- Email: [email protected]
The Rho's progress: a potential role during neuritogenesis for the Rho family of GTPases
REVIEW
R. Nixon
Acknowledgements The authors are gratetid to DTs Jennifer LaVail, Peter Hollenbeck, Douglas LauffenbergeT and Paul Mathews for helpful commenb on the manuscTipt, Katharine Nixon for aTtwoTk, and Johanne Khan for assisting in preparation of the manuscTi$t. Cited TeSeaTCh from this laboratory was supported by a LEAD (Leadership and Excellence in Alzheimer Disease) grant from the National Institute on Aging (AG10916).
and A.Cataldo-
The neuronal lysosomalsystem
30 Nixon, R.A. and Cataldo, A.M. (1993) Ann. NY Acad. Sci. 679, 87-109 31 Dice, J.F. et al. (1990) Semin. Cell Biol. 1, 449-455 32 Bhattacharya, R. and von Mayersbach, H. (1976) Acta Histochem. (Suppl.) 16, 109-l 15 33 Pannese, E. et al. (1976) Acta NeuropaNtoZ. 36, 209-220 34 Roberts, V.J. and Gorenstein, C. (1987) Dev. N~UTOSC~. 9,2X-264 35 Koenig, E. et al. (1985) I. Neurosci. 5, 715-729 36 Hollenbeck, P.J. and Bray, D. (1986) J. Cell Biol. 105, 2827-2835 37 Hollenbeck, P.M. (1993) J. Cell Biol. 121, 305-315 38 Overly, C.O. et al. (1995) PTOC. Nat1 Acad. Sci. USA 92,
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39 LaVail, J.H. and LaVail, M.M. (1974) 1. Comp. N~uTo~. 175, 303-358 40 Hirokawa, N. (1993) Neurosci. Res. 18, l-9 41 Corder, E.H. et al. (1993) Science 261, 921-923 42 Bu, G. et al. (1994) I. Biol. Chem. 269, 18521-18528 43 Lombardi, P. et al. (1993) Biochem. I. 290, 509-514 44 Muller, H.W. et al. (1985) Science 228, 499-501 45 ldriss, J.M. and Jonas, A.J. (1991) J. Biol. Chem. 266,9438-9441 46 Wells, A. et al. (1990) Science 247, 962-964 47 French, A.R. et al. (1994) 1. Biol. Chem. 269, 15749-15755 48 Mayor, S., Presley, J. and Maxfield, F. (1993) 1. Cell Biol. 121, 1257-1269
49 Cupp, M., Bensadoun, A. and Melford, K. (1987) I. Biol. Chem. 262,6383-6388 50 Fukushima, D. et al. (1993) Biochem. Biophys. Res. Commun. 194, 202-207 51 Renfrew, C.A. and Hubbard, A.L. (1991) J. Biol. Chem. 266, 4348-4356 52 Roederer, M., Bowser, R. and Murphy, R. (1987) J. Cell. Physiol. 131,200-209
53 Bowser, R. and Murphy, R. (1990) J. Cell. Physiol. 143, 110-117 54 Waheed, A. et al. (1988) EMBO 1. 7, 2351-2358 55 Diment, S., Leech, M.S. and Stahl, P.D. (1988) J. Biol. Chem. 263, 6901-6907 56 Casciola-Rosen, L.A.F. and Hubbard, A.L. (1991) J. Biol. Chem.
The Rho’s progress: a potential role during neuritogenesis for the Rho family of GTPases DeborahJ.G. Mackay, Catherine D. Nobes and Alan Hall Growth
cones navigate
actin cytoskeleton
through
basis of this coupling family
of small
fibroblasts,
growth
link between adhesion Trends
Deborah J.G. Mackay, Cathm’ne D. Nobes and Alan Hall are at the MRC Laboratory fOT MOkCuluT
cell
Biology, University College London, Gower Street, London, UK WClE 6BT.
496
GTPases
regulate
are striking
incoming (1995)
extracellular extension
unknown.
morphological
and lamellipodia,
of filopodia, similarities
lamellipodia between
suggests that the Rho family
outgrowth
of both
actin
but the biochemical of the Rho
and stress fibres
spreading
fibroblasts
of proteins
dynamics
of the
in and
could be the
and cell-substratum
cone.
18, 496-501
EURITES EXTENDED by developing neurones folN low long and tortuous routes to their targets, and painstaking research on the pathfinding of neurites
in vivo and in culture has shown that this navigation
relies on several kinds of environmental signals. First, morphogenetic gradients of diffusible chemoattractants (for example, netrins’) and chemorepellants (for example, collapsin’) have been shown to act on developing neurites over distances of hundreds of micrometres. Second, signals are derived from the substratum surrounding the migrating neurite; for example, in model systems, neurites presented with a choice TINS Vol. 18, No. 11,1995
cues to directed
Recent studies have shown that members
signals and the regulation growth
guidance
of filopodia
the formation
cones. This resemblance
in the neuronal
Neurosci.
cyclical
is at present
and there
advancing
by coupling
between more- or less-adhesive substrata tend to transfer to the more adhesive (for reviews, see Refs 3 and 4). A third source of guidance information is found in other cells since neurites can extend even across regions of non-adhesive substratum by extending processes towards intermediate target cells or ‘guideposts’; if a single process contacts the guidepost, the neurite can reorientate and travel towards it?. In vivo, ablation of a guidepost can prevent the neurite from making a course correction required for completion of its stereotyped pathway. On the other hand, heterotypic neurones can act as ‘anti-guidepostsf6, which cause 8 1995, Elsevier Science Ltd
D. Mackay,
C. Nobes
REVIEW
and A. Hall - Rho GTPases and neuritogenesis
retreat and reorientation of impinging neurites and assist in the formation of neuronal territories. If the actin cytoskeleton growth cones...
controls
pathfinding
by
To detect and respond to these various signals, the distal tip of the neurite forms a specialized bulbous structure - the growth cone (Fig. 1A; see Refs 7 and 8). The flattened leading edge of the growth cone is filled by a population of dynamic actin filaments (see Fig. 2), some 50% of which is organized with the barbed (polymerizing) ends orientated distally. These filaments undergo treadmilling, with polymerization at the leading edge, retrograde transport (probably via myosin motors, at some 200-300 pm h-‘; see Ref. 9) and depolymerization in the body of the growth cone. The remainder of the actin forms a dense isotropic mesh, which appears to exclude microtubules and organelles from the leading edge. These excluded structures occupy instead the dilated body of the growth cone, and the shaft of the axon is supported by a microtubular cytoskeleton (see Ref. 8). Careful observation of moving growth cones has suggested the scheme of locomotion represented in Fig. 1 (Refs 10 and 11). Filopodia grow outwards from the leading edge, in an arc centred on the direction of travel (Fig. 1B). These extensions are extremely dynamic: they might explore and retract with a lifetime of minutes, and, in vitro, they are often seen to detach from the substratum and extend into the medium. However, time-lapse studies show that the filopodia that extend in the direction of positive stimuli are stabilized, and the actin filaments in these filopodia thicken and move down into the body of the growth cone. Lamellipodia subsequently form between stable filopodia (Fig. lC), and remain stationary while the body of the growth cone advances (Fig. 1D) by dilation of the proximal end of the lamella and admission of microtubules. This results eventually in extension of the shaft of the axon. New filopodia are then extended, frequently from the region of the newly consolidated leading edge. This slow mode of advance (4 pm h-l; Ref. 12) seems to be preferred in neurites responding to weak or diffuse information. However, neurites in the vicinity of guideposts, which are strong localized guidance cues, can advance at 10 pm h-’ by extending and then dilating filopodia, without the intermediacy of lamellipodia (see Ref. 12). The growth cone appears to advance, therefore, by extension of the actin cytoskeleton, followed by consolidation that involves microtubules. Axon branches extending towards the strongest stimuli accumulate actin and thrive, while others going in different directions lose actin filaments and retract. Axons receiving guidance information from two sources always follow only one, the stronger13; that is, growth cones can integrate quantitative information from filopodia to make qualitative decisions about which stimuli to follow. Treatment of neurones with cytochalasins (inhibitors of actin polymerization) does not prevent the neurones from extending, but it does lead to disappearance of the actin structures at the leading edge of the growth cone, which is then dilated and invaded by microtubules and organelles’. This can leave the neurites literally going round in circles, as shown by Bentley and co-workers’4. This strongly suggests that guidance information is indeed interpreted and acted upon intracellularly by
*
D
* Fig. 1. Actin and microtubules in outgrowth ofneurites. (A) An axon branch terminating in a growth cone. The flattened leading edge is occupied by a dense network of actin filaments, and the inflated body of the growth cone (shaded) is taken up by microtubules. (B) The growth cone extends filopodia (fine arrows) mainly in the direction of travel. The target region is represented by a star in the bottom-right corner of the diagram. (C) The filopodia directed towards the target are more stab/e than their neighbours, which are retracting. Lamellipodia (broad arrows) are extending between three stab/e, slightly dilated filopodia. (D) The /ameNpodium remains stationary, while the body of the growth cone behind it dilates to admit microtubules, and the growth cone advances. New filopadia are extending from the lamellipodium in the direction of the target.
regulation of the actin cytoskeleton. The biochemical mechanism modulating actin dynamics is not clear in neurones, but recent advances in analogous areas of fibroblast research have suggested that a key role might be played by members of the Rho branch of the Ras superfamily of small GTP-binding proteins. . ..and Rho proteins cytoskeleton...
regulate
the actin
Members of the Ras superfamily are involved in the regulation of a wide range of cellular activities, from intracellular protein trafficking to growth control (for review, see Ref. 15). The Rho branch of the superfamily TIN.5 Vol. 18, No. 11, 1995
497
REVIEW
D. Mackay,
C. Nobes
and A. Hall - Rho GTPases and neuritogenesis
Actin polymerisation
Microtubules l in dilated body of growth cone
Leading edge of growth cone
Fig. 2. The cytoskeleton of the growth cone. Actin filaments
assembled at the leading edge are transported into the body of the growth cone and disassembled there. The net result of this is continuous retrograde flux of fikmentous actin. The leading edge a/so contains shorter filaments in random orientation, while microtubules and organelles are confined to the dilated body of the growth cone and the axon shaft.
(for reviews, see Refs 16 and 17) currently has five members, which share SO-?&% amino-acid similarity: Rho (A, B and C isoforms), Rat (two isoforms), cdc42 (two isoforms), Rho G and TClO. They, like all proteins of the Ras superfamily, are active in the GTPbound form, and hydrolysis of GTP by activity of their intrinsic GTPase returns them to the inactive state. Activation of Rho proteins requires GDP-GTP exchange, and is catalysed by various guaninenucleotide exchange factors (GEFs). Their activity is also regulated by GTPase-activating proteins (GAPS), which stimulate their intrinsic GTPase activity. In addition, the guanine-nucleotide dissociation inhibitor RhoGDI interacts with all the proteins of the Rho family, and might well link nucleotide exchange with the cytosol-membrane redistribution that is essential for their activity. In the Rho family, Rho, Rat and cdc42 have been shown to exert specific effects on the actin cytoskeleton of Swiss 3T3 cells?, and their activities are schematized in Fig. 3. Swiss 3T3 fibroblasts grown to confluence and then deprived of serum lose much of their actin cytoskeleton; very little polymerised actin is detectable with phalloidin, and focal complexes, visualized with antibodies against the focal-adhesion protein, vinculin, are small or absent. Addition of serum (or its active principle, lysophosphatidic acid) to these cells causes activation of Rho and dramatic reorganisation of the actin cytoskeleton. Within a few minutes, the cell is placed under tension by stress fibres of bundled actin filaments, whose ends are associated with large, arrowhead-shaped focal adhesions at the plasma membrane”. Activation of Rat by various growth factors (for example, platelet-derived growth factor) leads to the formation of circumferential lamellipodia underpinned by a ring of focal complexes smaller than those generated by Rho, but with the same range of protein 498
TINS vol.
18, NO. 11, 1995
constituents20,21. The lamellipodia spread on the substratum, and then curl up from it and are drawn centripetally over the dorsal surface of the cell to produce membrane ruffles. This gives a visual impression a little like the breaking of waves, but in reverse. An additional and later effect of Rat is to activate the Rho pathway. The effects of cdc42 were elusive until they were investigated in fibroblasts growing at low density, and hence with space to extend and move’l. In such cells, cdc42 first directs the formation of dynamic filopodia associated with small focal complexes, and then activates Rat, which leads to the formation of lamellipodia between the filopodia. This enables an initially rounded cell to spread out over the substratum. Figure 3 summarizes the activities of cdc42, Rat and Rho in the formation of filopodia, lamellipodia, and stress fibres, respectively. . ..do Rho proteins regulate pathfinding by growth cones?
There are clear morphological similarities between fibroblasts that contain active cdc42 or Rat and the growth cones of advancing neurites: both can spread by extension of filopodia and subsequent generation of lamellipodia between the filopodia (see Fig. 4). This prompts the suggestion that cdc42 and Rat regulate actin dynamics in the growth cone, and a model for how they might work is outlined in Fig. 5. Filopodia would first be extended by local, transient activation of cdc42. The presence of a positive stimulus at a filopodial tip would lead to sustained local activation of cdc42, increased polymerization of actin and enlargement of focal complexes. This would result in stabilization of the filopodium. Since formation of focal complexes in fibroblasts is known to be dependent on integrins (the receptors for the extracellular matrix) filopodia would only be expected to be stabilized on a favourable substratum. Extrapolation from the data on fibroblasts suggests that, subsequently, sustained activation of cdc42 would promote activation of Rat, and lamellipodia would therefore be extended in the direction of the strongest stimuli. Is there, then, any evidence that focal complexes play a role in the dynamics of the growth cones? An elegant study on ‘cultured PC12 cells provides a link between the stability of focal complexes and the stability of the filopodia and lamellipodia in growth cones. Varnum-Finney and Reichardt” generated PC12 lines that expressed only one-fifth of the normal levels of vinculin. Filopodia in normal PC12 growth cones show staining of vinculin along their lengths and at their tips, whereas filopodia in the mutant PC12 cells showed much less staining of vinculin. Analysis of the vinculin-deficient cells indicated that their filopodia were extended at the same rate and were of the same length as those of wildtype PC12 cells, but had a much shorter lifespan. Moreover, in the mutant cells, developing lamellipodia were almost
D. Mackay,
-
REVIEW
and A. Hall - Rho GTPases and neuritogeneris
PDGF Insulin
GEF
cdc42 GDP
C. Nobes
cdc42 GTP
GAP
1
GEF
rat GTP
rat GDP
Filopodia Focal contacts
LPA
GEF
GAP
rho GTP
rho GDP
Lamellipodia Focal contacts
GAP
Stress fibres Focal adhesions
Fig. 3. The effects of the proteins of the Rho family, Rho, Rat and cdc42, on the actin cytoskeleton of fibroblasts. Each of the three proteins is active in the CTP-bound state and inactive in the GDP-bound form. Activation of the proteins requires guanine-nucleotide exchange factors (GEFs), and deactivation occurs through an intrinsic CTPase-activity that can be stimulated by C JPase-activating proteins (GAPS). Cuoninenucleotide dissociation inhibitors (CD/s) increase the stability of both G JP-bound and GDP-bound forms. While the proteins are activated by extracellular signals, they a/so participate in a cascade of activation from cdc42 through Rat to Rho.
always retracted before advance of growth cones could take place. As a result, the vinculin-deficient axons grew much more slowly than controls. This study suggests that integrity of focal complexes is required for the stability of both filopodia and lamellipodia in migrating growth cones. Wu and Goldberg23 used an anti-phosphotyrosine antibody to visualize a tyrosine phosphoprotein at the tips of filopodia in Aplysia neurones that were either
migrating slowly on a polylysine-coated substratum or more rapidly on one coated with Aplysia haemolymph. The neurones that were migrating on polylysine had short filopodia, 80% of which showed staining phosphotyrosine. By contrast, the filopodia of the neurones that were migrating on haemolymph were only 15% phosphotyrosine-positive, but twice as long as, and more stable than, the filopodia extended on polylysine. Similarly, extension of neurites in TINS Vol. 18, NO. 11, 1995
499
REVIEW
D. Mackay,
C. Nobes
and A. Hall - Rho GTPases and neuritogenesis
Fig. 4. Morphological similurity between an advancing growth cone (kft) ond Swiss 3T3 fibroblosts that are extending fi/opodia (upper right) or filopodia and lamellipodia [lower right). The cell on the upper right was injected with activated cdc42 to promote formation of fi/opodia, along with dominantnegative Rat and C3 transferose to suppress the activation of endogenous Rat and Rho, respective/y. The cell on the lower right was injected with activated cdc42 and C3, and has undergone octivotion of endogenous Rot. Stole bars, I Ohm.
developing chick neurones was potentiated by the tyrosine-kinase inhibitor genisteinz4. Tyrosine phosphoproteins could regulate extension of filopodia by one of two mechanisms: they could regulate elongation of filaments at the tips of the fllopodia or they could regulate the association between cell-substratum adhesions and the retrograde flux of actin in the leading edge of the growth cone. This latter suggestion is borne out by the observation that the rate of motility in various cells is inversely related to the rate of flux of actirP, and suggests that advance of growth cones is controlled, in part, by regulatable cell adhesion (for review of the model, see Ref. 9). Direct evidence for a role of Rat and cdc42 in formation of axons has been reported in Drosophilaz6. Active and inactive mutants of the rat and cdc42 genes of Drosophila were expressed at various sites and stages of embryogenesis by the use of developmentally regulated promoters. In almost all cases, the neurones expressing the mutant proteins were correctly localized. However, the axons of the various rut mutants either did not extend at all or were stalled halfway to their targets, while mutations in cdc42 resulted in hindrance of the development of both axons and dendrites. These observations connecting formation of focal complexes with the length and stability of filopodia and lamellipodia have been complemented by studies on the behaviour of actin in growth cones in vivo and in vitro. One series of studies on grasshopper embryos examined the navigation of pioneer neurones along a stereotyped pathway marked out by guidepost cells (see Refs 12 and 13). On contact with a guidepost cell, 500
TINS Vol. 18, No. 11, 1995
the filopodia of the pioneer neurones are dilated by thickening of bundles of actin, which are then transported back into the body of the growth conez7. A related study examined growth cones arriving at their targets in an in vitro systemz8. The filopodia contacting the target were dilated by formation of new actin filaments and then by microtubules, before formation of a tight contact. By contrast, if positively charged polystyrene beads were applied to the growth cone as mock targets, they became associated with its actin cytoskeleton but did not promote subsequent accretion of microtubules; instead, the beads were swept back along the growth cone at the speed of the actin flux. It appeared that the treatment accorded to the target and the target-like microbead depended on their relative size and tractability. The actin filaments that accumulated at the target contracted to place it under tension, and only then did microtubules invade the contact site. This observation leads to the interesting possibility that one of the major intracellular signals in the growth cone is mechanical tension in actin filaments (see Refs 9 and 29). It also brings up the subject of the effects of Rho on neurites. Rho: the growth
cone
meets
its match
Rho appears to be required not for the outgrowth of neurites, but for their retraction (Fig. 5). Contact of the filopodium of a growth cone with a repellant such as another neurone can provoke collapse of the filopodium or of the whole growth cone6t30.The cone then remains frozen for some time before extension of new filopodia, which are normally directed away from the site of the repulsive contact. Even more repellent to neurites is exposure to serum or thrombin on traumatic injury of the nervous system: serum-treated axons will retract completely within seconds. Moolenaar and co-workers31,32studied this process using the cell line NlE-115, which has a fibroblast-like morphology when growing in serum, but, after serum-starvation, differentiates to generate axons and growth cones. Addition of serum or thrombln to these neurone-like cells caused rapid collapse of the axons by contraction of actomyosin. This collapse could be prevented by pretreatment of the cells either with specific inhibitors of myosin light-chain kinase, or with C3 transferase, a Clostridium boiulinum exoenzyme that specifically inactivates Rho. It is possible that the role of Rho is to test the interaction between growth cone and target by generation of mechanical tension in actin filaments. If adhesion were strong enough to withstand tension, then docking would proceed, and if not, the neurite would be retracted. Inappropriate activation of Rho during outgrowth of neurites would lead to their retraction. It is possible that tensile strength is also used to integrate the signalling inputs of filopodia during outgrowth of
D. Mackay,
neurites (for review, see Ref. 29); to compare the tension developed in various filopodia would be one means of determining the quantity of actin accumulated in each one. Future cones
directions
activity
1 2 3 4
Rat
b-l
GDP
eXClUSiVelY in ner-
of
the
Serine/threonine
cdc42 GTP
-
Filopodia
I Collapse
Rho GTP
A
-
Rho GDP
-
Fig. 5. The proposed roles for cdc42, Rot and Rho in navigation of growth cones. Cdc42 triggers formation and stabilization of filopodia in response to extracellular guidance cues, and also leads to activation of Rat and formation of filopodia. Active Rat might be able to sustain activity of cdc42 loco//y since filopodia arise frequent/y from stable lame/lipodia of growth cones. Cdc42 or Rat might also activate Rho, leoding to tension in actin filaments, but activation of Rho seems to suppress formation of filopodia, at /east in the short term.
p2J-aCiiVated
kinase3* is stimulated bvI GTP-bound Rat. while mvr5. , I a novel myosin-like actin-binding protein, interacts with Rho, Rat and cdc42 through a GAP domain39. Another candidate effector is phosphatidylinositol 5-kinase, which generates the second messenger phosphatidylinositol (4,5)-bisphosphate (PIP,) (Ref. 40). This is of interest since a number of actin-binding proteins are also PIP,-binding proteins (for example, gelsolin’l, profilin4’ and cofilin43) and the activity of the kinase in cell extracts is modulated by Rho4’. Lipid metabolism might represent an important link between the proteins of the Rho family and actin dynamics in both neuronal and non-neuronal cells. In conclusion, those interested in how growth cones move towards their targets should seriously consider a role for the Rho family of GTPases. Selected
f7(
cdc42 GDP
vous tissue. Differential expression and activity of these proteins might well turn out to be pivotal in the development of the nervous system. Second, as mentioned above, Rho, Rat and cdc42 all appear to promote the clustering of integrins and the formation of focal complexes’l, and adhesion complexes are likely to turn out to be central to the movement of the growth cone over the substratum. Third, it is currently a matter of great interest to identify effecters for Rho proteins, and a number of possibilities have already been suggested. For example, ihe
Rat GTP
A
REVIEW
and A. Hall - Rho GTPases and neuritogenesis
for growth
The intracellular signalling pathways that regulate the remarkably complex behaviour of growth cones are somewhat unclear at present, but a number of interesting observations might indicate some future directions for research. First, the activation of the Rho proteins requires GEFs (see Fig. 3), and these would then be the primary targets of signal transduction. A paradigm for this is the recruitment of the exchange factor SOSto signalling complexes, leading to activation of Ras (for review, see Ref. 33). A number of exchange factors for the Rho family, for example, db134and ost3’, and GAPS such as N-chimaerin3’j and ABR [active bcr-related (bcr breakpoint cluster region, a Rat GTPase-activating protein)]37, are ex-
Presseda1most
Lamellipodia
C. Nober
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Acknowledgements The authors would like to thank the Wellcome Trust (D/GM) and the CRC (AH, CDN) for their generous financial support. We are very grateful to Dr Dennis Bray for the photograph of a growth cone shown in Fig. 4, and to Dr Paul Martin for his critical reading of the manuscriit.
501
R. Nixon
Acknowledgements The authors are gratetid to DTs Jennifer LaVail, Peter Hollenbeck, Douglas LauffenbergeT and Paul Mathews for helpful commenb on the manuscTipt, Katharine Nixon for aTtwoTk, and Johanne Khan for assisting in preparation of the manuscTi$t. Cited TeSeaTCh from this laboratory was supported by a LEAD (Leadership and Excellence in Alzheimer Disease) grant from the National Institute on Aging (AG10916).
and A.Cataldo-
The neuronal lysosomalsystem
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49 Cupp, M., Bensadoun, A. and Melford, K. (1987) I. Biol. Chem. 262,6383-6388 50 Fukushima, D. et al. (1993) Biochem. Biophys. Res. Commun. 194, 202-207 51 Renfrew, C.A. and Hubbard, A.L. (1991) J. Biol. Chem. 266, 4348-4356 52 Roederer, M., Bowser, R. and Murphy, R. (1987) J. Cell. Physiol. 131,200-209
53 Bowser, R. and Murphy, R. (1990) J. Cell. Physiol. 143, 110-117 54 Waheed, A. et al. (1988) EMBO 1. 7, 2351-2358 55 Diment, S., Leech, M.S. and Stahl, P.D. (1988) J. Biol. Chem. 263, 6901-6907 56 Casciola-Rosen, L.A.F. and Hubbard, A.L. (1991) J. Biol. Chem.
The Rho’s progress: a potential role during neuritogenesis for the Rho family of GTPases DeborahJ.G. Mackay, Catherine D. Nobes and Alan Hall Growth
cones navigate
actin cytoskeleton
through
basis of this coupling family
of small
fibroblasts,
growth
link between adhesion Trends
Deborah J.G. Mackay, Cathm’ne D. Nobes and Alan Hall are at the MRC Laboratory fOT MOkCuluT
cell
Biology, University College London, Gower Street, London, UK WClE 6BT.
496
GTPases
regulate
are striking
incoming (1995)
extracellular extension
unknown.
morphological
and lamellipodia,
of filopodia, similarities
lamellipodia between
suggests that the Rho family
outgrowth
of both
actin
but the biochemical of the Rho
and stress fibres
spreading
fibroblasts
of proteins
dynamics
of the
in and
could be the
and cell-substratum
cone.
18, 496-501
EURITES EXTENDED by developing neurones folN low long and tortuous routes to their targets, and painstaking research on the pathfinding of neurites
in vivo and in culture has shown that this navigation
relies on several kinds of environmental signals. First, morphogenetic gradients of diffusible chemoattractants (for example, netrins’) and chemorepellants (for example, collapsin’) have been shown to act on developing neurites over distances of hundreds of micrometres. Second, signals are derived from the substratum surrounding the migrating neurite; for example, in model systems, neurites presented with a choice TINS Vol. 18, No. 11,1995
cues to directed
Recent studies have shown that members
signals and the regulation growth
guidance
of filopodia
the formation
cones. This resemblance
in the neuronal
Neurosci.
cyclical
is at present
and there
advancing
by coupling
between more- or less-adhesive substrata tend to transfer to the more adhesive (for reviews, see Refs 3 and 4). A third source of guidance information is found in other cells since neurites can extend even across regions of non-adhesive substratum by extending processes towards intermediate target cells or ‘guideposts’; if a single process contacts the guidepost, the neurite can reorientate and travel towards it?. In vivo, ablation of a guidepost can prevent the neurite from making a course correction required for completion of its stereotyped pathway. On the other hand, heterotypic neurones can act as ‘anti-guidepostsf6, which cause 8 1995, Elsevier Science Ltd
D. Mackay,
C. Nobes
REVIEW
and A. Hall - Rho GTPases and neuritogenesis
retreat and reorientation of impinging neurites and assist in the formation of neuronal territories. If the actin cytoskeleton growth cones...
controls
pathfinding
by
To detect and respond to these various signals, the distal tip of the neurite forms a specialized bulbous structure - the growth cone (Fig. 1A; see Refs 7 and 8). The flattened leading edge of the growth cone is filled by a population of dynamic actin filaments (see Fig. 2), some 50% of which is organized with the barbed (polymerizing) ends orientated distally. These filaments undergo treadmilling, with polymerization at the leading edge, retrograde transport (probably via myosin motors, at some 200-300 pm h-‘; see Ref. 9) and depolymerization in the body of the growth cone. The remainder of the actin forms a dense isotropic mesh, which appears to exclude microtubules and organelles from the leading edge. These excluded structures occupy instead the dilated body of the growth cone, and the shaft of the axon is supported by a microtubular cytoskeleton (see Ref. 8). Careful observation of moving growth cones has suggested the scheme of locomotion represented in Fig. 1 (Refs 10 and 11). Filopodia grow outwards from the leading edge, in an arc centred on the direction of travel (Fig. 1B). These extensions are extremely dynamic: they might explore and retract with a lifetime of minutes, and, in vitro, they are often seen to detach from the substratum and extend into the medium. However, time-lapse studies show that the filopodia that extend in the direction of positive stimuli are stabilized, and the actin filaments in these filopodia thicken and move down into the body of the growth cone. Lamellipodia subsequently form between stable filopodia (Fig. lC), and remain stationary while the body of the growth cone advances (Fig. 1D) by dilation of the proximal end of the lamella and admission of microtubules. This results eventually in extension of the shaft of the axon. New filopodia are then extended, frequently from the region of the newly consolidated leading edge. This slow mode of advance (4 pm h-l; Ref. 12) seems to be preferred in neurites responding to weak or diffuse information. However, neurites in the vicinity of guideposts, which are strong localized guidance cues, can advance at 10 pm h-’ by extending and then dilating filopodia, without the intermediacy of lamellipodia (see Ref. 12). The growth cone appears to advance, therefore, by extension of the actin cytoskeleton, followed by consolidation that involves microtubules. Axon branches extending towards the strongest stimuli accumulate actin and thrive, while others going in different directions lose actin filaments and retract. Axons receiving guidance information from two sources always follow only one, the stronger13; that is, growth cones can integrate quantitative information from filopodia to make qualitative decisions about which stimuli to follow. Treatment of neurones with cytochalasins (inhibitors of actin polymerization) does not prevent the neurones from extending, but it does lead to disappearance of the actin structures at the leading edge of the growth cone, which is then dilated and invaded by microtubules and organelles’. This can leave the neurites literally going round in circles, as shown by Bentley and co-workers’4. This strongly suggests that guidance information is indeed interpreted and acted upon intracellularly by
*
D
* Fig. 1. Actin and microtubules in outgrowth ofneurites. (A) An axon branch terminating in a growth cone. The flattened leading edge is occupied by a dense network of actin filaments, and the inflated body of the growth cone (shaded) is taken up by microtubules. (B) The growth cone extends filopodia (fine arrows) mainly in the direction of travel. The target region is represented by a star in the bottom-right corner of the diagram. (C) The filopodia directed towards the target are more stab/e than their neighbours, which are retracting. Lamellipodia (broad arrows) are extending between three stab/e, slightly dilated filopodia. (D) The /ameNpodium remains stationary, while the body of the growth cone behind it dilates to admit microtubules, and the growth cone advances. New filopadia are extending from the lamellipodium in the direction of the target.
regulation of the actin cytoskeleton. The biochemical mechanism modulating actin dynamics is not clear in neurones, but recent advances in analogous areas of fibroblast research have suggested that a key role might be played by members of the Rho branch of the Ras superfamily of small GTP-binding proteins. . ..and Rho proteins cytoskeleton...
regulate
the actin
Members of the Ras superfamily are involved in the regulation of a wide range of cellular activities, from intracellular protein trafficking to growth control (for review, see Ref. 15). The Rho branch of the superfamily TIN.5 Vol. 18, No. 11, 1995
497
REVIEW
D. Mackay,
C. Nobes
and A. Hall - Rho GTPases and neuritogenesis
Actin polymerisation
Microtubules l in dilated body of growth cone
Leading edge of growth cone
Fig. 2. The cytoskeleton of the growth cone. Actin filaments
assembled at the leading edge are transported into the body of the growth cone and disassembled there. The net result of this is continuous retrograde flux of fikmentous actin. The leading edge a/so contains shorter filaments in random orientation, while microtubules and organelles are confined to the dilated body of the growth cone and the axon shaft.
(for reviews, see Refs 16 and 17) currently has five members, which share SO-?&% amino-acid similarity: Rho (A, B and C isoforms), Rat (two isoforms), cdc42 (two isoforms), Rho G and TClO. They, like all proteins of the Ras superfamily, are active in the GTPbound form, and hydrolysis of GTP by activity of their intrinsic GTPase returns them to the inactive state. Activation of Rho proteins requires GDP-GTP exchange, and is catalysed by various guaninenucleotide exchange factors (GEFs). Their activity is also regulated by GTPase-activating proteins (GAPS), which stimulate their intrinsic GTPase activity. In addition, the guanine-nucleotide dissociation inhibitor RhoGDI interacts with all the proteins of the Rho family, and might well link nucleotide exchange with the cytosol-membrane redistribution that is essential for their activity. In the Rho family, Rho, Rat and cdc42 have been shown to exert specific effects on the actin cytoskeleton of Swiss 3T3 cells?, and their activities are schematized in Fig. 3. Swiss 3T3 fibroblasts grown to confluence and then deprived of serum lose much of their actin cytoskeleton; very little polymerised actin is detectable with phalloidin, and focal complexes, visualized with antibodies against the focal-adhesion protein, vinculin, are small or absent. Addition of serum (or its active principle, lysophosphatidic acid) to these cells causes activation of Rho and dramatic reorganisation of the actin cytoskeleton. Within a few minutes, the cell is placed under tension by stress fibres of bundled actin filaments, whose ends are associated with large, arrowhead-shaped focal adhesions at the plasma membrane”. Activation of Rat by various growth factors (for example, platelet-derived growth factor) leads to the formation of circumferential lamellipodia underpinned by a ring of focal complexes smaller than those generated by Rho, but with the same range of protein 498
TINS vol.
18, NO. 11, 1995
constituents20,21. The lamellipodia spread on the substratum, and then curl up from it and are drawn centripetally over the dorsal surface of the cell to produce membrane ruffles. This gives a visual impression a little like the breaking of waves, but in reverse. An additional and later effect of Rat is to activate the Rho pathway. The effects of cdc42 were elusive until they were investigated in fibroblasts growing at low density, and hence with space to extend and move’l. In such cells, cdc42 first directs the formation of dynamic filopodia associated with small focal complexes, and then activates Rat, which leads to the formation of lamellipodia between the filopodia. This enables an initially rounded cell to spread out over the substratum. Figure 3 summarizes the activities of cdc42, Rat and Rho in the formation of filopodia, lamellipodia, and stress fibres, respectively. . ..do Rho proteins regulate pathfinding by growth cones?
There are clear morphological similarities between fibroblasts that contain active cdc42 or Rat and the growth cones of advancing neurites: both can spread by extension of filopodia and subsequent generation of lamellipodia between the filopodia (see Fig. 4). This prompts the suggestion that cdc42 and Rat regulate actin dynamics in the growth cone, and a model for how they might work is outlined in Fig. 5. Filopodia would first be extended by local, transient activation of cdc42. The presence of a positive stimulus at a filopodial tip would lead to sustained local activation of cdc42, increased polymerization of actin and enlargement of focal complexes. This would result in stabilization of the filopodium. Since formation of focal complexes in fibroblasts is known to be dependent on integrins (the receptors for the extracellular matrix) filopodia would only be expected to be stabilized on a favourable substratum. Extrapolation from the data on fibroblasts suggests that, subsequently, sustained activation of cdc42 would promote activation of Rat, and lamellipodia would therefore be extended in the direction of the strongest stimuli. Is there, then, any evidence that focal complexes play a role in the dynamics of the growth cones? An elegant study on ‘cultured PC12 cells provides a link between the stability of focal complexes and the stability of the filopodia and lamellipodia in growth cones. Varnum-Finney and Reichardt” generated PC12 lines that expressed only one-fifth of the normal levels of vinculin. Filopodia in normal PC12 growth cones show staining of vinculin along their lengths and at their tips, whereas filopodia in the mutant PC12 cells showed much less staining of vinculin. Analysis of the vinculin-deficient cells indicated that their filopodia were extended at the same rate and were of the same length as those of wildtype PC12 cells, but had a much shorter lifespan. Moreover, in the mutant cells, developing lamellipodia were almost
D. Mackay,
-
REVIEW
and A. Hall - Rho GTPases and neuritogeneris
PDGF Insulin
GEF
cdc42 GDP
C. Nobes
cdc42 GTP
GAP
1
GEF
rat GTP
rat GDP
Filopodia Focal contacts
LPA
GEF
GAP
rho GTP
rho GDP
Lamellipodia Focal contacts
GAP
Stress fibres Focal adhesions
Fig. 3. The effects of the proteins of the Rho family, Rho, Rat and cdc42, on the actin cytoskeleton of fibroblasts. Each of the three proteins is active in the CTP-bound state and inactive in the GDP-bound form. Activation of the proteins requires guanine-nucleotide exchange factors (GEFs), and deactivation occurs through an intrinsic CTPase-activity that can be stimulated by C JPase-activating proteins (GAPS). Cuoninenucleotide dissociation inhibitors (CD/s) increase the stability of both G JP-bound and GDP-bound forms. While the proteins are activated by extracellular signals, they a/so participate in a cascade of activation from cdc42 through Rat to Rho.
always retracted before advance of growth cones could take place. As a result, the vinculin-deficient axons grew much more slowly than controls. This study suggests that integrity of focal complexes is required for the stability of both filopodia and lamellipodia in migrating growth cones. Wu and Goldberg23 used an anti-phosphotyrosine antibody to visualize a tyrosine phosphoprotein at the tips of filopodia in Aplysia neurones that were either
migrating slowly on a polylysine-coated substratum or more rapidly on one coated with Aplysia haemolymph. The neurones that were migrating on polylysine had short filopodia, 80% of which showed staining phosphotyrosine. By contrast, the filopodia of the neurones that were migrating on haemolymph were only 15% phosphotyrosine-positive, but twice as long as, and more stable than, the filopodia extended on polylysine. Similarly, extension of neurites in TINS Vol. 18, NO. 11, 1995
499
REVIEW
D. Mackay,
C. Nobes
and A. Hall - Rho GTPases and neuritogenesis
Fig. 4. Morphological similurity between an advancing growth cone (kft) ond Swiss 3T3 fibroblosts that are extending fi/opodia (upper right) or filopodia and lamellipodia [lower right). The cell on the upper right was injected with activated cdc42 to promote formation of fi/opodia, along with dominantnegative Rat and C3 transferose to suppress the activation of endogenous Rat and Rho, respective/y. The cell on the lower right was injected with activated cdc42 and C3, and has undergone octivotion of endogenous Rot. Stole bars, I Ohm.
developing chick neurones was potentiated by the tyrosine-kinase inhibitor genisteinz4. Tyrosine phosphoproteins could regulate extension of filopodia by one of two mechanisms: they could regulate elongation of filaments at the tips of the fllopodia or they could regulate the association between cell-substratum adhesions and the retrograde flux of actin in the leading edge of the growth cone. This latter suggestion is borne out by the observation that the rate of motility in various cells is inversely related to the rate of flux of actirP, and suggests that advance of growth cones is controlled, in part, by regulatable cell adhesion (for review of the model, see Ref. 9). Direct evidence for a role of Rat and cdc42 in formation of axons has been reported in Drosophilaz6. Active and inactive mutants of the rat and cdc42 genes of Drosophila were expressed at various sites and stages of embryogenesis by the use of developmentally regulated promoters. In almost all cases, the neurones expressing the mutant proteins were correctly localized. However, the axons of the various rut mutants either did not extend at all or were stalled halfway to their targets, while mutations in cdc42 resulted in hindrance of the development of both axons and dendrites. These observations connecting formation of focal complexes with the length and stability of filopodia and lamellipodia have been complemented by studies on the behaviour of actin in growth cones in vivo and in vitro. One series of studies on grasshopper embryos examined the navigation of pioneer neurones along a stereotyped pathway marked out by guidepost cells (see Refs 12 and 13). On contact with a guidepost cell, 500
TINS Vol. 18, No. 11, 1995
the filopodia of the pioneer neurones are dilated by thickening of bundles of actin, which are then transported back into the body of the growth conez7. A related study examined growth cones arriving at their targets in an in vitro systemz8. The filopodia contacting the target were dilated by formation of new actin filaments and then by microtubules, before formation of a tight contact. By contrast, if positively charged polystyrene beads were applied to the growth cone as mock targets, they became associated with its actin cytoskeleton but did not promote subsequent accretion of microtubules; instead, the beads were swept back along the growth cone at the speed of the actin flux. It appeared that the treatment accorded to the target and the target-like microbead depended on their relative size and tractability. The actin filaments that accumulated at the target contracted to place it under tension, and only then did microtubules invade the contact site. This observation leads to the interesting possibility that one of the major intracellular signals in the growth cone is mechanical tension in actin filaments (see Refs 9 and 29). It also brings up the subject of the effects of Rho on neurites. Rho: the growth
cone
meets
its match
Rho appears to be required not for the outgrowth of neurites, but for their retraction (Fig. 5). Contact of the filopodium of a growth cone with a repellant such as another neurone can provoke collapse of the filopodium or of the whole growth cone6t30.The cone then remains frozen for some time before extension of new filopodia, which are normally directed away from the site of the repulsive contact. Even more repellent to neurites is exposure to serum or thrombin on traumatic injury of the nervous system: serum-treated axons will retract completely within seconds. Moolenaar and co-workers31,32studied this process using the cell line NlE-115, which has a fibroblast-like morphology when growing in serum, but, after serum-starvation, differentiates to generate axons and growth cones. Addition of serum or thrombln to these neurone-like cells caused rapid collapse of the axons by contraction of actomyosin. This collapse could be prevented by pretreatment of the cells either with specific inhibitors of myosin light-chain kinase, or with C3 transferase, a Clostridium boiulinum exoenzyme that specifically inactivates Rho. It is possible that the role of Rho is to test the interaction between growth cone and target by generation of mechanical tension in actin filaments. If adhesion were strong enough to withstand tension, then docking would proceed, and if not, the neurite would be retracted. Inappropriate activation of Rho during outgrowth of neurites would lead to their retraction. It is possible that tensile strength is also used to integrate the signalling inputs of filopodia during outgrowth of
D. Mackay,
neurites (for review, see Ref. 29); to compare the tension developed in various filopodia would be one means of determining the quantity of actin accumulated in each one. Future cones
directions
activity
1 2 3 4
Rat
b-l
GDP
eXClUSiVelY in ner-
of
the
Serine/threonine
cdc42 GTP
-
Filopodia
I Collapse
Rho GTP
A
-
Rho GDP
-
Fig. 5. The proposed roles for cdc42, Rot and Rho in navigation of growth cones. Cdc42 triggers formation and stabilization of filopodia in response to extracellular guidance cues, and also leads to activation of Rat and formation of filopodia. Active Rat might be able to sustain activity of cdc42 loco//y since filopodia arise frequent/y from stable lame/lipodia of growth cones. Cdc42 or Rat might also activate Rho, leoding to tension in actin filaments, but activation of Rho seems to suppress formation of filopodia, at /east in the short term.
p2J-aCiiVated
kinase3* is stimulated bvI GTP-bound Rat. while mvr5. , I a novel myosin-like actin-binding protein, interacts with Rho, Rat and cdc42 through a GAP domain39. Another candidate effector is phosphatidylinositol 5-kinase, which generates the second messenger phosphatidylinositol (4,5)-bisphosphate (PIP,) (Ref. 40). This is of interest since a number of actin-binding proteins are also PIP,-binding proteins (for example, gelsolin’l, profilin4’ and cofilin43) and the activity of the kinase in cell extracts is modulated by Rho4’. Lipid metabolism might represent an important link between the proteins of the Rho family and actin dynamics in both neuronal and non-neuronal cells. In conclusion, those interested in how growth cones move towards their targets should seriously consider a role for the Rho family of GTPases. Selected
f7(
cdc42 GDP
vous tissue. Differential expression and activity of these proteins might well turn out to be pivotal in the development of the nervous system. Second, as mentioned above, Rho, Rat and cdc42 all appear to promote the clustering of integrins and the formation of focal complexes’l, and adhesion complexes are likely to turn out to be central to the movement of the growth cone over the substratum. Third, it is currently a matter of great interest to identify effecters for Rho proteins, and a number of possibilities have already been suggested. For example, ihe
Rat GTP
A
REVIEW
and A. Hall - Rho GTPases and neuritogenesis
for growth
The intracellular signalling pathways that regulate the remarkably complex behaviour of growth cones are somewhat unclear at present, but a number of interesting observations might indicate some future directions for research. First, the activation of the Rho proteins requires GEFs (see Fig. 3), and these would then be the primary targets of signal transduction. A paradigm for this is the recruitment of the exchange factor SOSto signalling complexes, leading to activation of Ras (for review, see Ref. 33). A number of exchange factors for the Rho family, for example, db134and ost3’, and GAPS such as N-chimaerin3’j and ABR [active bcr-related (bcr breakpoint cluster region, a Rat GTPase-activating protein)]37, are ex-
Presseda1most
Lamellipodia
C. Nober
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Acknowledgements The authors would like to thank the Wellcome Trust (D/GM) and the CRC (AH, CDN) for their generous financial support. We are very grateful to Dr Dennis Bray for the photograph of a growth cone shown in Fig. 4, and to Dr Paul Martin for his critical reading of the manuscriit.
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