The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia.

n-Chimaerin is a GTPase-activating protein (GAP) mainly for Rac1 and less so for Cdc42Hs in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of Rac1 and Cdc42Hs into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia. These structures undergo cycles of dissolution and formation, resembling natural morphological events occurring at the leading edge of fibroblasts and neuronal growth cones. The effects of n-chimaerin on formation of lamellipodia and filopodia were inhibited by dominant negative Rac1(T17N) and Cdc42Hs(T17N), respectively. n-Chimaerin's effects were also inhibited by coinjection with Rho GDP dissociation inhibitor or by treatment with phorbol ester. A mutant n-chimaerin with no GAP activity and impaired p21 binding was ineffective in inducing morphological changes, while a mutant lacking GAP activity alone was effective. Microinjected n-chimaerin colocalized in situ with F-actin. Taken together, these results suggest that n-chimaerin acts synergistically with Rac1 and Cdc42Hs to induce actin-based morphological changes and that this action involves Rac1 and Cdc42Hs binding but not GAP activity. Thus, GAPs may have morphological functions in addition to downregulation of GTPases.     

Autonomous right-screw rotation of growth cone filopodia drives neurite turning

The direction of neurite elongation is controlled by various environmental cues. However, it has been reported that even in the absence of any extrinsic directional signals, neurites turn clockwise on two-dimensional substrates. In this study, we have discovered autonomous rotational motility of the growth cone, which provides a cellular basis for inherent neurite turning. We have developed a technique for monitoring three-dimensional motility of growth cone filopodia and demonstrate that an individual filopodium rotates on its own longitudinal axis in the right-screw direction from the viewpoint of the growth cone body. We also show that the filopodial rotation involves myosins Va and Vb and may be driven by their spiral interactions with filamentous actin. Furthermore, we provide evidence that the unidirectional rotation of filopodia causes deflected neurite elongation, most likely via asymmetric positioning of the filopodia onto the substrate. Although the growth cone itself has been regarded as functionally symmetric, our study reveals the asymmetric nature of growth cone motility.     

Galanin stimulates neurite outgrowth from sensory neurons by inhibition of Cdc42 and Rho GTPases and activation of cofilin

We and others have previously shown that the neuropeptide galanin modulates neurite outgrowth from adult sensory neurons via activation of the second galanin receptor; however, the intracellular signalling pathways that mediate this neuritogenic effect have yet to be elucidated. Here, we demonstrate that galanin decreases the activation state in adult sensory neurons and PC12 cells of Rho and Cdc42 GTPases, both known regulators of filopodial and growth cone motility. Consistent with this, activated levels of Rho and Cdc42 levels are increased in the dorsal root ganglion of adult galanin knockout animals compared with wildtype controls. Furthermore, galanin markedly increases the activation state of cofilin, a downstream effector of many of the small GTPases, in the cell bodies and growth cones of sensory neurons and in PC12 cells. We also demonstrate a reduction in the activation of cofilin, and alteration in growth cone motility, in cultured galanin knockout neurons compared with wildtype controls. These data provide the first evidence that galanin regulates the Rho family of GTPases and cofilin to stimulate growth cone dynamics and neurite outgrowth in sensory neurons. These findings have important therapeutic implications for the treatment of peripheral sensory neuropathies.

     

Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin- deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.     

Substrate-bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation.

The surfaces on which neurons grow greatly affect neurite elongation, but it is unclear how substrates influence the events within the growth cone that bring about elongation. Neurite elongation by Aplysia californica neurons in culture occurs through a series of transformations of the structures of the growth cone (Goldberg and Burmeister, J. Cell Biol., 103:1921-1931, 1986). The growth cone produces actin-rich protrusions, veils, and lamellipodia, which can then mature into the central body of the growth cone through the net advance of microtubules and membranous organelles from contiguous central regions, a process called "engorgement." Aplysia neurons form growth cones on poly-l-lysine-treated substrates, but their rate of neurite elongation is greatly enhanced on substrates additionally exposed to Aplysia hemolymph. The acute application of hemolymph to slowly growing neurites brings about a rapid acceleration of neurite elongation and engorgement. The enhancement of engorgement was effected with material eluted from hemolymph-treated substrates and was not seen when hemolymph was added to neurons cultured on hemolymph-treated substrates inactivated by exposure to UV radiation. Thus, we conclude that the rapid acceleration of engorgement caused by hemolymph is, in large part, a substrate-mediated effect. We propose that extracellular substrate molecules can modulate the rate of neurite growth through the regulation of the engorgement of lamellipodia. The microtubule disrupters colcemid and nocodazole inhibit the advance of vesicular elements into the lamellipodia following hemolymph treatment, but taxol, which promotes the polymerization and stabilization of microtubules, does not itself enhance engorgement. The microfilament disrupter cytochalasin B, however, stimulates engorgement. Our results suggest that regulating the resistance of the peripheral actin meshwork to penetration by microtubules and vesicles may be a mechanism by which substrate-attached molecules regulate neurite advance.     

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

Background

The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth.

Methodology/Principal Findings

We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth.

Conclusions/Significance

We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.     

The neurite outgrowth multiadaptor RhoGAP, NOMA-GAP, regulates neurite extension through SHP2 and Cdc42

Neuronal differentiation involves the formation and extension of neuronal processes. We have identified a novel regulator of neurite formation and extension, the neurite outgrowth multiadaptor, NOMA-GAP, which belongs to a new family of multiadaptor proteins with RhoGAP activity. We show that NOMA-GAP is essential for NGF-stimulated neuronal differentiation and for the regulation of the ERK5 MAP kinase and the Cdc42 signaling pathways downstream of NGF. NOMA-GAP binds directly to the NGF receptor, TrkA, and becomes tyrosine phosphorylated upon receptor activation, thus enabling recruitment and activation of the tyrosine phosphatase SHP2. Recruitment of SHP2 is required for the stimulation of neuronal process extension and for sustained activation of ERK5 downstream of NOMA-GAP. In addition, we show that NOMA-GAP promotes neurite outgrowth by tempering activation of the Cdc42/PAK signaling pathway in response to NGF. NOMA-GAP, through its dual function as a multiadaptor and RhoGAP protein, thus plays an essential role downstream of NGF in promoting neurite outgrowth and extension.     

Cdc42 is involved in PKCepsilon- and delta-induced neurite outgrowth and stress fibre dismantling

We have shown that protein kinase C (PKC)epsilon, independently of the catalytic domain, induces outgrowth of cellular processes via its regulatory domain in both neural cells and fibroblasts. This was accompanied by stress fibre loss. Here, we have examined the role of the small GTPases, Rac1, and Cdc42, in these PKC-mediated morphological and cytoskeletal changes. Both constitutively active and dominant negative Rac1 and Cdc42 attenuated the PKC-mediated outgrowth of processes. The suppression was larger for Cdc42 than for Rac1. The PKC-mediated dismantling of the stress fibres in both HiB5 and fibroblasts was inhibited by the expression of the Cdc42 mutants whereas they had smaller effects on the stress fibre dismantling induced by the ROCK inhibitor, Y-27632, indicating a more crucial role for Cdc42 in the PKC-mediated pathway. We conclude that Cdc42 is an important downstream factor in the pathway through which PKC mediates morphological and cytoskeletal effects.     

Control of growth cone motility and neurite outgrowth by SPIN90

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.     

Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. It is widely accepted that Rac1 and Cdc42, members of the Rho GTPase family, positively regulate neurite extension through reorganization of the actin cytoskeleton; however, it remains largely unknown when and where Rac1 and Cdc42 are activated during neuritogenesis. This study visualized the spatio-temporal regulation of Rac1 and Cdc42 activities during nerve growth factor (NGF)-induced neurite outgrowth in living PC12 cells by using probes based on the principle of fluorescence resonance energy transfer (FRET). Immediately after the addition of NGF, Rac1 and Cdc42 were transiently activated in broad areas of the cell periphery; a repetitive activation and inactivation cycle was then observed at the motile tips of protrusions. This localized activation, which was more evident in PC12 cells treated with NGF for more than 24 h, might facilitate neurite extension, because the expression of constitutively active mutants of Rac1 and Cdc42 abrogated NGF-induced neurite outgrowth. FRET imaging also delineated a difference between the localization of activated Rac1 and that of Cdc42 within the neurite tips. Experiments with dominant-negative mutants suggested that Rac1 and Cdc42 were activated by a common guanine nucleotide exchange factor(s) in an early stage of the activation phase. Therefore, the difference between Rac1- and Cdc42-activated areas possibly came from the differential localization between Rac1-specific GTPase-activation proteins (GAPs) and Cdc42-specific GAPs. It was concluded that the localized activation of Rac1 and Cdc42 was caused by both guanine nucleotide exchange factors and GAPs, and was important for neurite extension.     

Cdc42 participates in the regulation of ADF/cofilin and retinal growth cone filopodia by brain derived neurotrophic factor

Rho family GTPases have important roles in mediating the effects of guidance cues and growth factors on the motility of neuronal growth cones. We previously showed that the neurotrophin BDNF regulates filopodial dynamics on growth cones of retinal ganglion cell axons through activation of the actin regulatory proteins ADF and cofilin by inhibiting a RhoA-dependent pathway that phosphorylates (inactivates) ADF/cofilin. The GTPase Cdc42 has also been implicated in mediating the effects of positive guidance cues. In this article we investigated whether Cdc42 is involved in the effects of BDNF on filopodial dynamics. BDNF treatment increases Cdc42 activity in retinal neurons, and neuronal incorporation of constitutively active Cdc42 mimics the increases in filopodial number and length. Furthermore, constitutively active and dominant negative Cdc42 decreased and increased, respectively, the activity of RhoA in retinal growth cones, indicating crosstalk between these GTPases in retinal growth cones. Constitutively active Cdc42 mimicked the activation of ADF/cofilin that resulted from BDNF treatment, while dominant negative Cdc42 blocked the effects of BDNF on filopodia and ADF/cofilin. The inability of dominant negative Cdc42 to block ADF/cofilin activation and stimulation of filopodial dynamics by the ROCK inhibitor Y-27632 indicate interaction between Cdc42 and RhoA occurs upstream of ROCK. Our results demonstrate crosstalk occurs between GTPases in mediating the effects of BDNF on growth cone motility, and Cdc42 activity can promote actin dynamics via activation of ADF/cofilin.     

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons. These authors contributed equally     

Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.     

Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase

Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.     

Retinoic acid induces neurite outgrowth and growth cone turning in invertebrate neurons

Identification of molecules involved in neurite outgrowth during development and/or regeneration is a major goal in the field of neuroscience. Retinoic acid (RA) is a biologically important metabolite of vitamin A that acts as a trophic factor and has been implicated in neurite outgrowth and regeneration in many vertebrate species. Although abundant in the CNS of many vertebrates, the precise role of RA in neural regeneration has yet to be determined. Moreover, very little information is available regarding the role of RA in invertebrate nervous systems. Here, we demonstrate for the first time that RA induces neurite outgrowth from invertebrate neurons. Using individually identified neurons isolated from the CNS of Lymnaea stagnalis, we demonstrated that a significantly greater proportion of cells produced neurite outgrowth in RA. RA also extended the duration of time that cells remained electrically excitable in vitro, and we showed that exogenously applied RA acted as a chemoattractive factor and induced growth cone turning toward the source of RA. This is the first demonstration that RA can induce turning of an individual growth cone. These data strongly suggest that the actions of RA on neurite outgrowth and cell survival are highly conserved across species.     

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.     

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.     

PTP mu-dependent growth cone rearrangement is regulated by Cdc42

PTP mu is expressed in the developing nervous system and promotes growth and guidance of chick retinal ganglion cells. Using a newly developed growth cone rearrangement assay, we examined whether the small G-proteins were involved in PTP mu-dependent signaling. The stimulation of retinal cultures with purified PTP mu resulted in a striking morphological change in the growth cone, which becomes dominated by filopodia within 5 min of addition. This rearrangement in response to PTP mu stimulation was mediated by homophilic binding. We perturbed GTPase signaling using Toxin B, which inhibits Cdc42, Rac, and Rho, as well as the toxin Exoenzyme C3 that inhibits Rho. The PTP mu-induced growth cone rearrangement was blocked by Toxin B, but not by Exoenzyme C3. This result suggests that either Cdc42 or Rac are required but not Rho. To determine which GTPase was involved in PTP mu signaling, we utilized dominant-negative mutants of Cdc42 and Rac. Dominant-negative Cdc42 blocked PTP mu-induced rearrangement, while wild-type Cdc42 and dominant-negative Rac did not. Together, these results suggest a molecular signaling cascade beginning with PTP mu homophilic binding at the plasma membrane and the activation of Cdc42, which acts on the actin cytoskeleton to result in rearrangement of the growth cone.     

Stochastic dynamics of the nerve growth cone and its microtubules during neurite outgrowth

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345-363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of approximately 4 min, meaning that growth cone advances tended to be followed by growth cone retractions approximately 4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cone's microtubule array. Cross-correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of approximately 0-2 min, while microtubules tended to precede growth cone dynamics on a approximately 0-20-s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean-squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance. (c) 1996 John Wiley & Sons, Inc.     

EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42

Subversion of the eukaryotic cell cytoskeleton is a virulence strategy employed by many bacterial pathogens. Due to the pivotal role of Rho GTPases in actin dynamics they are common targets of bacterial effector proteins and toxins. IpgB1, IpgB2 ( Shigella ), SifA, SifB ( Salmonella ) and Map and EspM (attaching and effacing pathogens) constitute a family of type III secretion system effectors that subverts small GTPase signalling pathways. In this study we identified and characterized EspT from Citrobacter rodentium that triggers formation of lamellipodia on Swiss 3T3 and membrane ruffles on HeLa cells, which are reminiscent of the membrane ruffles induced by IpgB1. Ectopic expression of EspT and IpgB1, but not EspM, resulted in a mitochondrial localization. Using dominant negative constructs we found that EspT-induced actin remodelling is dependent on GTP-bound Rac-1 and Cdc42 but not ELMO or Dock180, which are hijacked by IpgB1 in order to form a Rac-1 specific guanine nucleotide exchange factor. Using pull-down assays with the Rac-1 and Cdc42 binding domains of Pak and WASP we demonstrate that EspT is capable of activating both Rac-1 and Cdc42. These results suggest that EspT modulates the host cell cytoskeleton through coactivation of Rac-1 and Cdc42 by a distinct mechanism.