early times after touching, actin accumulates in the lamella at the site of contact (Figure 3). Furthermore, actin is depleted from adjacent regions toward the central portion of the growth cone. In these situations, the stable attachment of a filopodia or lamella to a favorable substrate stimulates local accumulation of actin. How then is engagement of cell surface receptors linked with changes in actin distribution? Recently, Lin and Forscher (1995) showed in the Aplysia system that these changes in actin distribution coincide with the slowing down of the retrograde flow of actin in that region of the lamella. They suggest that the binding of cell surface receptors in that region engages these receptors with the actin cytoskeleton and inhibits its retrograde flow. If assembly of actin continued, but its rate of retrograde flow was reduced, then it would ostensibly accumulate at the periphery as seen. The depletion of actin in the adjacent area is consistent with the continued depolymerization of actin toward the cell center. Microtubules Invade the Selected Site Microtubules, which are generally spread into all areas of the growth cone, also begin to localize to the contact site during the site selection stage (Figure 3). When Sabry
Cell 174
et al. (1991) observed microtubules in grasshopper Til neurons during turning decisions, they found that microtubules always invaded the branch that would eventually become the new axon. At this early stage, whether microtubules invaded other branches that would not be stabilized depended on the type of turning decision it made. At segment boundary turns, where the growth cone was faced with a boundary of cells expressing a less preferable growth surface, microtubules sometimes entered several branches, eventually only being retained in one, and at other times only invaded the branch eventually chosen. When turning at a guidepost cell, where the growth cone encountered a discrete, positive cue, microtubules entered only the branch that had contacted the guidepost cell. Similar events were observed in more dynamic frog neurons turning at a substrate boundary (Tanaka and Kirschner, 1995). In Aplysia, most microtubules are confined to the central region of the growth cone, with only a small number of single microtubules extending into the peripheral actin rich lamella. At the earliest time after contact is made with another growth cone, microtubules extend specifically toward the region of contact (Lin and Forscher, 1993). In all of these systems, microtubule invasion toward the region of future growth was seen as an early event in growth cone steering. During the exploration stage, the microtubulesthat enter the peripheral lamella are dynamic. Are these early invading microtubules important for site selection? Experiments inhibiting the dynamic microtubules in growth cones suggest that they are (Tanaka et al., 1995). Treatment of growing neurons with low concentrations of the microtubuleinhibiting drug vinblastine blocks dynamic instability of microtubules without eliminating growth cone microtubules. Under such conditions, axons still grow, and growth cones still extend lamella and move, but the overall motion is meandering and not directed, and microtubules in the growth cone do not efficiently form into axonal bundles. This suggests that the dynamic exploratory microtubules are important in choosing a direction of growth and organizing cellular components. It will be important to establish whether such treatments inhibit the site selection of microtubules during turning using systems such as Aplysia or grasshopper. How do microtubules selectively invade the selected region? Does the breakdown of the actin network allow more microtubules into the lamella, or are microtubules captured and stabilized by the contact site? In Aplysia, where the changes in actin that occur upon cell-cell contact are visualized most dramatically, the dense lamellipodial actin network is depleted in the area adjacent to the contact site (Lin and Forscher, 1993). Forscher and Smith (1988) argue that these changes allow microtubules to penetrate that site since depleting actin throughout the growth cone using cytochalasin allows microtubules to enter all of the lamella. This does not exclude the possibility that microtubules are selectively stabilized by factors that accumulate at the contact site. One such protein might be ezrin, which associates with both tubulin and actin structures and, more impressively, requires microtubules to maintain its distribution
in the growth
cone
(Goslin
et al., 1989).
Again,
with the development should now be possible ezrin or other proteins and whether disruption bule localization.
Stage Three: Formation-The
of reproducible to study more are concentrated of these proteins
turning assays, it precisely whether in contact sites prevents microtu-
Site Stabilization and Axon Ultimate Commitment?
In the last stage of turning, microtubules, which are spread, kinked, and looped in the growth cone, organize to form a highly ordered, parallel bundle oriented toward the selected site. Furthermore, the membrane of the growth cone collapses around the microtubule bundle to generate an axon tube. Time-lapse recording of microtubules in Xenopus neurons showed that existing microtubules in the growth cone coalesce into a bundle, often zippering from the base of the growth cone to its edge and thus assuming an essentially axonal organization while the growth cone was still spread (Tanaka and Kirschner, 1991). Samples fixed after site selection suggest that microtubules also zipper in Aplysia neurons (Lin and Forscher, 1993). This zippering involves the translocation of spread microtubules toward the contact site. It is not known whether microtubule-based motors power this movementorwhetherthe bundling of microtubulesaround those that first invade the contact site is sufficient to generate zippering. How does the growth conespatiallyand temporally regulate the stability and bundling of microtubules so that in the early stages of turning, microtubules are dynamic, but in the later stages, they can become bundled and stabilized? The axon-specific stability of microtubules is attributable to microtubule-associated proteins (MAPS), such asTau, MAPl, and MAP2. These proteins bind to microtubules and suppress their intrinsic dynamic instability, rendering them more stable (Drechsel et al., 1992). It is not yet clear how the neuron maintains these distinct domains. One possibility is the local and temporal regulation of MAPS by phosphorylation. It is known that MAPS can be phosphorylated on many sites by many kinases, including CDC2 kinase, MAP kinase, and pl 1 Omark, a MAP/ microtubule affinity-regulating kinase (Drewes et al., 1995). When certain MAPS are phosphorylated, they bind to microtubules less well and hence are less effective at stabilizing microtubule structures. It is possible that MAPS are phosphorylated specifically in the growth cone, allowing microtubules to retain their dynamic instability. However, in these later stages of turning, as new axon is formed, the MAPS would be dephosphorylated, allowing microtubule bundling. After the microtubules bundle, the cell cortex collapses around it to form the axon tube. It is not known whether the same signals that cause microtubules to bundle also induce cortical collapse or whether the organization of microtubules itself induces the cortex to conform to it.
Turning
Is a Variable
and Flexible
Process
While we have described growth cone turning as if it were a stereotyped sequence of events, it must be remembered that in reality it is a flexible progression that uses many
Re;iew:
Growth
Cone Guidance
steps. It appears that even the same stereotyped turn within an organism, for instance, the segment boundary turn in the grasshopper limb, when examined carefully, is somewhat variable from individual to individual (Sabry et al., 1991). Furthermore, turning seems able to be reset at any stage of the process. For instance, it is easy to see how site selection might be a fairly ephemeral and changeable attribute by which actin accumulates at the tip of one lamella and microtubules extend toward that tip, but if another lamella then establishes a stronger attachment with the substrate and actin accumulation, growth might eventually be redirected, probably what happens during the random outgrowth of neurons on a substrate where slight inhomogeneities in the substrate might influence the growth cone behavior. It might seem that once the major proportion of microtubules infiltrate a lamella and form a bundle, the axon would be irreversibly committed to that route. However, even at this late stage, neurons can be redirected. When cultured Aplysia neurons turn at glasslpolylysine boundaries, they form a mature axon branch on each of the substrates, with one of the branches eventually being pruned (Burmeister and Goldberg, 1988). New branches can even be formed from the sides of mature axons (O’Leary and Terashima, 1988). It is not yet known how these back-branching signals affect the cytoskeleton; however, it seems likely that they act by locally reactivating microtubule dynamics in the axon (Bray et al., 1978). Do All Guidance Molecules Act by the Same Signaling Mechanism? Considering the complexity and flexibility of growth cone turning, it is likely that guidance molecules can intervene to regulate turning at any stage. For instance, if aguidance molecule regulated the initial exploration by biasing where filopodia and lamella were made, then it could bias growth in that direction. This could be achieved by locally activating the Rho family GTPases in response to tropic factors. Biased exploration would obviate the need for directed site selection since filopodia and lamella would only form in the proper direction. Such biased filopodial extension is observed during turning of the Ql neuron toward the midline of the grasshopper central nervous system (Myers and Bastiani, 1993). Alternatively, guidance cues could act to stabilize selected lamella by promoting actin accumulation and tethering. This would be expected for those cues that bind to integrins and cause accumulation of focal adhesion proteins. Random initial exploration followed by localized actin accumulation would promote microtubule invasion toward the location of the actin spot. This type of guidance has been seen in turning at guidepost cells and at substrate boundaries (O’Connor et al., 1990). In contrast, collapsin, one of a growing family of inhibitory guidance cues, appears to block or reverse such actin accumulation (Fan et al., 1993). This might be achieved by inhibiting the coupling of extracellular receptors to the actin network or by actively causing actin depolymerization. Interestingly, the guidance molecule netrin has both attractive and repellent activities on different neuronal types, suggesting that the
same molecule can affect the cytoskeleton in different ways (Colamarino and Tessier-Lavigne, 1995). Whether this reflects different receptors or different signaling pathways in different neuronal types is not known. It is also possible that some guidance cues act by localizing microtubules in the growth cone without having to affect actin accumulation. For instance, the APC protein is known to bind both microtubules and B-catenin, a protein that binds to the cytoplasmic domain of the cadherin cell adhesion molecule (Munemitsu et al., 1994). In this way, cadherin-mediated guidance may act by directly affecting microtubule location. Some inhibitory cues could act by inhibiting microtubule invasion of lamella or bundling. Conclusion Considering the wide variety of guidance signals present in the embryo, it will be interesting to determine how many different ways these signals cause growth cones to turn and how the balance of multiple signals impinging on the growth cone allow it to find the right path. Identifying the key changes in actin and microtubules that occur during growth cone turning represents a primary step in unraveling how extracellular cues cause neurons to turn. A full understanding of growth cone guidance still requires a deeper knowledge of the basic mechanisms of cell movement. It also will be important to identifythe key factors that modulate actin and microtubule distribution in response to extracellular guidance cues.
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Tanaka, E., and Kirschner, M. (1991). Microtubule behavior in the growth cones of living neurons during axon elongation. J. Cell Biol. 115, 345-363. Tanaka, E., and Kirschner, M. W. (1995). The role of microtubules in growth cone turning at substrate boundaries. J. Cell Biol. 128, 127137. Tanaka, E., Ho, T., and Kirschner, M. W. (1995). The role of microtubule dynamics in growth cone motility and axonal growth. J. Cell Biol. 728,139-155. Vasiliev, J. M., Gelfand, I. M., Dominina, L. V., Ivanova, 0. Y., Komm, S. G., and Olshevskaja, L. V. (1970). Effect of clocemid on the locomotory behavior of fibroblasts. J. Embryol. Exp. Morph. 24, 625-640. Yamada, K. M., Spooner, B. S., and Wessells, N. K. (1970). Axon growth: role of microfilaments and microtubules. Proc. Natl. Acad. Sci. USA 66, 1206-1212.
October
20, 1995, Copyright
0 1995 by Cell
Press
Making the Connection: Cytoskeletal Rearrangements during Growth Cone Guidance Elly Tanaka’ and James Sabryt *Ludwig Institute of Cancer Research London WlP 8BT England tDepartment of Biochemistry Stanford University Medical School Stanford, California 94305
One of the fundamental questions of neurobiology is how neurons acquire the intricate yet stereotyped pattern of connections characteristic of the adult nervous system. A century ago, Ramon y Cajal hypothesized that neurons grow by extending axons and dendrites through embryonic tissues guided by the expanded terminal structure that he named the growth cone. Two decades later, Granville Harrison directly observed the dynamic nature of the growth cone in vitro, thus confirming Ramon y Cajal’s hypothesis. Since then, it has become clear that neurites grow toward their targets and that this growth is regulated by the interaction of this growth cone with environmental cues. However, the path of the neuron is not asimple one. The growth cone is likely to be receiving signals simultaneously from molecules on the surface of other neuronal and nonneuronal cells, molecules in the extracellular matrix, and diffusible chemoattractants and chemorepellents (Grenningloh and Goodman, 1992; Keynes and Cook, 1995; Dodd and Schuchardt, 1995). How does the growth cone reliably interpret these signals to generate a change in its shape and motility that results in neurite extension in the correct direction? It has become clear that the cytoskeleton plays a central role during axonal guidance. The internal organization of actin filaments and microtubules changes rapidly within the growth cone before large-scale changes in growth cone shape can be seen. These cytoskeletal changes predict the direction of future growth, indicating that environmental cues steer neurites by stabilizing local changes of cytoskeletal polymers in the growth cone. Here we will review the changes in actin and microtubule organization that occur when growth cones turn toward a favorable cue and the mechanism by which these changes occur. Based on observations from diverse systems, we have subdivided turning into three stages-exploration, site selection, and the final stage, site stabilization and axon formation. It is unlikely that these steps form an obligatory sequence of events. Growth cones at turning decisions explore many options and assume many shapes before making a choice. Therefore, the process of growth cone steering is a highly flexible one, and the multitude of extracellular guidance cues probably exert their effects on different cytoskeletal elements at different stages of the turning decision. Stage One: The Growth Cone Explores Its Environment Growth Cone Shape Is Highly Dynamic Turning growth cones are characterized by the constant protrusion and retraction of several morphologically dis-
Review
tinct features from its cell surface. Filopodia are thin (0.20.5 urn) spike-like projections up to 40 frrn long that grow out at rates of up to 12 pmlmin and retract at similar rates. Lamella are web-like veils of cytoplasm that spread and retract, often between filopodia, as depicted by the arrow in Figure 1 (Bray and Chapman, 1985; Goldberg and Burmeister, 1986). So, while a growth cone maintains a fairly constant volume over time, it is continuously changing its shape. Filopodia and lamella are likely to serve several different functions: to detect the extracellular environment and to mediate motility and adhesion. Growth cones function as cellular antennae to sample a large volume of the environment and to increase the sensitivity to subtle gradients across the growth cone. In a typical cultured neurite, the axonal diameter measures intherangeof2-lOurn, butthewidthspanned byagrowth cone (including filopodia) can range from 5 to 50 km (Bray and Chapman, 1985; Goldberg and Burmeister, 1986). Contact of the filopodia with a cue is sufficient to cause neurons to turn (O’Connor et al., 1990). Furthermore, it has been shown that temporal retinal ganglion cells decrease their rate of outgrowth in the presence of an increasing gradient of a growth inhibitory factor (posterior tectal membranes) of as little as 1% over 25 urn (Baier and Bonhoeffer, 1992). Filopodia and lamella often mediate attachment of the growth cone to the substrate or cell surfaces and in some instances can generate the force necessary to pull the growth cone forward (Lamoureux et al., 1989). In addition, the protrusion of lamella provides new cytoplasmicvolume that can be invaded by organelles and expand to form the new growth cone (Goldberg and Burmeister, 1986). Therefore, these structures provide the basic components for the growth cone to move and sense its environment, and they are intimately linked to new axon formation. Actin Dynamics Control Growth Cone Shape The shape of filopodia and lamella is largely determined by the organization of the actin cytoskeleton. At the core of each filopodium is a dense, cross-linked bundle of actin filaments that extend into the lamella, while in lamella long actin filaments criss cross, appearing like woven fabric (Figure 1). The actin filaments in both filopodia and lamella are predominantly oriented with their faster growing ends at the periphery and their slower growing ends toward the center of the cell (Lewis and Bridgman, 1992). Drugs that disrupt F-actin cause lamellipodial and filopodial collapse and block the ability of neurons to pathfind (Chien et al., 1993). Although precisely how these structures are produced and what powers their movement are still controversial, their dynamic properties are generally thought to be determined by three main processes: the assembly of actin at the membrane, the disassembly of actin at sites in the growth cone center, and the translocation of the actin network from the leading edge of the cell toward the center in a process called retrograde flow (Forscher and Smith, 1988) (Figures 1 and 2). The size of the lamella appears to be determined by the balance of these three parameters. it
Cell 172
Figure
1. Growth
Cone
Exploration
Lamellipodia and filopodia protrude and retract, leading to rapid changes in growth cone shape. Lamellipodia often form between existing filopodia (arrow). Actin (red barbed filaments) fill the lamellipodia and filopodia and are mostly oriented with their faster growing ends toward the membrane. Assembly of actin occurs at the membrane (green subunits in right growth cone). Most microtubules (yellow lines) are found in the growth cone center, but some extend deep into the lamellipodia. They also change their distribution rapidly. This redistribution occurs by polymerization (black segments in right growth cone), movement, and bending.
has been suggested that when cell surface receptors bind extracellular matrix ligands, they recruit a multiprotein complex that links the receptor to the actin meshwork. This retards the retrograde flow of actin relative to the substrate, allowing the continued assembly of actin at the leading edge, or the action of actin-based motors to cause a
retrograde
Figure
2. Protrusion
of Lamella
fbw
by Inhibiting
Retrograde
Actin
Flow
(a) Retrograde flow is likely driven by myosin-type motors, which are hypothesized to bind to the membrane. Actin assembly (green subunits) occurs at the membrane, and actin disassembly occurs toward the center of the growth cone. (b) When ligands engage extracellular matrix receptors, it is proposed that a protein complex can then bind to actin (black boxes), retarding retrograde flow relative to the substrate. Continued assembly of actin and the continued action of myosin lead to forward protrusion of membrane.
protrusion of the lamella and cell movement in that direction (Figure 2) (Lin and Forscher, 1995). This mechanism clearly links extracellular cues with cell motility. But what powers retrograde flow in the first place? It has been proposed that this flux can be powered simply by the assembly of actin at the edge of the cell (Hill and Kirschner, 1982). However, if this assembly is blocked in growth cones by cytochalasin, flux is not inhibited (Forscher and Smith, 1988). Therefore, it is likely that an actin-based motor, probably a myosin, is responsible for retrograde flux. In nonneuronal cells, the unconventional type I myosins have been localized to lammella (Fukui et al., 1989). Interestingly, many of these myosins have recently been found in growth cones (Ruppert et al., 1993). Rho Family GTPases LCnk Extracellular Signals to the Actin Cytoskeleton While growth cones may have some intrinsic ability to form lamella and filopodia, their formation seems to be regulated by extracellularcues (Holt, 1989; Myers and Bastiani, 1993). These extracellular signals are likely to be transduced via the Rho subfamily of Ras-related GTPases, which includes CDC42, Rat, and Rho. In fibroblasts, growth factors signal through these GTPases to elicit changes in the actin cytoskeleton. Microinjection of activated Rat into fibroblasts rapidly stimulated the formation of lamellipodial ruffles and stress fibers while microinjection of a dominant negative mutant of Rat inhibited the ability of growth factors to stimulate ruffle formation (Ridley et al., 1992). Interestingly, the three GTPasesseem to be responsible for generating distinct actin-containing structures. Microinjection of activated Rho stimulates the formation of stress fibers while microinjection of Rat stimulates both lamella and stress fibers, and CDC42 stimulates filopodia, lamella, and stress fibers. The coordinated formation of all three actin structures by CDC42 occurs by activating endogenous Rat, which in turn stimulates Rho since injection of activated CDC42 in the presence of Rat and Rho inhibitors results in the formation only of filopodia. Hence, each GTPase induces a different structure: CDC42 stimulates the formation of filopodia, Rat stimulates the formation of stress fibers, and Rho stimulates the formation of lamella, but since CDC42, Rat, and Rho function in a signaling hierarchy, multiple actin structures can be formed from a single signal (Nobes and Hall, 1995). Work in Drosophila points to a potential role of these proteins in neuronal pathfinding. Rat and CDC42 homologs are abundantly expressed in the nervous system. Furthermore, if dominant negative or constituitively active mutants of Rat are expressed in certain peripheral neurons, those axons fail to elongate normally and actin organization in the axons is abnormal (Luo et al., 1994). It has not yet been shown how these GTPases elicit changes in the actin cytoskeleton-whether they modulate actin polymerization, translocation, or bundling. Their activation is associated with the accumulation of a complex of proteins, including paxillin, vinculin, and focal adhesion kinase (Nobes and Hall, 1995). However, it is not yet clear whether these focal adhesion complexes act solely as sites of attachment or whether they also constitute sites where new actin is assembled.
Review: 173
Growth
Cone
Guidance
et al., 1993) or by modulation thaler et al., 1988).
Figure
3. Site Selection
and Site Stabilization
in Growth
ConeTurning
During site selection, actin accumulates at the point where the growth cone contacts the guidance cue and is depleted in the zone adjacent to it. The growth cone remains spread in other directions. Microtubules extend toward the contact site. At this stage, microtubules may occupy other regions of the growth cone as well. During site stabilization, microtubules have coalesced toward the contact site and form a bundle in the growth cone. The growth cone membrane has begun to collapse around it.
Microtubules Probe the Intracellular Environment At first there appeared to be a clear separation between the function of actin in driving growth cone motility and of microtubules for providing structural support and for acting as tracks for vesicular transport in the axon (Yamada et al., 1970). Yet recent visualizations of microtubules in growing neurons indicate they are intimately associated with the dynamic actin-based protrusions in the growth cone and play a critical role in turning decisions. Microtubules in the axon form highly stable cross-linked bundles (Schnapp and Reese, 1982), but as they emerge from the axon into the growth cone, they spread into single filaments that continuously extend into and retract from the peripheral areas of the lamella and the bases of filopodia (see Figure 1) (Tanaka and Kirschner, 1991). Thisconstant exploration is attributable to a property of microtubules called dynamic instability, by which individual microtubules randomly transit between phases of polymerization (in growth cones, at 11 Bmlmin) and depolymerization (10 pm/min)(Mitchison and Kirschner, 1984). In growth cones, the more dynamic (positive) ends of microtubules are pointed toward the periphery of the growth cone (Heidemann et al., 1981). It has been proposed that dynamic instability enables microtubules to explore cytoplasmic space more efficiently than other forms of polymer kinetics (Holy and Leibler, 1994). The precise role of microtubules in growth cone motility is not yet clear. Disrupting microtubule assembly and inhibiting microtubule motors in moving cells reduce lamellipodial protrusions and persistent migration (Rodionov et al., 1993; Vasiliev et al., 1970). This suggests that the penetration of microtubules into the lamella promotes their formation either by local membrane insertion (Martenson
of actin organization
(Rinner-
Stage Two: Doing the Right or Left ThingSite Selection In the exploration stage, actin-driven protrusions are extending and retracting rapidly in multiple directions while inside the growth cone, the microtubules are exploring the intracellular environment created by those protrusions. How does this dynamic growth cone orient its growth and motility in response to extracellular cues? Observations in several systems indicate that during the early stages of turning, while the growth cone shape may still appear spread and uncommitted to a new direction, the internal actin and microtubule cytoskeletons are undergoing dramatic rearrangements resulting in a branch or region of the growth cone being chosen for the site of future growth. Two major changes occur: the accumulation of actin polymer at the site of future growth and the invasion of microtubules toward that site (Figure 3). Actin Marks the Spot Actin distribution was found to be highly dynamic within growth cones of grasshopper Til pioneer neurons as they turned toward natural cues (O’Connor and Bentley, 1993). When filopodia contacted certain guidepost cells, those filopodia showed a specific accumulation of F-actin. The spots of F-actin were observed to move down and accumulate at the base of the filopodia. Cultured Aplysia growth cones growing on polylysine assume very different morphologies from grasshopper neurons, elaborating a large fan-like lamella rather than long filopodia, but similar cytoskeletal changes occur during contact mediated turning (Lin and Forscher, 1993). When two Aplysia growth cones touch, they reorient their growth toward each other and their growth rate increases lo-fold. At early times after touching, actin accumulates in the lamella at the site of contact (Figure 3). Furthermore, actin is depleted from adjacent regions toward the central portion of the growth cone. In these situations, the stable attachment of a filopodia or lamella to a favorable substrate stimulates local accumulation of actin. How then is engagement of cell surface receptors linked with changes in actin distribution? Recently, Lin and Forscher (1995) showed in the Aplysia system that these changes in actin distribution coincide with the slowing down of the retrograde flow of actin in that region of the lamella. They suggest that the binding of cell surface receptors in that region engages these receptors with the actin cytoskeleton and inhibits its retrograde flow. If assembly of actin continued, but its rate of retrograde flow was reduced, then it would ostensibly accumulate at the periphery as seen. The depletion of actin in the adjacent area is consistent with the continued depolymerization of actin toward the cell center. Microtubules Invade the Selected Site Microtubules, which are generally spread into all areas of the growth cone, also begin to localize to the contact site during the site selection stage (Figure 3). When Sabry
Cell 174
et al. (1991) observed microtubules in grasshopper Til neurons during turning decisions, they found that microtubules always invaded the branch that would eventually become the new axon. At this early stage, whether microtubules invaded other branches that would not be stabilized depended on the type of turning decision it made. At segment boundary turns, where the growth cone was faced with a boundary of cells expressing a less preferable growth surface, microtubules sometimes entered several branches, eventually only being retained in one, and at other times only invaded the branch eventually chosen. When turning at a guidepost cell, where the growth cone encountered a discrete, positive cue, microtubules entered only the branch that had contacted the guidepost cell. Similar events were observed in more dynamic frog neurons turning at a substrate boundary (Tanaka and Kirschner, 1995). In Aplysia, most microtubules are confined to the central region of the growth cone, with only a small number of single microtubules extending into the peripheral actin rich lamella. At the earliest time after contact is made with another growth cone, microtubules extend specifically toward the region of contact (Lin and Forscher, 1993). In all of these systems, microtubule invasion toward the region of future growth was seen as an early event in growth cone steering. During the exploration stage, the microtubulesthat enter the peripheral lamella are dynamic. Are these early invading microtubules important for site selection? Experiments inhibiting the dynamic microtubules in growth cones suggest that they are (Tanaka et al., 1995). Treatment of growing neurons with low concentrations of the microtubuleinhibiting drug vinblastine blocks dynamic instability of microtubules without eliminating growth cone microtubules. Under such conditions, axons still grow, and growth cones still extend lamella and move, but the overall motion is meandering and not directed, and microtubules in the growth cone do not efficiently form into axonal bundles. This suggests that the dynamic exploratory microtubules are important in choosing a direction of growth and organizing cellular components. It will be important to establish whether such treatments inhibit the site selection of microtubules during turning using systems such as Aplysia or grasshopper. How do microtubules selectively invade the selected region? Does the breakdown of the actin network allow more microtubules into the lamella, or are microtubules captured and stabilized by the contact site? In Aplysia, where the changes in actin that occur upon cell-cell contact are visualized most dramatically, the dense lamellipodial actin network is depleted in the area adjacent to the contact site (Lin and Forscher, 1993). Forscher and Smith (1988) argue that these changes allow microtubules to penetrate that site since depleting actin throughout the growth cone using cytochalasin allows microtubules to enter all of the lamella. This does not exclude the possibility that microtubules are selectively stabilized by factors that accumulate at the contact site. One such protein might be ezrin, which associates with both tubulin and actin structures and, more impressively, requires microtubules to maintain its distribution
in the growth
cone
(Goslin
et al., 1989).
Again,
with the development should now be possible ezrin or other proteins and whether disruption bule localization.
Stage Three: Formation-The
of reproducible to study more are concentrated of these proteins
turning assays, it precisely whether in contact sites prevents microtu-
Site Stabilization and Axon Ultimate Commitment?
In the last stage of turning, microtubules, which are spread, kinked, and looped in the growth cone, organize to form a highly ordered, parallel bundle oriented toward the selected site. Furthermore, the membrane of the growth cone collapses around the microtubule bundle to generate an axon tube. Time-lapse recording of microtubules in Xenopus neurons showed that existing microtubules in the growth cone coalesce into a bundle, often zippering from the base of the growth cone to its edge and thus assuming an essentially axonal organization while the growth cone was still spread (Tanaka and Kirschner, 1991). Samples fixed after site selection suggest that microtubules also zipper in Aplysia neurons (Lin and Forscher, 1993). This zippering involves the translocation of spread microtubules toward the contact site. It is not known whether microtubule-based motors power this movementorwhetherthe bundling of microtubulesaround those that first invade the contact site is sufficient to generate zippering. How does the growth conespatiallyand temporally regulate the stability and bundling of microtubules so that in the early stages of turning, microtubules are dynamic, but in the later stages, they can become bundled and stabilized? The axon-specific stability of microtubules is attributable to microtubule-associated proteins (MAPS), such asTau, MAPl, and MAP2. These proteins bind to microtubules and suppress their intrinsic dynamic instability, rendering them more stable (Drechsel et al., 1992). It is not yet clear how the neuron maintains these distinct domains. One possibility is the local and temporal regulation of MAPS by phosphorylation. It is known that MAPS can be phosphorylated on many sites by many kinases, including CDC2 kinase, MAP kinase, and pl 1 Omark, a MAP/ microtubule affinity-regulating kinase (Drewes et al., 1995). When certain MAPS are phosphorylated, they bind to microtubules less well and hence are less effective at stabilizing microtubule structures. It is possible that MAPS are phosphorylated specifically in the growth cone, allowing microtubules to retain their dynamic instability. However, in these later stages of turning, as new axon is formed, the MAPS would be dephosphorylated, allowing microtubule bundling. After the microtubules bundle, the cell cortex collapses around it to form the axon tube. It is not known whether the same signals that cause microtubules to bundle also induce cortical collapse or whether the organization of microtubules itself induces the cortex to conform to it.
Turning
Is a Variable
and Flexible
Process
While we have described growth cone turning as if it were a stereotyped sequence of events, it must be remembered that in reality it is a flexible progression that uses many
Re;iew:
Growth
Cone Guidance
steps. It appears that even the same stereotyped turn within an organism, for instance, the segment boundary turn in the grasshopper limb, when examined carefully, is somewhat variable from individual to individual (Sabry et al., 1991). Furthermore, turning seems able to be reset at any stage of the process. For instance, it is easy to see how site selection might be a fairly ephemeral and changeable attribute by which actin accumulates at the tip of one lamella and microtubules extend toward that tip, but if another lamella then establishes a stronger attachment with the substrate and actin accumulation, growth might eventually be redirected, probably what happens during the random outgrowth of neurons on a substrate where slight inhomogeneities in the substrate might influence the growth cone behavior. It might seem that once the major proportion of microtubules infiltrate a lamella and form a bundle, the axon would be irreversibly committed to that route. However, even at this late stage, neurons can be redirected. When cultured Aplysia neurons turn at glasslpolylysine boundaries, they form a mature axon branch on each of the substrates, with one of the branches eventually being pruned (Burmeister and Goldberg, 1988). New branches can even be formed from the sides of mature axons (O’Leary and Terashima, 1988). It is not yet known how these back-branching signals affect the cytoskeleton; however, it seems likely that they act by locally reactivating microtubule dynamics in the axon (Bray et al., 1978). Do All Guidance Molecules Act by the Same Signaling Mechanism? Considering the complexity and flexibility of growth cone turning, it is likely that guidance molecules can intervene to regulate turning at any stage. For instance, if aguidance molecule regulated the initial exploration by biasing where filopodia and lamella were made, then it could bias growth in that direction. This could be achieved by locally activating the Rho family GTPases in response to tropic factors. Biased exploration would obviate the need for directed site selection since filopodia and lamella would only form in the proper direction. Such biased filopodial extension is observed during turning of the Ql neuron toward the midline of the grasshopper central nervous system (Myers and Bastiani, 1993). Alternatively, guidance cues could act to stabilize selected lamella by promoting actin accumulation and tethering. This would be expected for those cues that bind to integrins and cause accumulation of focal adhesion proteins. Random initial exploration followed by localized actin accumulation would promote microtubule invasion toward the location of the actin spot. This type of guidance has been seen in turning at guidepost cells and at substrate boundaries (O’Connor et al., 1990). In contrast, collapsin, one of a growing family of inhibitory guidance cues, appears to block or reverse such actin accumulation (Fan et al., 1993). This might be achieved by inhibiting the coupling of extracellular receptors to the actin network or by actively causing actin depolymerization. Interestingly, the guidance molecule netrin has both attractive and repellent activities on different neuronal types, suggesting that the
same molecule can affect the cytoskeleton in different ways (Colamarino and Tessier-Lavigne, 1995). Whether this reflects different receptors or different signaling pathways in different neuronal types is not known. It is also possible that some guidance cues act by localizing microtubules in the growth cone without having to affect actin accumulation. For instance, the APC protein is known to bind both microtubules and B-catenin, a protein that binds to the cytoplasmic domain of the cadherin cell adhesion molecule (Munemitsu et al., 1994). In this way, cadherin-mediated guidance may act by directly affecting microtubule location. Some inhibitory cues could act by inhibiting microtubule invasion of lamella or bundling. Conclusion Considering the wide variety of guidance signals present in the embryo, it will be interesting to determine how many different ways these signals cause growth cones to turn and how the balance of multiple signals impinging on the growth cone allow it to find the right path. Identifying the key changes in actin and microtubules that occur during growth cone turning represents a primary step in unraveling how extracellular cues cause neurons to turn. A full understanding of growth cone guidance still requires a deeper knowledge of the basic mechanisms of cell movement. It also will be important to identifythe key factors that modulate actin and microtubule distribution in response to extracellular guidance cues.
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