Making all the right moves: chemotaxis in neutrophils and Dictyostelium

Making all the right moves: chemotaxis in neutrophils and Dictyostelium Carole A Parent Neutrophils and Dictyostelium discoideum share the ability to migrate directionally in response to external chemoattractant gradients. The binding of chemoattractants to specific receptors that are coupled to heterotrimeric G proteins leads to a wide range of biochemical responses that become highly localized as cells polarize and migrate by chemotaxis. The signaling mechanisms that lead to the predominant polymerization of Factin at the front of cells for propulsion and to myosin II assembly at the sides to suppress lateral pseudopod formation and at the back for retraction are now beginning to emerge. Addresses Laboratory of Cellular and Molecular Biology, National Cancer Institute, NIH, 37 Convent Drive, Bldg37/Rm1E24, Bethesda MD 20892-4255, USA e-mail: [email protected]

Current Opinion in Cell Biology 2004, 16:4–13 This review comes from a themed issue on Cell structure and dynamics Edited by John A Cooper and Margaret A Titus 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2003.11.008

Abbreviations CRAC cytosolic regulator of adenylyl cyclase Gbp cGMP binding protein GEF guanine nucleotide exchange factor PAK p21-activated kinases PH Pleckstrin homology PhdA PH-domain-containing protein A PIXa PAK-interacting exchange factor P-Rex PtdIns(3,4,5)P3-dependent Rac exchanger PTX pertussis toxin SCAR suppressor of cAR SHIP 50 -phosphoinositide phosphatase VASP vasodilator-stimulated phosphoprotein WASP Wiskott–Aldrich syndrome protein

exquisitely regulated amoeboid motion, which can be achieved in the presence of very shallow attractant gradients, gives rise to speeds that can reach up to 20 mm/ min. This behavior has withstood the pressure of millions of years of evolution: Dictyostelium discoideum behave in a virtually identical fashion when they respond chemotactically to form aggregates that will differentiate into multicellular organisms (Figure 1) [1,2]. Most remarkably, this conservation is also observed in the underlying biochemical machinery as neutrophils and D. discoideum share common signaling mechanisms to decipher chemoattractant gradients. Indeed, many fundamental aspects of chemotaxis have been derived from studies using D. discoideum. For both neutrophils and D. discoideum, chemotactic movement is initiated when chemoattractants bind to transmembrane receptors that couple to heterotrimeric G proteins. This leads to the dissociation of the G protein into a- and bg-subunits, which activate a plethora of effectors [3]. These in turn orchestrate a signaling cascade that ultimately gives rise to cellular polarization and movement. The acquisition of polarity is accompanied by a dramatic redistribution of cytoskeletal components, during which F-actin and numerous actin-binding proteins are enriched at the front or leading edge and myosin II is assembled on the sides and at the back or trailing edge. In this review I will focus on the extensive progress that has recently been made in understanding the events that transduce shallow extracellular gradients of chemoattractants into highly polarized intracellular signaling responses. Because they provide a wonderful example of evolutionary conservation, emphasis will be given to chemotactic signaling in neutrophils and D. discoideum. The important role of adhesion in chemotaxis will not be discussed; the reader is directed to recent excellent reviews on the subject [4,5].

Signaling to the front Introduction A wide variety of cells exhibit the capacity to respond and migrate directionally in response to external gradients. This behavior is essential for a variety of processes including angiogenesis, nerve growth, wound healing and embryogenesis. But perhaps the most distinguished chemotactic response is exemplified by neutrophils as they navigate to sites of inflammation. When exposed to an attractant gradient, these cells quickly orient themselves and move using anterior pseudopod extension together with posterior contraction and retraction. This Current Opinion in Cell Biology 2004, 16:4–13

Studies in neutrophils and D. discoideum have established that chemoattractant receptors and their downstream heterotrimeric G proteins are not spatially restricted during chemotaxis. GFP-labeled receptors are uniformly distributed at the periphery of neutrophils and D. discoideum cells undergoing chemotaxis [6,7]. Similarly, GFP-labeled Gb-subunits show a mostly uniform plasma membrane distribution in D. discoideum undergoing chemotaxis [8]. Moreover, using FRET (fluorescence resonance energy transfer), Janetopoulos et al. showed that the activation of G proteins reflects receptor occupancy in D. discoideum and that G proteins remain dissociated as long www.sciencedirect.com

Making all the right moves: chemotaxis in neutrophils and Dictyostelium Parent 5

Figure 1

are exposed to gradients, the membrane recruitment is persistently restricted to the side of the cell facing the highest concentration of attractant. Remarkably, this redistribution occurs in the presence of inhibitors of actin polymerization, demonstrating that the actin cytoskeleton is not required for signal sensing. It was therefore proposed that cells sense external gradients by generating binding sites for PH-domain-containing proteins at their leading edge. Targeted PH-domain-containing proteins at the leading edge then serve as nucleation sites to activate various downstream effectors, including actin polymerization. From these findings two obvious questions arise: first, how are PH-domain binding sites spatially confined to the cell anterior, and second, how do PH-domain-containing proteins target actin polymerization?

How are PH-domain binding sites spatially confined to the leading edge of cells undergoing chemotaxis?

D. discoideum and neutrophils migrating in response to chemoattractant gradients. (a) Differentiated wild-type D. discoideum cells were plated on a chambered cover glass, and subjected to a cAMP gradient (supplied by a micropipette containing 1 mM cAMP). The bottom panel shows a high-magnification image of a polarized cell. (b) Peripheral blood neutrophils were plated on a chambered cover glass and subjected to an fMLP gradient (supplied by a micropipette containing 1 mM fMLP). The bottom panel shows a high magnification image of a polarized cell. For both (a) and (b), the numbers on the lower right corner indicate elapsed time in seconds. The images on the upper panels were taken using 20X objectives and the high magnification bottom panel images were captured using 63X objectives.

as receptors are occupied [9]. Taken together, these results suggest that the spatial segregation of signals occurs downstream of receptor activation. The observation that pleckstrin homology (PH)-domaincontaining proteins specifically translocate to the leading edge of cells undergoing chemotaxis provided the first clue into how signals become spatially restricted and how the cell anterior becomes organized. In D. discoideum, several such proteins have been identified, including CRAC (cytosolic regulator of adenylyl cyclase), PhdA (PH-domain-containing protein A), and Akt, which also translocates to the leading edge of neutrophils, again underscoring the evolutionary conservation of the signaling cascade (Figure 2) [10–13]. Under basal conditions, these proteins are cytosolic; their recruitment to the plasma membrane occurs when cells are exposed to chemoattractants. In the presence of a uniform stimulus of chemoattractant, a global but transient translocation occurs around the cell periphery. However, when cells www.sciencedirect.com

As the subset of PH-domain-containing proteins that redistribute during chemotaxis specifically bind PtdIns(3,4)P2/PtdIns(3,4,5)P3, attention has shifted to establishing the role of the enzymes that control the synthesis (PI3K) and degradation (PTEN) of these lipids during chemotaxis [14]. Chemoattractant-mediated translocation of PH-domain-containing proteins is inhibited in the presence of PI3K inhibitors and studies using pharmacological inhibitors or cells derived from null-mutant animal models have implicated Class I PI3K in the control of polarity and motility [15,16]. In neutrophils, chemoattractants could stimulate more than one isoform of Class I PI3K. Although studies using neutrophils derived from PI3Kg-null mice show that the bulk of PtdIns(3,4,5)P3 is generated by this Gbg-sensitive isoform of PI3K, it was recently demonstrated that a selective inhibitor of PI3Kd, which is preferentially expressed in hematopoietic cells, has deleterious effects on neutrophil polarity as well as fMLP-induced chemotaxis [17–19,20]. As PI3Kd is activated via tyrosine kinases, it was suggested that Srcfamily tyrosine kinases could be part of an amplification signal that controls PtdIns(3,4,5)P3 levels [20,21,22]. Indeed, in vitro analyses have shown that Gai–GTP, the Ga subunit that couples to chemoattractant receptors, can bind and activate Src and Hck kinases [23]. The involvement of PI3Kd could explain the residual chemotaxis response and chemoattractant-mediated actin polymerization observed in neutrophils derived from PI3Kg-null mice (Figure 2) [17–19]. Cytosolic inositol pyrophosphates could also be involved in regulating how cells sense gradients. Work performed by Luo et al. provides evidence that IP7 competes for PHdomain binding with PtdIns(3,4,5)P3 in D. discoideum [24]. These authors show that chemoattractants trigger elevations in levels of IP7 and IP8 and that cells lacking the IP6-kinase, which converts IP6 to IP7, have no measurable levels of IP7 or IP8, are significantly more Current Opinion in Cell Biology 2004, 16:4–13

6 Cell structure and dynamics

Figure 2

F-actin

PIP2/3

Gbγ PI3K

Arp2/3

Akt PhD

Gβγ PIP2/3

Rac-GEF? CRAC

Rac?

F-actin

PI3Kδ/PI3Kγ PIXα

Akt

?

P-Rex

Arp2/3

Cdc42

Gαi SCAR/ PIR121

PAK1

Rac SCAR/ PIR121

WASP

cGMP

RhoGEF RhoA

PAKa ROCK

ACA

MyoII MyoII

PTEN cAMP

D. discoideum

PTEN

Neutrophil Current Opinion in Cell Biology

Schematic representation of polarized signaling events in D. discoideum cells (left panel) and neutrophils (right panel). The cartoon depicts cells as they are moving in response to a gradient of chemoattractant; the leading edge of the cells is at the top of the figure. The dashed lines represent pathways that have yet to be fully characterized. Although both chemoattractant receptors (not shown) and heterotrimeric G proteins are mostly uniformly distributed, the majority of the downstream effectors become segregated as cells polarize (see text for details). The sustained levels of PtdIns(3,4)P2/PtdIns(3,4,5)P3 (PIP2/3) to the leading edge is caused by the reciprocal cellular distribution of PI3K at the front and PTEN at the back. In neutrophils, Gai could activate a Gbg-insensitive PI3K, PI3Kd, via activation of a Src kinase. The polarized distribution of PIP2/3 leads to the leading edge recruitment of the PH-domain-containing proteins PhD and CRAC for D. discoideum, and Akt for both D. discoideum and neutrophils. Although the PhdA protein has been shown to be involved in chemotaxis, the mechanisms by which this occurs remain to be established. CRAC is essential for the activation of the adenylyl cyclase ACA. ACA is highly enriched at the backs of cells undergoing chemotaxis and involved in signal relay to neighboring cells. The role of Akt in neutrophil chemotaxis remains to be determined. In D. discoideum, Akt phosphorylates and activates PAKa, which is then involved in the regulation of myosin II assembly. Myosin II assembly is also regulated by cGMP in D. discoideum, but the upstream events that spatially regulate the synthesis and degradation of cGMP have yet to be determined. In neutrophils, myosin II assembly is regulated by the activation of RhoA and its downstream effector ROCK. The RhoGEF is proposed to be activated by Ga12/13 (not shown). At the leading edge, the RhoGEFs are boxed and represent links to the actin cytoskeleton. In neutrophils, P-Rex possesses GEF activity towards Rac and requires both Gbg and PtdIns(3,4,5)P3 to be activated; PtdIns(3,4,5)P3 is proposed to act as an allosteric regulator of P-Rex. The Cdc42 GEF, PIXa, forms a complex with Gbg, PAK1, and Cdc42. PIP2/3 is thought to be required for the proper cellular localization of Ccd42 and for PIXa activity. In neutrophils, Rac and Cdc42 regulate actin polymerization via the SCAR/PIR121 multi-protein complex and WASP, respectively. Rac is also proposed to be part of a positive feedback loop where it amplifies PIP2/3 polarity, possibly by activating PI3K. In D. discoideum, the identification of the Rac and its GEF has yet to be determined but the SCAR/PIR121 multi-protein complex has been identified and characterized. However, in D. discoideum other components must be involved in regulating actin polymerization since cells lacking either SCAR or PIR121 retain the capacity to polymerize actin in response to chemoattractants. Finally, recent and still-uncharacterized signaling pathways emanating from Rac at the front and RhoA at the back of neutrophils are proposed to serve as mutual inhibitory signals aimed at reinforcing cellular polarity.

Current Opinion in Cell Biology 2004, 16:4–13

www.sciencedirect.com

Making all the right moves: chemotaxis in neutrophils and Dictyostelium Parent 7

sensitive to chemoattractants, and show enhanced PHdomain translocation in response to chemoattractants. It therefore appears that PH-domain-containing proteins are actively being sequestered in the cytoplasm by IP7 via a chemoattractant-dependent process and that this has an impact on the chemotactic response. Although the tumor suppressor PTEN displays protein tyrosine phosphatase activity in vitro, its major in vivo activity is to dephosphorylate the 30 phosphate on PtdIns(3,4)P2/PtdIns(3,4,5)P3. Several inactivating mutations of PTEN have been identified in a wide variety of cancers and the phosphatase has been demonstrated to be involved in a variety of cellular functions, including migration [25]. While PTEN-null alleles are lethal in mice, PTEN-deficient mammalian cell lines exhibit elevated levels of PtdIns(3,4,5)P3 and increased activity of the actin cytoskeleton, and display more invasive behaviors. The role of PTEN in chemotaxis recently emerged from work performed in D. discoideum. Cells in which the PTEN gene has been disrupted display a broader leading edge with increased actin polymerization responses, extend pseudopods more rapidly and randomly, and do not show persistent and directional movement in gradients of chemoattractants [26]. It therefore appears that the loss of PTEN results in increased activity of the actin cytoskeleton and that, in chemotactic cells, this leads to the inability to properly sense and respond to external gradients. Indeed, upon uniform chemoattractant stimulation, PH-domain translocation is dramatically prolonged in D. discoideum pten cells, remaining at the plasma membrane several minutes after chemoattractant addition. Interestingly, this defect does not perfectly correlate with changes in PtdIns(3,4,5)P3 levels. In response to uniform increases of chemoattractant, wild-type cells show a PtdIns(3,4,5)P3 increase that peaks at 5 s, and returns to basal levels by 40–60 s. While pten cells do show increased basal PtdIns(3,4,5)P3 levels, the response still returns close to basal levels by 60 s [27]. The discrepancy between the kinetics of PH-domain translocation and PtdIns(3,4,5)P3 levels could be explained by 50 -phosphoinositide phosphatase (SHIP) activity, which converts PtdIns(3,4,5)P3 into PtdIns(3,4)P2. As CRAC and Akt bind both PtdIns(3,4)P2 and PtdIns(3,4,5)P3, SHIP activity could give rise to the sustained PH-domain membrane association observed in pten cells. In D. discoideum, a bona fide SHIP has yet to be identified. In neutrophils, biochemical analyses have suggested that 50 -phosphatase activity is mainly responsible for the metabolism of PtdIns(3,4,5)P3 [28]. Mammalian genomes carry two isoforms of SHIP and hematopoietic cells derived from mice lacking SHIP-1 show enhanced chemotaxis and actin polymerization to SDF-1 [29]. The cellular distribution of PTEN and PI3K provided further insight into how cells can spatially regulate the www.sciencedirect.com

localization of signaling components involved in the establishment and maintenance of polarity. Studies performed in D. discoideum showed that the cellular distribution of PTEN and the Class I PI3K1 and PI3K2 is highly dynamic. Like PH-domain-containing proteins, PI3K1– GFP and PI3K2–CFP are cytosolic and specifically recruited to the leading edge of D. discoideum cells undergoing chemotaxis. Conversely, a fraction of PTEN– GFP is bound to the plasma membrane in resting cells. In a chemoattractant gradient, PTEN–GFP selectively dissociates from the front and accumulates at the back and sides of cells [26,30]. This exclusive cellular distribution leads to sustained levels of PtdIns(3,4)P2/ PtdIns(3,4,5)P3 at the leading edge and to the anterior recruitment of PH-domain-containing proteins. Although the cellular distribution of PI3Kg has yet to be established in neutrophils, it was recently shown that PTEN is also distributed at the opposite end of F-actin in neutrophils undergoing chemotaxis [31]. A glimpse of the mechanism(s) involved in the recruitment of PTEN to the membrane has surfaced in recent studies. It appears that PtdIns(4,5)P2 is involved: D. discoideum PTEN mutants lacking the putative PtdIns(4,5)P2-binding motif do not bind the plasma membrane or rescue pten cells [26]. Moreover, using purified mammalian PTEN, Campbell et al. recently showed that PtdIns(4,5)P2 acts as an allosteric activator of PTEN [32]. The mechanisms that direct the cellular distribution of PI3K remain elusive. PtdIns(3,4,5)P3 does not appear to be involved, as treatment of D. discoideum with the PI3K inhibitor LY29 4002 does not alter PI3K1 or PI3K2 recruitment to the plasma membrane [30]. Moreover, although it is essential for activation, the Ras-binding domain of D. discoideum PI3K is also not required. Similarly, Ras activation does not result in the translocation of PI3Kg to neutrophil membranes [33]. In D. discoideum, the nonconserved N-terminal domain of PI3K has been shown to be necessary and sufficient for translocation, although the mechanism by which this occurs has not been established [30]. It has been shown that Gbg subunits directly activate mammalian PI3Kg by a mechanism that does not appear to involve translocation to the plasma membrane [34,35]. This is not surprising as Gbg subunits are uniformly distributed and activated during chemotaxis (see above). Clearly, other mechanisms must be involved.

How do PH-domain-containing proteins target actin polymerization? It is well established that Rho-family GTPases are essential regulators of cell polarity and motility [36]. They control the cytoskeleton by relaying signals to the Arp2/3 complex, which is composed of seven proteins that contribute to nucleate actin polymerization from existing filaments at the leading edge. The WASP/SCAR (Wiskott–Aldrich syndrome protein/suppressor of cAR) family of proteins bind the Arp2/3 complex and are Current Opinion in Cell Biology 2004, 16:4–13

8 Cell structure and dynamics

thought to represent the major signaling link between RhoGTPases and Arp2/3 [37]. Rac and Cdc42 have also been shown to control actin dynamics by activating p21activated kinases (PAK), which co-localizes with F-actin at the leading edge of neutrophils [38]. As with other GTPases, RhoGTPases exist in two forms: a GDP-bound inactive state or a GTP-bound active state. A variety of activators, known as GEFs (guanine nucleotide exchange factors), and inhibitors, known as GAPs (GTPase-activating proteins) and GDIs (guanine exchange inhibitors), tightly control their activation state. Chemoattractantmediated GDP/GTP exchange for both Rac and Cdc42 has been shown to be PI3K-dependent in neutrophils [39]. Moreover, activated Rac localizes to the leading edge of neutrophils [40]. As RhoGEFs harbor PH domains, it has been postulated that the spatial activation of RhoGTPases occurs via the recruitment and/or activation of GEFs by PtdIns(3,4)P2/PtdIns(3,4,5)P3 at the leading edge of cells undergoing chemotaxis (Figure 2). Although a handful of mammalian RhoGEFs have been shown to be activated by PtdIns(3,4,5)P3, the molecular mechanisms by which PtdIns(3,4)P2/PtdIns(3,4,5)P3 targets or activates GEFs are still emerging [41].

Another GEF, PIXa (PAK-interacting exchange factor), has also been implicated as a regulator of RhoGTPase function in neutrophils [31]. The activation of PIXa, which shows specificity for Cdc42, involves Gbg subunits as well as PAK1. Li et al. show that PIXa and PAK1 form a complex that is recruited to the plasma membrane by binding Gbg subunits [31]. Once at the membrane, PIXa activates Cdc42, which then stimulates PAK1 activity. Although PI3Kg deficiency does not affect Cdc42 or PAK1 activation, it does lead to their mislocalization. This does not necessarily mean that PtdIns(3,4)P2/PtdIns(3,4,5)P3 are not required for PIXa activation, as Yoshii et al. previously showed that PtdIns(3,4)P2/PtdIns(3,4,5)P3 directly activate PIXa, albeit weakly [45]. Perhaps the basal PI3K activity in neutrophils lacking PI3Kg is enough to stimulate PIXa. Intriguingly, Li and collaborators also found that PIXa deficiency leads to the mislocalization of PTEN: in these cells, in contrast to wild-type cells, it co-localizes with F-actin. It therefore appears that the Gbg/PAK1/ PIXa/Cdc42 complex is an important component in the proper co-ordination of PI3K/PTEN signals during chemotaxis.

The identification of a novel Rac-GEF from neutrophils, P-Rex (PtdIns(3,4,5)P3-dependent Rac exchanger), could represent the link between G-protein-coupled receptors, PI3K and Rac [42]. P-Rex was purified as a PtdIns(3,4,5)P3-sensitive activator of Rac and represents 65% of the total Rac-GEF activity measured in neutrophils. Most remarkably, P-Rex, which is partly associated with the plasma membrane in non-stimulated cells, is directly and synergistically activated by PtdIns(3,4,5)P3 and Gbg. PtdIns(3,4,5)P3 is proposed to act as an allosteric regulator of P-Rex, stimulating GEF activity by causing a conformational change or reorientation at the plasma membrane, perhaps analogously to the effect of PtdIns(3,4,5)P3 on Vav1 or Sos1 [43]. Although the role of P-Rex in chemotaxis has yet to be determined, it has been shown that PDGF (platelet-derived growth factor) stimulation of cells expressing P-Rex induces lamellipodia reminiscent of those observed in cells expressing constitutively activated Rac.

The components that link PtdIns(3,4)P2/PtdIns(3,4,5)P3 and actin polymerization in D. discoideum have yet to be identified. Fifteen Rac homologues and several RacGEFs have been identified in D. discoideum and deletion as well as overexpression analyses of a subset of these established their role in regulating various cytoskeletalmediated responses, including cell polarity and chemotaxis [46]. However, intense database searches have yet to uncover Rho or Cdc42 homologues. This is intriguing because D. discoideum and neutrophils show a high degree of conservation. It nevertheless suggests that Cdc42 and Rho activities can be replaced by other components. D. discoideum cells express VASP (vasodilator-stimulated protein), SCAR and WASP homologues [47,48]. As expected, cells lacking either VASP or SCAR display chemotaxis defects, but the signaling components that link chemoattractant receptor signals to their activation remain to be determined. It was recently shown that D. discoideum cells express a group of proteins that form a complex with SCAR in mammalian cell extracts [49,50]. Deletion of one of these, PIR121, gives rise to cells exhibiting migration and chemotaxis defects with increased pseudopod extensions and F-actin levels. It is proposed that PIR121 (possibly along with the other members of the complex) maintains SCAR in an inactive state and that addition of chemoattractants dissociates the complex, thereby releasing active SCAR to activate Arp2/ 3 (Figure 2). The spatial control of this cascade presumably occurs via RhoGTPases as the dissociation of the PIR121–SCAR complex is proposed to depend on Rac activation. Regardless, the pathways leading to actin polymerization in D. discoideum are more complex: Blagg et al. found that chemoattractant addition still gives rise to

The role of the Rac-GEF Vav1 in the control of neutrophil chemotaxis was recently investigated [44]. Although neutrophils isolated from mice lacking Vav1 did show reduced levels of actin polymerization and displayed weaker chemotaxis, these defects were specific for the chemoattractant fMLP; IL8 and LTB4 responses appeared unaltered. In addition, no detectable difference in fMLP-induced Rac1-GTP or Rac2-GTP formation in neutrophils isolated from mice lacking Vav1 was observed. These results suggest that Vav1 is responsible for a small proportion of the total Rac-GEF activity in neutrophils and that it is perhaps specific to a subset of chemoattractants. Current Opinion in Cell Biology 2004, 16:4–13

www.sciencedirect.com

Making all the right moves: chemotaxis in neutrophils and Dictyostelium Parent 9

increases in the proportion of polymerized F-actin in both pir121 and scar cells [50].

Signaling to the back The spatial cues that organize signaling cascades at the backs of cells are not well defined. Nevertheless, events that lead to the myosin-based contraction and retraction of the cell posterior are beginning to surface. Interestingly, neutrophils and D. discoideum appear to use different pathways to target myosin assembly. RhoA is important in monocyte and neutrophil tail retraction [51,52]. Remarkably, it was recently shown that, in contrast to other chemokine signaling events, RhoA activation is mostly insensitive to pertussis toxin (PTX), which specifically modifies and inactivates Gai [53]. PTXtreated neutrophils are apolar and lose most chemoattractant-mediated signaling responses, including activation of PI3K, Rac and F-actin polymerization. Xu et al. observed that, rather than being non-responsive, PTX-treated neutrophils behave as if they had ‘back’ around their entire periphery. When exposed to a chemoattractant gradient, PTX-treated cells form a uropod-like structure up the gradient [53]. These effects appear to be mediated via ROCK and myosin II function: inhibitors of either component caused cells to lose polarity and display multiple pseudopods when exposed to a chemoattractant gradient. In other words, these cells displayed ‘front’ around their entire periphery. Moreover, RhoA as well as the myosin heavy and light chains localize to the back of neutrophils (Figure 2) [53]. These results are in perfect agreement with earlier findings in D. discoideum, in which cells lacking myosin II or components that regulate its assembly are unable to suppress lateral pseudopods and display chemotaxis defects [54,55]. Interestingly, Xu et al. propose that Ga12 and Ga13, which have been shown to directly activate RhoGEFs, could be responsible for mediating RhoA activation at the back of cells [53]. Whereas expression of constitutively active Ga13 leads to ‘backness’ phenotypes, dominant-negative versions of Ga12 or Ga13 give rise to ‘frontness’ phenotypes similar to those observed with inhibitors of ROCK and myosin II. It therefore appears that in neutrophils chemoattractant receptors are coupled to two different heterotrimeric G proteins, Gi and G12/13, to control events occurring at the front and at the back, respectively. How the receptor–Gprotein coupling is spatially controlled remains to be established. As discussed, in D. discoideum Rho homologues have not been identified and these cells use alternative signaling pathways to regulate myosin II function. The link between myosin II phosphorylation and cGMP originally came from studies of a mutant, StmF, which shows elevated cGMP levels and a significant delay in the phosphorylation of myosin II in response to chemoattractant stimulation [56]. Since then, the effectors of cGMP and the molecular components involved in its metabolism www.sciencedirect.com

have been identified and genetic analyses have been possible. The results generally agree with a model in which elevated cGMP levels give rise to increased myosin II phosphorylation and cytoskeleton association as well as improved chemotaxis. Reduced cGMP levels produce the opposite effects, although some myosin II phosphorylation and cytoskeleton association persists in cells lacking guanylyl cyclase activity. No cGMP protein kinase (PKG)-like activity has been measured in D. discoideum and the effects of cGMP are proposed to be primarily mediated by two cGMP binding proteins (Gbp) [57,58]. In addition to their nucleotide binding domain, Gbps harbor RasGEF, Ras and MAPKKK domains and could represent functional homologues of the mammalian CNRasGEF [59]. Although the mechanism by which Gbps control myosin II function has yet to be determined, it has been proposed to involve the regulation of myosin light- and heavy-chain kinases. The role of cGMP in neutrophil function has not been extensively investigated. However, studies of the effector functions of nitric oxide (an activator of soluble guanylyl cyclase) have suggested a link between cGMP and neutrophil chemotaxis [60,61]. Another component involved in the regulation of myosin II function is the D. discoideum PAKa. Cells lacking PAKa show decreased levels of myosin II phosphorylation and assembly, do not show the characteristic myosin II enrichment at their backs, and display phenotypes that are similar to cells lacking myosin II [62]. In contrast to the mammalian PAK1, PAKa is highly enriched at the back of D. discoideum cells undergoing chemotaxis (Figure 2). Although the mechanisms by which PAKa regulates myosin II function have not been defined, it is known that PAKa does not phosphorylate myosin II. It is proposed to control myosin II assembly by regulating myosin heavy/light-chain kinases.

Feedback control The remarkable ability of cells undergoing chemotaxis to sense and respond to shallow attractant gradients is achieved by the elaborate spatial control of signaling cascades. The segregation of these responses allows cells to acquire a highly polarized shape, with an active anterior and a retracting posterior. Most impressively, the chemotactic responses are very dynamic and cells rapidly adjust their direction in response to changing gradients. It has been suggested that feedback loops are involved, not only to amplify local inputs but also to send long-range inhibitory signals, thereby restricting front and back domains. Recent work in neutrophils has begun to outline the details of such signals. First, it has been shown that the exogenous addition of membrane-permeable homologues of PtdIns(3,4,5)P3 to neutrophils bypasses the need for chemoattractants and induces polarity and motility as well as localized translocation of PH-domaincontaining proteins. Interestingly, agents that inhibit Current Opinion in Cell Biology 2004, 16:4–13

10 Cell structure and dynamics

PI3K or RhoGTPases significantly abrogate the activating capacities of the membrane-permeable homologues of PtdIns(3,4,5)P3 [63,64]. These results suggest that exogenously added PtdIns(3,4,5)P3 is not sufficient to give rise to robust polarity and that de novo PtdIns(3,4,5)P3 synthesis, possibly via a Rac-dependent activation of PI3K, is important to establish a steep PtdIns(3,4,5)P3 gradient and cellular polarity (Figure 2). Second, on the basis of work using PH-domain translocation and actin polymerization as read-outs, it has been proposed that anterior and posterior signaling pathways somehow exert signals that antagonize each other [40,53]. While the expression of dominant-negative Rac or constitutively active RhoA in neutrophil cell lines inhibits PH-domain translocation and actin polymerization, expression of constitutively active Rac or dominant-negative RhoA has the opposite effect. These inhibitory signals could therefore amplify the spatial segregation of the anterior/posterior signals and reinforce cellular polarity (Figure 2).

Figure 3

CRAC-GFP

ACA-YFP

The complexity of the signaling cross talk is also apparent from work in D. discoideum, where the PI3K pathway controls effectors located at the back of cells. The first example of this involves PAKa, which localizes to the back of cells undergoing chemotaxis (Figure 3). PAKa is phosphorylated by the anteriorly localized Akt on a conserved Akt-phosphorylation consensus sequence, and mutation of this site abolishes chemoattractant-mediated PAKa phosphorylation [54]. Moreover, cells lacking either PI3K or Akt do not phosphorylate or activate PAKa and display abnormal PAKa cellular distribution. The second example involves the regulation of the adenylyl cyclase ACA, the enzyme responsible for the synthesis of cAMP in early aggregation. In D. discoideum, cAMP acts as a chemoattractant, allowing cells to migrate by chemotaxis into aggregates that later differentiate into multicellular structures. ACA activation requires the cytosolic regulator CRAC; cells lacking CRAC do not activate ACA in response to chemoattractant addition [65]. CRAC was the first PH-domain-containing protein shown to translocate to the leading edge of cells undergoing chemotaxis [10]. Subsequent studies established that ACA is highly enriched at the backs of cells, where it is proposed to provide a compartment from which cAMP is secreted to locally attract neighboring cells (Figure 3) [66]. The cells then orient themselves in a head-to-tail fashion and form streams (an hallmark behavior of D. discoideum). For both PAKa and ACA, therefore, signals originating from the front of the cell, via Akt and CRAC, regulate effectors that are enriched at the back (Figure 2). These ‘signal relays’ between the cell anterior and posterior add yet another layer of regulation that allows D. discoideum, and presumably neutrophils, to transduce and integrate information obtained from chemoattractant gradients into persistent and directed migration.

Conclusions

Akt-GFP

PAKa-GFP

Current Opinion in Cell Biology

Fluorescent images depicting the cellular distribution of CRAC, ACA, Akt, or PAKa in D. discoideum cells undergoing chemotaxis. Differentiated cells expressing GFP or YFP fusions of the signaling molecules were placed in a cAMP gradient and allowed to undergo chemotaxis. The arrow indicates the direction of migration. Images depicting the PH domain of Akt fused to GFP and the N-terminal domain of PAKa fused to GFP were kindly provided by R A Firtel. Current Opinion in Cell Biology 2004, 16:4–13

The chemotactic signals that transduce shallow extracellular attractant gradients into polarized responses and directed migration show a high degree of complexity and coordination. The accumulation of PtdIns(3,4)P2/ PtdIns(3,4,5)P3 at the leading edge of cells undergoing chemotaxis and the subsequent translocation of PHdomain-containing proteins appear to represent the initial amplification events during chemotaxis. Evidence now suggests that this signaling polarity is reinforced by feedback loops involving components that regulate the actin cytoskeleton. Moreover, close interplay between the front and the back of cells is now emerging as a key factor in the control of directed cell migration. Notwithstanding this significant progress, critical links in the signaling pathways are still missing. First, the upstream signals that direct the reciprocal cellular distribution of PI3K and PTEN are unknown and their identification is critical to understanding gradient sensing. Second, the precise roles of the various Ga subunits and the exact mechanisms that govern how they segregate ‘front’ and www.sciencedirect.com

Making all the right moves: chemotaxis in neutrophils and Dictyostelium Parent 11

‘back’ signals remain to be determined and their unveiling will provide more insight into the signaling cross-talk involved in deciphering chemotactic signals. Third, although components that connect localized PtdIns(3,4)P2/PtdIns(3,4,5)P3 to cytoskeletal rearrangements are emerging, a clear path is still missing. Finally, the various feedback loops that act both locally and distally are mainly undefined and their clarification will certainly open new avenues. Indeed, inhibitory/adaptive signals represent essential components of the signaling machinery that cells use to detect chemoattractant gradients and migrate directionally [67]. The future will undoubtedly provide an even broader view of how signals are integrated to amplify responses both at the front and at the back of cells undergoing chemotaxis.

Acknowledgements I would like to thank Frank Comer, Paul Kriebel, Dana Mahadeo as well as Joe Brzostowski, Alan Kimmel, Robert Insall, and Pierre Coulombe for insightful discussions and for their very helpful comments on the manuscript. Frank Comer and Dana Mahadeo provided Figures 1 and 3, respectively.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Parent CA, Devreotes PN: A cell’s sense of direction. Science 1999, 284:765-770.

2.

Weiner OD: Regulation of cell polarity during eukaryotic chemotaxis: the chemotactic compass. Curr Opin Cell Biol 2002, 14:196-202.

3. Kimmel AR, Parent CA: The signal to move: D. discoideum go  orienteering. Science 2003, 300:1525-1527. This review accompanies the D. discoideum cAMP signaling pathway described in Science’s online STKE site. On this site, the D. discoideum chemoattractant-mediated pathways are described in great detail, with specific comments and a complete set of references for every entry. A must if you need a more in-depth analysis. 4.

Laudanna C, Kim JY, Constantin G, Butcher E: Rapid leukocyte integrin activation by chemokines. Immunol Rev 2002, 186:37-46.

5.

DeMali KA, Wennerberg K, Burridge K: Integrin signaling to the actin cytoskeleton. Curr Opin Cell Biol 2003, 15:572-582.

6.

Servant G, Weiner OD, Neptune ER, Sedat JW, Bourne HR: Dynamics of a chemoattractant receptor in living neutrophils during chemotaxis. Mol Biol Cell 1999, 10:1163-1178.

7.

Xiao Z, Zhang N, Murphy DB, Devreotes PN: Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation. J Cell Biol 1997, 139:365-374.

8.

9.

Jin T, Zhang N, Long Y, Parent CA, Devreotes PN: Localization of the G protein bc complex in living cells during chemotaxis. Science 2000, 287:1034-1036. Janetopoulos C, Jin T, Devreotes PN: Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science 2001, 291:2408-2411.

10. Parent CA, Blacklock BJ, Froehlich WM, Murphy DB, Devreotes PN: G protein signaling events are activated at the leading edge of chemotactic cells. Cell 1998, 95:81-91. 11. Meili R, Ellsworth C, Lee S, Reddy TB, Firtel RA: Chemoattractantmediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J 1999, 18:2092-2105. www.sciencedirect.com

12. Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR: Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 2000, 287:1037-1040. 13. Funamoto S, Milan K, Meili R, Firtel RA: Role of phosphatidylinositol 30 kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in Dictyostelium. J Cell Biol 2001, 153:795-810. 14. Iijima M, Huang YE, Devreotes P: Temporal and spatial regulation of chemotaxis. Dev Cell 2002, 3:469-478. 15. Stephens L, Ellson C, Hawkins P: Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Curr Opin Cell Biol 2002, 14:203-213. 16. Chung CY, Funamoto S, Firtel RA: Signaling pathways controlling cell polarity and chemotaxis. Trends Biochem Sci 2001, 26:557-566. 17. Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, Sozzani S, Mantovani A, Altruda F, Wymann MP: Central role for G-protein-coupled phosphoinositide 3-kinase c in inflammation. Science 2000, 287:1049-1053. 18. Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D: Roles of PLC-b2 and -b3 and PI3Kc in chemoattractant-mediated signal transduction. Science 2000, 287:1046-1049. 19. Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzki I et al.: Function of PI3Kc in thymocyte development, T cell activation, and neutrophil migration. Science 2000, 287:1040-1046. 20. Sadhu C, Masinovsky B, Dick K, Sowell CG, Staunton DE:  Essential role of phosphoinositide 3-kinase d in neutrophil directional movement. J Immunol 2003, 170:2647-2654. These authors have developed an isoform-specific PI3Kd inhibitor that inhibits neutrophil chemotaxis, but not chemokinesis, to fMLP. In addition, fMLP-mediated actin polymerization remains unchanged in the presence of the inhibitor. 21. Ptasznik A, Traynor-Kaplan A, Bokoch GM: G-protein-coupled chemoattractant receptors regulate Lyn tyrosine kinase/Shc adapter protein signaling complexes. J Biol Chem 1995, 270:19969-19973. 22. Stephens L, Eguinoa A, Corey S, Jackson T, Hawkins PT: Receptor-stimulated accumulation of phosphatidylinositol (3,4,5)-trisphosphate by G-protein-mediated pathways in human myeloid-derived cells. EMBO J 1993, 12:2265-2273. 23. Ma YC, Huang J, Ali S, Lowry W, Huang XY: Src tyrosine kinase is a novel direct effector of G proteins. Cell 2000, 102:635-646. 24. Luo HR, Huang YE, Chen JC, Saiardi A, Iijima M, Ye K, Huang Y,  Nagata E, Devreotes P, Snyder SH: Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions. Cell 2003, 114:559-572. This paper provides an interesting new twist to the control of D. discoideum chemotaxis. The authors propose that the inositol pyrophophate IP7 competes for PH-domain binding with PtdIns(3,4,5)P3, therefore providing an added level of regulation during chemotaxis. 25. Sulis ML, Parsons R: PTEN: from pathology to biology. Trends Cell Biol 2003, 13:478-483. 26. Iijima M, Devreotes P: Tumor suppressor PTEN mediates  sensing of chemoattractant gradients. Cell 2002, 109:599-610. See annotation to [30]. 27. Huang YE, Iijima M, Parent CA, Funamoto S, Firtel RA, Devreotes P:  Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells. Mol Biol Cell 2003, 14:1913-1922. This paper provides a thorough biochemical analysis of chemoattractantmediated PtdIns(3,4,5)P3 levels in wild-type D. discoideum cells as well as in various signaling mutants. 28. Stephens LR, Hughes KT, Irvine RF: Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature 1991, 351:33-39. 29. Kim CH, Hangoc G, Cooper S, Helgason CD, Yew S, Humphries RK, Krystal G, Broxmeyer HE: Altered responsiveness Current Opinion in Cell Biology 2004, 16:4–13

12 Cell structure and dynamics

to chemokines due to targeted disruption of SHIP. J Clin Invest 1999, 104:1751-1759.

units in vivo and in vitro. It could therefore act as a coincidence detector for chemoattractant signaling.

30. Funamoto S, Meili R, Lee S, Parry L, Firtel RA: Spatial and  temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 2002, 109:611-623. This paper and [26] are two outstanding papers that extensively characterize the role and cellular distribution of PI3K and PTEN in D. discoideum cells undergoing chemotaxis.

43. Das B, Shu X, Day GJ, Han J, Krishna UM, Falck JR, Broek D: Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding. J Biol Chem 2000, 275:15074-15081.

31. Li Z, Hannigan M, Mo Z, Liu B, Lu W, Wu Y, Smrcka AV, Wu G,  Li L, Liu M et al.: Directional sensing requires G bc-mediated PAK1 and PIXa-dependent activation of Cdc42. Cell 2003, 114:215-227. This remarkable paper describes the existence of a complex between Gnbg/PIXa/Cdc42/PAK1 that is important for the proper polarized localization of F-actin and PTEN and for chemotaxis in neutrophils. 32. Campbell RB, Liu F, Ross AH: Allosteric activation of PTEN  phosphatase by phosphatidylinositol 4,5-bisphosphate. J Biol Chem 2003, 278:33617-33620. Using purified mammalian PTEN, this paper presents data suggesting that the binding of monomeric PtdIns(4,5)P2 to PTEN induces a conformational change that leads to the activation of PTEN. 33. Suire S, Hawkins P, Stephens L: Activation of phosphoinositide  3-kinase c by Ras. Curr Biol 2002, 12:1068-1075. This paper shows that Ras can potently activate mammalian PI3Kg. This occurs in the absence of any detectable PI3Kg translocation to membranes. 34. Brock C, Schaefer M, Reusch HP, Czupalla C, Michalke M,  Spicher K, Schultz G, Nurnberg B: Roles of G bc in membrane recruitment and activation of p110c/p101 phosphoinositide 3-kinase c. J Cell Biol 2003, 160:89-99. HEK cells expressing various combinations of the regulatory (p101) and catalytic (p110g) subunits of PI3Kg provide evidence that Gbg subunits activate PI3Kg by two mechanisms: via membrane recruitment mediated by p101 and by direct stimulation of p110g. The requirement for p101 may not be essential for PI3Kg function as monomeric p110g could also be active in vivo. 35. Krugmann S, Cooper MA, Williams DH, Hawkins PT, Stephens LR:  Mechanism of the regulation of type IB phosphoinositide 3OHkinase by G-protein bc subunits. Biochem J 2002, 362:725-731. Purified mammalian PI3Kg regulatory and catalytic subunits can associate with lipid vesicles in the absence or presence of Gbg. Moreover, membrane-targeted PI3Kg catalytic subunit can be super-activated by Gbg subunits. 36. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature 2002, 420:629-635. 37. Pollard TD, Borisy GG: Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003, 112:453-465. 38. Bokoch GM: Biology of the p21-activated kinases. Annu Rev Biochem 2003, 72:743-781. 39. Benard V, Bohl BP, Bokoch GM: Characterization of Rac and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 1999, 274:13198-13204.

44. Kim C, Marchal CC, Penninger J, Dinauer MC: The hemopoietic  Rho/Rac guanine nucleotide exchange factor Vav1 regulates N-formyl-methionyl-leucyl-phenylalanine-activated neutrophil functions. J Immunol 2003, 171:4425-4430. Using neutrophils derived from mice lacking Vav1, the authors show that this exchange factor is specifically involved in controlling fMLP-mediated superoxide production and F-actin generation. They also report weak defects in chemotaxis to fMLP. 45. Yoshii S, Tanaka M, Otsuki Y, Wang DY, Guo RJ, Zhu Y, Takeda R, Hanai H, Kaneko E, Sugimura H: aPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase. Oncogene 1999, 18:5680-5690. 46. Wilkins A, Insall RH: Small GTPases in Dictyostelium: lessons from a social amoeba. Trends Genet 2001, 17:41-48. 47. Han YH, Chung CY, Wessels D, Stephens S, Titus MA, Soll DR,  Firtel RA: Requirement of a vasodilator-stimulated phosphoprotein family member for cell adhesion, the formation of filopodia, and chemotaxis in Dictyostelium. J Biol Chem 2002, 277:49877-49887. The authors characterize the function of VASP in D. discoideum cells and find that cells lacking VASP have chemotaxis defects that are probably due to their defects in adhesion to the substratum. 48. Bear JE, Rawls JF, Saxe CL III: SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. J Cell Biol 1998, 142:1325-1335. 49. Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW:  Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 2002, 418:790-793. The authors have purified a SCAR/WAVE-containing complex from bovine brain composed of PIR121, Nap125, WAVE1 and HSPC300. They show that Rac-GTP or the adaptor Nck can activate WAVE1 activity. Interestingly, they find that while recombinant WAVE1 is active, the WAVE1 complex is inactive. They propose that Rac-GTP or Nck activates WAVE1 by complex dissociation. 50. Blagg SL, Stewart M, Sambles C, Insall RH: PIR121 regulates  pseudopod dynamics and SCAR activity in Dictyostelium. Curr Biol 2003, 13:1480-1487. The authors identify the four members of the SCAR complex described in  [49 ] in D. discoideum and generate cells lacking PIR121. These cells show excessive levels of polymerized actin and display severe motility and chemotaxis defects. Genetic analyses show that PIR121 is acting downstream of SCAR. 51. Alblas J, Ulfman L, Hordijk P, Koenderman L: Activation of RhoA and ROCK are essential for detachment of migrating leukocytes. Mol Biol Cell 2001, 12:2137-2145. 52. Worthylake RA, Lemoine S, Watson JM, Burridge K: RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 2001, 154:147-160.

40. Srinivasan S, Wang F, Glavas S, Ott A, Hofmann F, Aktories K,  Kalman D, Bourne HR: Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J Cell Biol 2003, 160:375-385. By expressing constitutively active and dominant-negative versions of Rac and Cdc42 in a neutrophil cell line, the authors propose that Rac represents a key regulator in the formation of the leading edge. Conversely, Cdc42 would be involved in locally directing and stabilizing the leading edge.

53. Xu J, Wang F, Van Keymeulen A, Herzmark P, Straight A, Kelly K,  Takuwa Y, Sugimoto N, Mitchison T, Bourne HR: Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 2003, 114:201-214. Using inhibitors as well as expression of constitutively active and dominant-negative versions of various G proteins and GTPases in a neutrophil cell line, the authors present data suggesting that chemoattractants segregate signals by triggering Gai/Rac-dependent events at the front of cells and Ga12-13/RhoA-dependent events at the back.

41. Welch HC, Coadwell WJ, Stephens LR, Hawkins PT: Phosphoinositide-3-kinase-dependent activation of Rac. FEBS Lett 2003, 546:93-97.

54. Chung CY, Potikyan G, Firtel RA: Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. Mol Cell 2001, 7:937-947.

42. Welch HC, Coadwell WJ, Ellson CD, Ferguson GJ, Andrews SR,  Erdjument-Bromage H, Tempst P, Hawkins PT, Stephens LR: PRex1, a PtdIns(3,4,5)P3- and Gbc-regulated guanine-nucleotide exchange factor for Rac. Cell 2002, 108:809-821. This remarkable paper describes the purification and characterization of a novel Rac-GEF, called P-Rex, from neutrophil cytosol. The authors show that P-Rex is synergistically activated by PtdIns(3,4,5)P3 and Gbg sub-

55. Wessels D, Soll DR, Knecht D, Loomis WF, De Lozanne A, Spudich J: Cell motility and chemotaxis in Dictyostelium amoebae lacking myosin heavy chain. Dev Biol 1988, 128:164-177.

Current Opinion in Cell Biology 2004, 16:4–13

56. Newell PC, Liu G: Streamer F mutants and chemotaxis of Dictyostelium. Bioessays 1992, 14:473-479. www.sciencedirect.com

Making all the right moves: chemotaxis in neutrophils and Dictyostelium Parent 13

57. Roelofs J, Smith JL, Van Haastert PJ: cGMP signalling: different ways to create a pathway. Trends Genet 2003, 19:132-134. 58. Bosgraaf L, Van Haastert PJ: A model for cGMP signal transduction in Dictyostelium in perspective of 25 years of cGMP research. J Muscle Res Cell Motil 2002, 23:781-791. 59. Pham N, Cheglakov I, Koch CA, de Hoog CL, Moran MF, Rotin D: The guanine nucleotide exchange factor CNrasGEF activates Ras in response to cAMP and cGMP. Curr Biol 2000, 10:555-558. 60. Shibata K, Warbington ML, Gordon BJ, Kurihara H, Van Dyke TE: Nitric oxide synthase activity in neutrophils from patients with localized aggressive periodontitis. J Periodontol 2001, 72:1052-1058.

64. Weiner OD, Neilsen PO, Prestwich GD, Kirschner MW, Cantley LC,  Bourne HR: A PtdInsP(3)- and Rho-GTPase-mediated positive feedback loop regulates neutrophil polarity. Nat Cell Biol 2002, 4:509-513. With the paper by Niggli [63], this paper shows that the exogenous addition of PtdIns(3,4,5)P3 homologues to neutrophils induces polarity and motility; the authors propose that positive feedback loops are involved in amplifying PtdIns(3,4,5)P3 polarity. 65. Insall R, Kuspa A, Lilly PJ, Shaulsky G, Levin LR, Loomis WF, Devreotes PN: CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G-proteinmediated activation of adenylyl cyclase in Dictyostelium. J Cell Biol 1994, 126:1537-1545.

62. Chung CY, Firtel RA: PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis. J Cell Biol 1999, 147:559-576.

66. Kriebel PW, Barr VA, Parent CA: Adenylyl cyclase localization  regulates streaming during chemotaxis. Cell 2003, 112:549-560. This paper provides a molecular mechanism to explain how D. discoideum cells align in a head-to-tail fashion to form streams when they undergo chemotaxis. The authors find that the adenylyl cyclase ACA is highly enriched at the back of cells undergoing chemotaxis and propose that this provides a compartment from which the chemoattractant cAMP is secreted to locally attract surrounding cells.

63. Niggli V: A membrane-permeant ester of phosphatidylinositol 3,4,5-trisphosphate (PIP[3]) is an activator of human neutrophil migration. FEBS Lett 2000, 473:217-221.

67. Devreotes P, Janetopoulos C: Eukaryotic chemotaxis: distinctions between directional sensing and polarization. J Biol Chem 2003, 278:20445-20448.

61. Kaplan SS, Billiar T, Curran RD, Zdziarski UE, Simmons RL, Basford RE: Inhibition of chemotaxis Ng-monomethyl-Larginine: a role for cyclic GMP. Blood 1989, 74:1885-1887.

www.sciencedirect.com

Current Opinion in Cell Biology 2004, 16:4–13