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Chemotaxis: thedirected migration ofcells toward higher orlower concentrationsof chemical stimuli

cAMP: 3′,5′-cyclicadenosinemonophosphate

GPCR: G-proteincoupled receptor

PIP3:phosphatidylinositol3,4,5-trisphosphate

cGMP: 3′5′-cyclicguanosinemonophosphate

Adaptation: thetendency of responsesto subside whenreceptor occupancy isheld constant

Contents

INTRODUCTION: CHEMOTAXISOCCURS IN MANY CELLTYPES. . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

MOTILITY, DIRECTIONALSENSING, AND POLARITY . . . . . 267Motility: Pseudopod Extension

in Migrating Cells . . . . . . . . . . . . . . 268Directional Sensing: Temporal and

Spatial Sensing ofChemoattractants . . . . . . . . . . . . . . . 268

Polarity: Selective Localizationof Molecules and Reactions to theFront or Back of Cells . . . . . . . . . . . 272

A NETWORK OF SIGNALINGPATHWAYS CONTROLSCHEMOTAXIS . . . . . . . . . . . . . . . . . . . 275Receptor and G-Protein Module . . . 277RasC and RasG Modules . . . . . . . . . . . 278PIP3 and PIP2 Modules . . . . . . . . . . . . 278PKB and PKB Substrate Modules . . . 279cAMP Module . . . . . . . . . . . . . . . . . . . . . 280cGMP/Myosin II Module . . . . . . . . . . 280RasB/Rap1/MHCK Module . . . . . . . . 281Linking the Signaling Network

to the Cytoskeleton . . . . . . . . . . . . . 281

INTRODUCTION: CHEMOTAXISOCCURS IN MANY CELL TYPES

Many cells have an internal compass that al-lows them to detect extracellular chemical gra-dients and move toward or away from higherconcentrations. This process is referred to aschemotaxis or directed cell migration. Duringembryogenesis, chemotaxis is important for in-dividual and group cell migration events, or-gan formation, and wiring of the nervous sys-tem. In the adult, chemotaxis is critical forthe trafficking of immune cells and in inflam-mation, regenerative processes such as woundhealing, and maintenance of tissue architec-ture. Evidence suggests that chemotaxis al-lows stem cells to target to and persist intheir niches. See Supplemental Sidebar 1 for

examples of the importance of chemotaxis indisease (follow the Supplemental Materiallink from the Annual Reviews home page athttp://www.annualreviews.org).

Although an increasing number of cell typesthat carry out chemotaxis are being discovered,the signal transduction events mediating di-rected migration have been most thoroughlystudied in Dictyostelium, neutrophils, and a vari-ety of transformed mammalian cells (100, 105,119; see also Related Resources). The amoe-boid movements of Dictyostelium toward 3′,5′-cyclic adenosine monophosphate (cAMP) andof neutrophils toward a variety of chemokinesare based on the extension of pseudopodia.In both cell types, chemoattractants activateG-protein coupled receptors (GPCRs), result-ing in the localized accumulation of signalingmolecules, such as phosphatidylinositol 3,4,5-trisphosphate (PIP3), toward the high side ofthe gradient. PIP3 accumulation leads to pseu-dopodia extension at the leading edge of cells,which is thought to be driven by localizedRac-mediated actin polymerization. Primordialgerm cells (PGCs) also use GPCRs to sensechemoattractants, but these cells maintain uni-form PIP3 levels throughout the membrane andmigrate by extending actin-free blebs (3, 30).These blebs may be generated by myosin-basedcontraction, which is also important at the lag-ging edge for migration in Dictyostelium andneutrophils. These contractions are generallydirected by Rho, and in Dictyostelium, myosinregulation also involves 3′,5′-cyclic guanosinemonophosphate (cGMP). The combination offorces at the leading and lagging edges gener-ates rapid movement. Adaptation to persistentstimulation in Dictyostelium and neutrophils al-lows for enhanced sensitivity to differences inchemoattractant concentrations across the cell.

Stimulation of fibroblasts or breast carci-noma cells with growth factors that bind toReceptor Tyrosine Kinases (RTKs) also resultsin PIP3 accumulation and Rac-mediated actinpolymerization at the leading edge (reviewedin References 64, 100, and 119). In carcinomacells, the activation of Cofilin through its re-lease from the membrane, which is mediated by

266 Swaney · Huang · Devreotes

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chemoattractant-induced reductions in phos-phatidylinositol 4,5-bisphosphate (PIP2) levels,generates actin barbed ends and contributesto actin polymerization (114). As with amoe-boid cells, the actin-mediated events of fibrob-lasts and carcinoma cells are coordinated withmyosin-based contraction at the lagging edge,which is regulated by Rho and calcium signal-ing. Together, these result in migration that oc-curs much more slowly than that of amoeboidcells. Because there is no adaptation in fibrob-lasts, these cells respond only to absolute con-centrations of chemoattractant.

Despite slight differences in the migra-tory behaviors and specific signaling compo-nents of Dictyostelium, neutrophils, and theother cell types, the overall pathways thatregulate chemotaxis are similar. Furthermore,many of the guidance mechanisms in these celltypes are evolutionarily conserved among mostmigratory eukaryotic cells, including neurons(reviewed in References 100 and 119). For theremainder of this review, we focus primarily onchemoattractant-mediated signaling events inDictyostelium, in which genetic analysis has fa-cilitated a thorough assessment of chemotacticbehavior (Supplemental Sidebar 2).

MOTILITY, DIRECTIONALSENSING, AND POLARITY

Chemotaxis can be conceptually divided intothe processes of motility, directional sensing,and polarity (Figure 1). In the absence of astimulus, cells provided with a suitable surfacewill crawl about, a process referred to as motil-ity. Amoeboid cells, such as Dictyostelium andneutrophils, extend pseudopodia rhythmically,propelling the cell in random directions. Whenthe cells are exposed to a gradient of chemoat-tractant or chemorepellent, their motilityis biased toward or away from, respectively,higher concentrations. The molecular mech-anisms that read the gradient and provide thischemotactic bias are referred to as directionalsensing and correspond to the cells’ internalcompass described above. However, motilityand directional sensing are separable, since

Shallow gradient

Steep gradient

c

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Motility PolarityDirectional

sensing

Figure 1Chemotaxis is composed of motility, polarity, and directional sensing. In thepresence of a chemoattractant (or chemorepellent) gradient, cells move toward(or away from) higher concentrations. (a) Left: Free amoeboid cellsrhythmically extend pseudopodia and move in random directions. Middle:Spatial sensing, a means of directional sensing, can be demonstrated by thegradient-mediated relocalization of proteins in cells immobilized by actininhibitors. Right: Chemotactic cells are often polarized, with a stable leadingedge from which pseudopodia are extended. (b) In a shallow gradient, polarizedcells display biased patterns of pseudopodia extension at the leading edge thatcause cells to turn gradually toward higher concentrations of chemoattractant.(c) Sufficiently steep gradients can trigger new projections anywhere along thecell periphery.

molecules within immobilized cells can movetoward external stimuli and can dynamicallytrack changes in gradient direction. Finally,chemotactic cells often display a relatively sta-ble axis of polarity, which restricts pseudopodiaextension to the cell anterior. Polarity is alsoseparable from directional sensing, as cells inuniform chemoattractant can be polarized.Although polarized cells move with more

www.annualreviews.org • Signaling Events in Chemotaxis 267

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Directional sensing:the molecularmechanisms that readthe direction ofchemoattractantgradients and providea bias to guide themotility ofchemotactic cells

Polarity: amorphological statewith stable,functionally distinctleading and laggingedges that arecharacterized by thepreferentiallocalization of specificmolecules

persistence than unpolarized cells, they do notmove in a specific direction. Chemotaxis typi-cally incorporates motility, directional sensing,and polarity and should not be confused withany one of these processes alone.

Motility: Pseudopod Extensionin Migrating Cells

Several recent reports analyze the motile behav-ior of Dictyostelium cells through observations ofpseudopod extension in the absence or presenceof shallow cAMP gradients (1, 8). These studiesconclude that gradients modify the basal behav-ior that unstimulated cells already display. In theabsence of chemoattractant, polarized cells ex-tend pseudopodia of uniform size and durationalternately from either side of the axis of mo-tion in a behavior reminiscent of “ice skating.”Occasionally, the alternation is skipped andseveral subsequent pseudopodia are extendedfrom the same side. Chemotactic gradientscause more pseudopodia to be extended to-ward the “correct” direction. In one model, thisis achieved by more often choosing to retractpseudopodia extended in the “wrong” direc-tion (1). In another model, the probability ofextending pseudopodia toward the gradient ishigher; in addition, the angle at which pseu-dopodia are extended is altered to favor move-ment in the correct direction (8). In both mod-els, the bias causes cells to turn toward andremain facing the source of chemoattractant(Figure 1). The ice skating behavior is less ob-vious in neutrophils, in which individual pseu-dopodia are not as readily separable, and an al-ternative mechanism may exist for lamellipodextension in fibroblasts.

Analyses of cells in shallow gradients suggestthat generation of pseudopodia is autonomousand that the gradient can only bias this behav-ior; however, a strong chemotactic stimulus canalso directly elicit de novo production of a pseu-dopod (106). Whether a chemotactic stimuluscauses turning or triggers a new projection de-pends on the relative polarity of the cell ver-sus the strength of the stimulus. In a weaklypolarized cell, a chemotactic stimulus applied

anywhere around the perimeter often triggersthe formation of a new front. In a highly po-larized cell, the side and rear are less sensitivethan the front, resulting in the turning behaviordescribed above. However, even in a highly po-larized cell, a sufficiently steep gradient appliedto the side or back can break the polarity andcreate a new front (Figure 1).

Recent observations of fluorescent cy-toskeletal proteins on the basal surfaces ofmigrating neutrophils or Dictyostelium cellsshow wave-like propagation through thecytoskeleton (10, 118, 123; see also Supple-mental Movie 1 and references therein).Actin-binding proteins or Scar/WAVE com-plex components are recruited sequentiallyfrom the cytosol to adjacent points on the basalsurface, giving rise to a propagated wave. Basicwave propagation has been modeled and re-quires the mechanisms of signal relay, positivefeedback, and reversible inhibition. Waves canoriginate at random on the basal surface, al-though in polarized cells they arise more oftentoward the anterior and move outward towardthe edge. The arrival of waves at the perimetercoincides with the onset of a protrusion and mayunderlie the spontaneous generation of pseu-dopodia. The cytoskeletal events that mediateactin polymerization for the formation of cellprotrusions have been studied extensively andare beyond the scope of this review; however,little is known about how these events link toreceptor signaling (27, 50). Recent observationssuggest that chemoattractants influence wavepropagation, which may provide insight intothe mechanisms by which signaling pathwaysregulate the cytoskeleton and motility.

Directional Sensing: Temporal andSpatial Sensing of Chemoattractants

Physiological responses triggered bychemoattractants. When differentiated Dic-tyostelium cells are exposed to the chemoattrac-tant cAMP, a series of morphological changesand physiological responses is triggered(Figure 2a,b; see also Related Resources).The network of signaling pathways mediating


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these responses is discussed in detail below. InSupplemental Table 1, we list the genes im-plicated in chemotaxis or in the transduction ofchemotactic signals. Within seconds of cAMPstimulation, the heterotrimeric G-protein G2linked to the cAMP receptor cAR1 is activated,as evidenced by a rapid decrease in the fluores-cence resonance energy transfer (FRET) signalbetween the Gα2- and Gβ-subunits (54, 129).In addition, cAR1 becomes phosphorylated,the mitogen-activated protein kinase (MAPK)Erk2 is activated, and a calcium influx istriggered (11, 86). These responses persistduring continuous stimulation. However, manyother responses, listed below, are transientlyactivated upon chemoattractant stimulation.These transient responses typically display aninitial rapid activation and a secondary, delayedpeak. For example, multiple Ras proteins withsimilar kinetics are activated (57, 61, 99). Theadenylyl cyclase ACA and both soluble andmembrane-bound guanylyl cyclases (sGC andGCA, respectively) are activated, resultingin cAMP and cGMP production (93, 98).Phosphoinositide 3-kinases (PI3Ks) are acti-vated, resulting in PIP3 production (34, 42).Many proteins are phosphorylated, includingthe Gα2-subunit, a series of protein kinase B(PKB) substrates, and Myosin II heavy and lightchains (36, 62, 104, 131). Actin is polymerizedand actin-binding proteins are recruited to thecortex (25, 59, 101). The phosphoinositide3-phosphatase PTEN (phosphatase and Tensinhomolog on chromosome ten) dissociates fromthe membrane, and Myosin II is lost from thecortex (45; C. Janetopoulos & P. Devreotes,unpublished observations). In growing undif-ferentiated cells, folic acid and pterines triggermany of these same responses (37).

The tendency of cellular responses tosubside during constant receptor occupancy isknown as adaptation. The responses triggeredby cAMP can be broadly divided into twogroups on the basis of whether or not they adaptto continuous stimulation (Figure 2b). Thepersistent responses, including receptor phos-phorylation, G-protein and Erk2 activation,

cAR: cAMP receptor

FRET: fluorescenceresonance energytransfer

PTEN: phosphataseand Tensin homologon chromosome ten

Spatial versustemporal sensing:the ability of cells todetect differences inreceptor occupancyacross the cell lengthversus over time

and the calcium influx, are nonadapting,whereas most of the other responses, whichare transient, are adapting (12, 54, 61, 86). Themolecular level at which adaptation occurs isan important open question that is addressedbelow.

Temporal versus spatial sensing. For direc-tional sensing, cells need to detect nonuniformdistributions of chemoattractants. To this end,cells must be able to sense changes in recep-tor occupancy over time, referred to as tempo-ral sensing, and/or space, referred to as spatialsensing. Moving cells can detect spatial gradi-ents using a temporal sensing mechanism be-cause their receptor occupancy changes as afunction of time. In fact, chemotactic bacte-ria rely on temporal sensing to bias their ran-dom walks and to detect spatial gradients (120).However, here, the term spatial sensing is re-served for the detection of differences in recep-tor occupancy across the cell length.

Observations of immobilized cells show thateukaryotic cells are capable of both temporaland spatial sensing (Supplemental Movies 2and 3 and references therein). In cells immo-bilized with Latrunculin, an inhibitor of actinpolymerization, responses that have been ex-amined, such as Ras activation or PIP3 produc-tion, adapt normally to constant uniform stim-uli (Supplemental Movie 2). If the stimuli arefurther increased, these responses can be reac-tivated but again adapt over time. Therefore,cells respond to temporal changes in recep-tor occupancy rather than the absolute level.In contrast, immobilized cells placed in sta-ble gradients show persistent Ras and PIP3 re-sponses toward the high side of the gradient,as detected by recruitment of Ras-binding do-mains (RBDs) and specific Pleckstrin homol-ogy (PH) domains, respectively (SupplementalMovie 3). Because these cells are not moving,the steady-state level of receptor occupancy ateach position on the membrane is constant overtime. Therefore, cells are able to compare re-ceptor occupancy across their lengths and selec-tively maintain responses only at the high side.


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The temporal phenomenon of adaptationleads to spatial sensing, as illustrated by con-sidering how the steady-state response evolves.When cells are first exposed to a gradient,receptor occupancy increases everywhere andcells show initial global responses similar tothose induced by uniform stimuli. Over time,

responses at the high side of the gradientarrive at a steady, nonzero level, whereas thoseat the low side eventually vanish. Thus, adap-tation still occurs, but instead of eliminatingthe signal, it allows cells to respond to thedifference in receptor occupancy rather thanthe absolute level, resulting in an internal

c

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Local Excitation Global Inhibition model

Time

Front Back

Spatial gradient

Response

Inhib

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Excitation atfront or back

Note persistentresponse at front

b

Ca2+ influx

G-protein loss of FRETcAR1 phosphorylation

Decreasing responses:Membrane PTENCortical myosin

Increasing responses:Ras activityPIP3

PKB substrate phosphorylationscAMPcGMPCortical MyoB, Dynacortin, LimEActin polymerization

Cell behavior

Erk2 activity

Time

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Nonadapting responses

Adapting responses

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amplification of the gradient. So far, mutantsthat fail to adapt have not been identified.However, defects in adaptation can be mim-icked by removing negative regulators such asPTEN or the Ras GTPase activating protein(GAP) NF1 (45, 134). These cells have pro-longed and poorly localized PIP3 responses,resulting in impaired chemotaxis.

Adaptation and the Local Excitation GlobalInhibition model. If responses such as Rasactivation and PIP3 accumulation adapt to con-stant receptor occupancy, how can they persistlocally in an immobilized cell in a stable gradi-ent? The apparent paradox posed by this ques-tion can be explained by the Local ExcitationGlobal Inhibition (LEGI) model (Figure 2c)(78, 91; reviewed in Reference 44). Accordingto the LEGI model, two processes are elicitedby chemotactic stimuli: a fast excitation thatreflects local receptor occupancy and a delayedinhibition that is broader and more closelyreflects the mean receptor occupancy. Neitherprocess adapts; both persist as long as stimuliare maintained. The balance between excitationand inhibition determines the magnitude of theobserved responses, such as Ras activation andPIP3 production. Because excitation is fasterthan inhibition, there is a positive response im-mediately after stimuli are applied. However,with a uniform stimulus at steady state, inhi-bition eventually equals excitation all over the

Local ExcitationGlobal Inhibition(LEGI) model: amodel, involving abalance between localexcitatory and globalinhibitory processes,that can explain thetemporal and spatialresponses ofimmobilized cells tochemoattractantstimulation

Dispersion length:the effective range of asignaling molecule,determined by thediffusion coefficientand half-life of themolecule

cell, and there is no response. That is, the cellsadapt. In a chemoattractant gradient at steadystate, local excitation is higher toward the gra-dient, whereas inhibition is nearly uniform andintermediate in strength. Therefore, excitationexceeds inhibition at the high side, whereasthe opposite occurs at the low side. In thiscase, adaptation results in a persistent responsetoward the gradient. The model thereforerecapitulates the observed temporal and spatialbehaviors of immobilized cells as outlinedabove. Two predictions of the LEGI modelhave been verified experimentally (55). Forexample, when gradients change direction, theresponses reorient to the new high side. Inaddition, localized responses can be inducedsimultaneously at two points on the membraneby applying two steep gradients.

Four points about the LEGI model need tobe clarified. First, the nonadapting property ofexcitation and inhibition does not imply that themolecules involved are stable. Because the exci-tatory molecules are constantly diffusing, theymust be continually inactivated for excitationto remain localized and adjust to directionalchanges in the gradient (94). Second, local andglobal are relative terms. The LEGI model pre-dictions hold true as long as the inhibitor hasa longer range of action, or dispersion length,than the excitor. The dispersion length of amolecule is defined as

√D/k, where D is its dif-

fusion coefficient and k is its rate constant forinactivation. Thus, the inhibitor should have

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Temporal and spatial responses triggered by chemoattractants and the Local Excitation Global Inhibition (LEGI) model. (a) Whenexposed to a sudden increase in cAMP, cells retract projections or cringe; then, they periodically extend and retract projections atrandom sites on the periphery until, after several minutes, they regain their polarized morphology. (b) The biochemical responsestriggered by cAMP can be divided into two groups on the basis of whether or not they adapt to constant stimuli. Some responses, suchas G-protein activation, are nonadapting and persist as long as the stimuli are maintained. Of the adapting responses, most, such asPIP3 production, transiently increase, whereas others, such as membrane-localized PTEN, transiently decrease. The timescales shownin panels a and b are the same so that the cell behavior in panel a can be directly compared to the response curves in panel b. (c) Toexplain the temporal and spatial adapting responses of immobilized cells, the LEGI model proposes that chemotactic stimuli elicit anexcitor that reflects local receptor occupancy, as well as an inhibitor that is broader and more closely reflects the mean receptoroccupancy. Excitation rises faster than inhibition, resulting in an initial response. Left: In a uniform stimulus at steady state, excitationequals inhibition throughout the cell, which explains the experimentally observed disappearance of the initial response. Right: In agradient at steady state, excitation exceeds inhibition at the high side and vice versa at the low side; therefore, the response persists onlyat the high side of the gradient, as seen experimentally and indicated by arrows. Abbreviations: cAMP, 3′,5′-cyclic adenosinemonophosphate; cGMP, 3′5′-cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; PIP3,phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; PTEN, phosphatase and Tensin homolog on chromosome ten.


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either a faster diffusion rate or a longer half-life (or both) than the excitor. Third, the LEGImodel can only fully explain responses in un-polarized cells such as those immobilized byLatrunculin treatment. In polarized cells, thedifferential sensitivities at the opposing ends ofthe cell must be taken into account (see below).Fourth, although the inhibitor is often assumedto be a different molecule than the excitor, thisdoes not have to be the case. Hypothetically, ifcAMP stimulation resulted in rapid activationand then slow inactivation of the receptor, thereceptor itself could act as the excitor when firststimulated and become the inhibitor as adapta-tion ensued. In this case, the inhibitor wouldhave a longer half-life and therefore dispersionlength than the excitor, thus satisfying the re-quirements of the LEGI model.

While the LEGI model successfully explainsthe responses of cells to uniform stimuli andgradients, it is not known where adaptation, thebalance between excitation and inhibition, oc-curs along the signaling pathway. FRET experi-ments of the heterotrimeric G-protein indicatethat dissociation of the α- and β-subunits,a readout for G-protein activation, does notadapt to persistent stimulation, whereas theactivation of the Ras proteins does adapt(Figure 2b; Supplemental Movie 4 andreferences therein). Furthermore, prolongedRas activation leads to prolonged downstreamresponses (134). Together, these results implythat the major site of adaptation occurs afterthe G-protein dissociates and before the signalreaches the Ras proteins. The molecular linksfrom the heterotrimeric G-protein to the Rasguanine nucleotide exchange factors (GEFs)are unknown, and exactly where and howadaptation occurs are still open questions.One possible link is an extracellular super-oxide dismutase (SodC) that was identifiedin a restriction enzyme-mediated insertional(REMI) mutagenesis screen (SupplementalSidebar 2) as having elevated PIP3 levels onthe membrane (117). Disruption of SodCleads to continuous recruitment of RBD to themembrane, indicating persistent activation ofRas proteins. The G-protein α9-subunit has

also been implicated as a potential negativeregulator of the pathway, as disruption of theα9-subunit causes prolonged activation ofACA and increases the size of multicellularaggregates (13). However, there is still nodirect evidence that Gα9 regulates Ras activity.

Models based on positive feedback mecha-nisms have also been proposed to explain theasymmetric responses of cells (48, 82, 94, 124).These models describe the observed responsesof polarized cells, which cannot be readily ex-plained by the LEGI model. However, the pos-itive feedback models cannot easily account forthe rapid reorientation of responses to changesin the direction of gradients or the dual re-sponses induced by two sharply localized stim-uli observed in Latrunculin-treated cells. It ispossible that some combination of the LEGIand the positive feedback models is necessaryto account for the entire set of physiologicalresponses of chemotactic cells.

Polarity: Selective Localizationof Molecules and Reactions to theFront or Back of Cells

The polarization component of chemotaxismanifests itself in two ways. First, cells takeon an elongated morphology that becomes in-creasingly pronounced as they differentiate.Mutants that cannot maintain this morphol-ogy have reduced directional persistence, re-sulting in poor chemotaxis (see below). Second,in polarized cells, certain molecules are spa-tially restricted to the leading or lagging edge.These localizations are maintained even in theabsence of a gradient. The clustering of sig-naling molecules generates regions at opposingends of the cell that have distinct functions anddifferent sensitivities to chemoattractant, whichalters the way the cell responds to a gradient(Figure 1). Less differentiated cells, which havelow or moderate polarity, can easily form newfronts, whereas fully differentiated cells, whichare extremely polarized, tend to persist along aconstant path with increased velocity.

Although it is thought that positive feed-back must be involved, the molecules that are


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responsible for initiating polarity are still un-known. In neutrophils, a positive feedback loopinvolving PIP3 and actin has been described (48,124). However, the factors mediating polariza-tion must be redundant with the PIP3 pathway,because neutrophils or Dictyostelium cells lack-ing PI3K activity can initiate and maintain rela-tively normal polarity (33, 40). cGMP signalingmay contribute to polarity as discussed below(9, 88). It is also apparent from studies of mu-tants and chemical inhibitors that the cytoskele-ton is required for polarity. Evidence for therequirement of microtubules comes from over-expression of Lissencephaly protein 1 (Lis1)or fragments of Dynein, as well as Lis1 hypo-morphic mutants (97). In all three cases, mi-crotubules are unable to attach to the corticalshell, resulting in a flattened morphology withincreased lateral protrusions and reduced polar-ity. Furthermore, cells lacking Tsunami (TsuA)have normal motility and directional sensingcapabilities but cannot properly orient theirmicrotubules, which causes defects in polarityand chemotaxis (107). In addition, drugs, suchas Benomyl, that depolymerize microtubuleseliminate polarity (107). The actin cytoskele-ton is also required for polarity. Treatment withLatrunculin not only immobilizes cells, but alsodisrupts their stable axis of polarity and abol-ishes spatially restricted protein localizations(55, 73). Furthermore, pten- and tsuA- cells havehigh levels of F-actin, resulting in the forma-tion of lateral pseudopodia, reduced polarity,and impaired chemotaxis (45, 107). From theseand other mutants, including disruptions inthe MAPKK MEK1, Tortoise (TorA), and theNa-H exchanger Nhe1, which also cause ratherspecific defects in polarity, it is apparent that anincrease in the number of lateral pseudopodia iscorrelated with impaired chemotaxis (Supple-mental Table 1 and references therein).

Many molecules involved in chemotaxis, in-cluding both lipids and proteins, are localizedon the membrane or in the cortex specifi-cally at either the leading or lagging edge ofpolarized cells, and examples of spatially re-stricted proteins are continuously being iden-tified (Supplemental Table 2 and references

TorC2: Tor (Targetof Rapamycin)Complex 2

therein). In polarized cells, PI3Ks are foundat the leading edge, whereas PTEN is foundat the lagging edge, creating a localized accu-mulation of PIP3 and PH domain-containingeffectors at the front (Figure 3). In addition,actin and many actin-binding proteins havebeen identified in the cortex at the front of thecell. Interestingly, S-adenosylhomocysteine hy-drolase (SAHH) and the Shwachman-Bodian-Diamond syndrome protein (SBDS) localize tothe entire pseudopod at the anterior of the cell.Several other proteins are localized uniformlyon the membrane or in the cytosol but are enzy-matically active specifically at the leading edge,including Ras, Target of Rapamycin Complex2 (TorC2) subunits, and the PKB-related pro-tein PKBR1. In contrast, both cAR1 and theheterotrimeric G-proteins, as well as their ac-tivities, are uniformly localized throughout themembrane in polarized cells (58, 128).

Although most asymmetrically localizedproteins are found at the leading edge, severalhave also been identified at the lagging edge(Figure 3; Supplemental Table 2). These pro-teins are found at the lateral and rear edges ofthe cell at the outset of differentiation but be-come more tightly localized specifically to theback as differentiation progresses (K. Swaney &P. Devreotes, unpublished observations). Theseproteins include PTEN, as described above, aswell as Myosin II and the actin-binding proteinCortexillin I, which localize in the cortex andmediate rear contractility. Furthermore, p21-activated protein kinase A (PakA) is at the lag-ging edge and is proposed to prevent MyosinII dissociation by inhibiting Myosin II HeavyChain (MHC) kinases (MHCKs). ACA is theonly known integral membrane protein in thegroup of lagging edge proteins. The distribu-tion of ACA has been proposed to create a lo-calized synthesis and release of cAMP at thelagging edge, which attracts other nearby cellsand is important for generating the head-to-tailmigration, or streaming, characteristic of polar-ized cells.

Proteins that localize asymmetrically inpolarized cells display characteristic behav-iors in cells stimulated by chemoattractants


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Uniform cAMP stimulationPolarized cells or

cAMP gradient

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Figure 3Localization of signaling components to the leading or lagging edge. The distributions of leading edge proteins are represented by aPIP3-specific PH domain tagged with GFP, and those of lagging edge proteins are represented by PTEN-GFP. (a) In polarized orchemotaxing cells, many proteins are recruited to the leading or lagging edge. Arrows reflect the direction of migration. (b) Whenunpolarized cells are stimulated globally with cAMP, “leading edge” proteins, such as PI3Ks and several actin-associated proteins,translocate uniformly to the plasma membrane or cortex and then return to the cytosol. Conversely, “lagging edge” proteins, such asPTEN or Myosin II, transiently fall off the membrane or cortex (arrows) and then return to the periphery. Time in seconds after theaddition of chemoattractant is indicated for each frame. (c) During cytokinesis, “leading edge” proteins localize to the poles, whereas“lagging edge” proteins are targeted to the cleavage furrow (arrows). Images in panel c are reproduced from Reference 53.Abbreviations: cAMP, 3′,5′-cyclic adenosine monophosphate; GFP, green fluorescent protein; PIP3, phosphatidylinositol3,4,5-trisphosphate; PTEN, phosphatase and Tensin homolog on chromosome ten.

(Figure 3; Supplemental Table 2 and Sup-plemental Movies 5 and 6). In less polarizedcells, “leading edge” proteins such as PI3Ksaccumulate in the cytosol and on membraneprotrusions, whereas “lagging edge” proteinssuch as PTEN are localized uniformly on theplasma membrane with the exception of mem-brane protrusions. Upon the addition of cAMP,both sets of proteins respond by transientlyrelocalizing with respect to the plasma mem-brane or cytoskeletal cortex. The leading edgeproteins translocate uniformly to the periph-ery within 10 s and then return to the cytosolroughly 30 s after stimulation (SupplementalMovie 5 and references therein). With the samekinetics, most of the lagging edge proteins tran-siently fall off the cell periphery and into thecytosol before returning to the membrane orcortex (Supplemental Movie 6 and referencestherein). These redistribution patterns change

as the cells become more polarized. Leadingedge proteins are found at the front of polar-ized cells but are additionally and transientlyrecruited to the membrane globally with uni-form stimuli. Lagging edge proteins, alreadysharply localized to the back of cells, displaylimited redistribution in response to uniformstimuli in polarized cells. Protein localizationsat the leading or lagging edge are maintained inpolarized cells even in the absence of a gradi-ent, but, as noted above, treatment with Latrun-culin eliminates these spatially restricted distri-butions (55, 73). In Latrunculin-treated cells,many of these proteins translocate in responseto uniform stimuli as they do during early dif-ferentiation and localize toward or away fromthe source of chemoattractant gradients (47, 55,90, 99).

Current evidence, although limited, impliesthat the preferential targeting of proteins to


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the leading or lagging edge of migrating cellsis important for polarity and chemotaxis. Forexample, removing the PIP2-binding domainof PTEN causes mislocalization of the proteinto the cytosol, resulting in a broader distribu-tion of PIP3, loss of polarity, and chemotac-tic impairment that resemble the phenotype ofpten- cells (see below) (47). Similarly, expressionof PI3Ks tagged with CAAX or myristoylationmotifs, which target the proteins uniformly tothe plasma membrane, in wild-type cells mimicsthe pten- cell phenotype (34, 41). Furthermore,the mislocalization of Myosin II and its regu-latory proteins causes cells to lose polarity andimpairs chemotaxis. For example, excessive re-cruitment of MHCKA to the membrane resultsin the overall cortical loss of Myosin II and theoverproduction of pseudopodia from the lat-eral edges of the cell (9, 87). In the future, itwill be important to disrupt the localizations ofother spatially restricted proteins and to char-acterize the effects of these changes on polarityand chemotaxis. However, the mechanisms thattarget many of these asymmetrically localizedproteins, including PI3Ks and PTEN, to theleading or lagging edge are still unclear, whichcurrently hinders the investigation of proteinmislocalizations.

Several intriguing observations suggest thatdifferent components of the same signalingpathway sometimes have different localizations.For example, consider the Ras-TorC2-PKBpathway. The RasGEF Aimless (AleA) activatesRasC, which activates TorC2, which in turnmediates the phosphorylation and activationof PKBs, which then phosphorylate a numberof substrates (49, 60, 62, 75). The AleA, Ras,TorC2, and PKB proteins are localized globallyon the membrane or in the cytosol, but their ac-tivities appear to be localized specifically at theleading edge (62, 99). The dispersion lengthsof the active forms of these molecules must besufficiently short to maintain localized down-stream responses, implying that these proteinsare deactivated before diffusing away from thecell anterior. More puzzling, there are casesin which an upstream activator and its down-stream effector are localized at opposite ends

of the cell. For example, the PKB substratePakA resides at the lagging edge despite therestriction of PKB kinase activity to the lead-ing edge (21). It is possible that phosphory-lation by PKBs is not required for or relatedto PakA function at the lagging edge, but thisapparent paradox requires further study. Simi-larly, the activation of the lagging edge integralmembrane protein ACA requires both Cytoso-lic Regulator of Adenylyl Cyclase (Crac), whichis recruited by its PH domain to PIP3 at theleading edge, and Pianissimo (PiaA), a TorC2component (18, 90). The mechanism of ACAactivation by two proteins that are active at theleading edge is still unclear.

The asymmetric distribution of proteins hasimplications beyond polarity and chemotaxis,because the same proteins display character-istic localization patterns when cells undergomorphological changes in general (Figure 3;Supplemental Table 2). For example, ingrowing cells, both “leading edge” and “lag-ging edge” proteins have localizations thatresemble those of early differentiation dis-cussed above. During phagocytosis, many ofthe leading edge proteins, such as Crac andthe LIM domain-containing protein, LimE,localize to the phagocytic cup and phagosome,whereas lagging edge proteins, such as PTEN,are excluded from these structures (22, 29).During cytokinesis, PI3Ks and other leadingedge proteins are localized to the poles,whereas lagging edge proteins, such as PTENand Myosin II, are restricted to the cleavagefurrow (53; see also Supplemental Table 2).It will be interesting to determine how thesignals that target these proteins change duringthese different morphological states.

A NETWORK OF SIGNALINGPATHWAYS CONTROLSCHEMOTAXIS

Studies of numerous Dictyostelium mutantswith single or multiple gene deletions, orexpressing various mutant proteins, have fa-cilitated a molecular analysis of chemotaxis(Supplemental Sidebar 2). With current


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cARs

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responses

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Actin polymerization

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MHCKA

GacQ, TalinB, PI5K,PakA, GEFS, GEFN

Positive links

Feedback loop

Less well-characterizedlinks

Inhibitory links

Small-moleculereactions

Figure 4A network of signal responses controls chemotaxis. The interactions among the signaling components that generate chemotacticresponses in Dicytostelium cells are shown. Symbols used to indicate positive or inhibitory links, small molecule reactions, and less-wellcharacterized connections are shown in the key located in the lower left corner of the figure. The network is divided into severalmodules, which are contained in shaded boxes of different colors. Experimental data supporting the links between different componentsare discussed in detail in the main text. Abbreviations: AleA, RasGEF Aimless; cAMP, 3′,5′-cyclic adenosine monophosphate; cARs,cAMP receptors; cGMP, 3′5′-cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; GEF, guanonucleotideexchange factors; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PLA2,phospholipase A2; PTEN, phosphatase and Tensin homolog on chromosome ten; TorC2, Target of Rapamycin Complex 2.

techniques, it appears that most of the iso-lated mutants display general chemotacticimpairments rather than defects specific tomotility, directional sensing, or polarity. InFigure 4, a subset of the genes listed inSupplemental Table 1 is organized into an in-ternally consistent network of pathways that can

account for many experimental observations.This network is characterized by redundancyand cross-talk among the pathways. For ease ofdiscussion, the network is divided into a seriesof overlapping modules where, to a rough ap-proximation, the output of one module servesas an input for the next.


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Receptor and G-Protein Module

All responses to cAMP are initiated by a setof similar cAMP receptors, cAR1-4, that areexpressed at different stages of differentiation(reviewed in Reference 85; see also Supple-mental Table 1). When the cAMP receptorsare deleted and cells are assessed in early de-velopment, car1- cells respond weakly to highdoses of cAMP, whereas car1-/car3- cells donot respond at all (51). When cAR1, cAR3,or cAR2 is expressed ectopically in car1-/car3-cells, each receptor can mediate the same setof physiological responses but requires a pro-portionally higher stimulus concentration cor-responding to its respective affinity for cAMP(67). Differences in affinity have been traced tosequence differences in the extracellular looplinking transmembrane domains four and five(69).

The properties of cAR1 have been exten-sively characterized. As with most GPCRs,there are low- and high-affinity cAMP bindingsites, the latter of which are sensitive to GTPin isolated membranes (111). The dissociationrate of cAMP from the receptor is on the or-der of a few seconds (108, 111). cAR1 under-goes robust agonist-induced phosphorylation,which rises to a steady-state level proportionalto the level of receptor occupancy, with a half-time of about 45 s (39). Upon removal of thestimulus, dephosphorylation ensues with a half-time of about 2 min (115). A phosphorylatedreceptor displays an average fivefold-loweredaffinity for cAMP compared with its nativecounterpart (14). The shift in affinity does notoccur if specific phosphorylated serines are re-moved or replaced by alanines. However, theremainder of downstream responses still adaptto constant stimuli, suggesting that receptorphosphorylation is not the mechanism control-ling adaptation (70). A series of point mutationsin cAR1 have been described that alter its affin-ity or lock it in constitutively active or variousintermediate functional states. All of these pointmutations result in corresponding chemotacticdefects that are consistent with the biochemicalproperties of the receptors (68, 132).

Evidence suggests that the cARs are directlylinked to the heterotrimeric G-protein, G2,consisting of α2-, β-, and γ-subunits (reviewedin Reference 17). The β- and γ-subunits havebeen shown experimentally to directly interact,and cAMP triggers a rapid loss of FRETbetween the α2-subunit and the βγ-complex(43, 54, 129). In gα2- or gβ- cells, cAMP doesnot activate actin polymerization, ACA, sGC orGCA, or PI3Ks, nor does it mediate chemotaxis(42, 74, 127, 135). Cells expressing a dominantnegative γ-subunit or gγ - cells display asimilar phenotype (133; M. Ueda, personalcommunication). Observations suggest that theβγ-complex mediates downstream signaling,whereas the α2-subunit is required to linkthe heterotrimeric G-protein to the cARs(127). The Dictyostelium genome contains 13additional Gα-subunits, and evidence suggeststhat heterotrimeric G-proteins, consisting ofeither α4- or α5-subunits and sharing the βγ-complex, link to a set of undefined nutrient re-ceptors (32, 37). Consistently, in growth-stagegα2- cells, but not gβ- cells, folic acid activatescGMP production and mediates chemotaxis(74, 127). The Dictyostelium genome has atleast seven Regulator of G-protein Signaling(RGS) domain-containing proteins, whichare typically negative regulators of G-proteinsignaling, although none has been connected tospecific Gα-subunits so far (32). Disruption oroverexpression of one of these, RGS domain-containing protein kinase 1 (RCK1), results ina corresponding enhancement or reduction ofchemotactic speed (Supplemental Table 1).

Expression of genes in early, but not late,development and progression through the en-tire developmental program require oscillatorycAMP signaling through the receptor (Sup-plemental Sidebar 2 and references therein).Cells that do not oscillate, such as those withdisruptions in ACA, PiaA, or Crac, can still dif-ferentiate when pulsed with exogenous cAMP.However, cells lacking cARs or G2 fail to enterthe developmental program even when supple-mented with exogenous cAMP (102, 127). Theinitiation and progression of the developmentalprogram also require the protein kinase YakA,


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which appears to act immediately downstreamof the heterotrimeric G-protein in the media-tion of signaling events (110; see also Supple-mental Table 1). Disruption of YakA results incells that cannot transduce signals in responseto cAMP or folic acid stimulation, the latter ofwhich does not require differentiation. Consti-tutive activation of cAMP-dependent proteinkinase A (PKA) can partially bypass the require-ment for cAMP oscillations in the developmen-tal program (121).

The cAMP receptors are capable of trig-gering certain physiological responses in theabsence of functional G-proteins (reviewedin Reference 11). In gα2- or gβ- cells,chemoattractant-induced phosphorylation ofcAR1 occurs normally and calcium influx andErk2 activation are reduced by only 50% (84,86). Abrogation of calcium influx by disruptingthe putative inositol 1,4,5-trisphosphate (IP3)receptor (IplA) has little effect on chemotaxisunder normal conditions, possibly owing to re-dundancy (Supplemental Table 1). Furtherstudy is necessary to determine the physiologi-cal importance of this response.

RasC and RasG Modules

The receptor- and G-protein-mediated tran-sient activations of Ras proteins appear to besignificant early steps in the network (reviewedin Reference 122). Although genetic analy-sis of these proteins is complicated by appar-ent redundancy and/or compensation amongthe pathways they trigger, some of the uniqueroles have been established. For example, AleAand GEFR appear to be the predominant ex-change factors for RasC and RasG, respectively(60). In addition, studies of rasC-, rasG-, andrasC-/G- cells suggest that RasC and RasGactivate TorC2 and PI3Ks, respectively, withpossible overlaps in specificity (62, 99). In rasC-or rasC-/G- cells, phosphorylation of PKBR1,a readout of TorC2 activation, is reduced by70% (62). The residual activation may be at-tributable to the upregulation of RasD in rasG-and rasC-/G- cells (66). It has been reported thatPKBA is not activated by chemoattractant in

rasG- cells, suggesting that PIP3 levels are notelevated (4). However, PIP3-binding PH do-mains are still recruited to the membrane (C.Janetopoulos, personal communication). Theseapparent discrepancies could be explained byvariations in experimental conditions or com-pensation by RasD. A link between RasG andPI3Ks is also supported by the disruption ofthe RasGAP NF1, which coincidently prolongsthe activation of both proteins (134). The re-sulting excessive PIP3 levels cause a chemo-tactic phenotype resembling that of pten- cells(see below). Disruption of RasG or inhibitionof PI3K activity partially rescues the nf1- cellphenotype. The prolonged PIP3 production innf1- cells is consistent with adaptation occur-ring upstream of the Ras proteins. Both TorC2and PI3Ks lead to multiple downstream re-sponses, including chemotaxis and the activa-tion of ACA. This can be partially reconciledwith early studies, which suggested that RasCregulates ACA activation, whereas RasG reg-ulates chemotaxis, although the actual roles ofthe Ras proteins are probably more complexthan originally thought (4).

PIP3 and PIP2 Modules

The activation of Ras proteins leads to increasesin PIP3, mediated by a burst in PI3K activityand a kinetically similar loss of PTEN from themembrane (28, 42, 45, 46, 83). These regula-tions are independent of PIP3 levels (47). Ev-idence suggests that PI3K activation requiresboth recruitment to the membrane and inter-action with Ras (34, 42). N-terminal regions ofPI3Ks, lacking the RBD and kinase domain, aresufficient for recruitment of GFP to the mem-brane in a chemoattractant-dependent manner.PI3Ks lacking the N-terminal regions cannotrescue the pi3k1-/pi3k2- cell phenotype, but ad-dition of a CAAX motif to a truncated proteincan produce a membrane-associated enzymethat is active in the absence of chemoattrac-tant (34, 41, 42). For PTEN, an N-terminal,15-residue PIP2-binding motif is required formembrane association, activity in cells, andreversal of the pten- phenotype (47). The


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essential function of this motif is conservedin human PTEN (hPTEN), which inciden-tally can also rescue Dictyostelium pten- cells(77, 116). In HeLa cells, rapid depletion ofPIP2 causes hPTEN to immediately dissoci-ate from the membrane, which further sug-gests that PIP2 anchors PTEN to the mem-brane (96). It is speculated that temporal andspatial regulation of PIP2 levels by chemoat-tractant occur primarily through PhospholipaseC (PLC) in Dictyostelium (71). In theory, activa-tion of PLC leads to a decrease in PIP2 levels,dissociation of PTEN from the membrane, andan enhanced increase in PIP3. In chemotaxingplc- cells, PTEN is not dissociated from themembrane at the front and PIP3 increases at theleading edge are reduced. The chemorepellent8-CPT-cAMP, a cAMP analog, acts through theG-protein α1-subunit to inhibit PLC activity,which is presumed to increase PIP2 and recruitPTEN toward the high side of the gradient,flipping the axis of polarization (65).

Local accumulations of PIP3 lead to the re-cruitment of multiple PH domain–containingproteins to the membrane (SupplementalTable 2). The PH domain–containing proteinsthat translocate to the membrane in responseto chemoattractant stimulation in Dictyosteliuminclude Crac, PKBA, and PH domain proteinA (PhdA), and cells lacking these proteins havebeen reported to display weak defects in chemo-taxis (23, 90; see also Supplemental Table 2).Characterization of the other translocating PHdomain-containing proteins is in progress, andmany more that have yet to be studied arecurrently being evaluated for responsiveness toPIP3 signaling.

Chemoattractant-induced PIP3 productionis a highly conserved signature of chemotacticsignaling in many cell types, yet inhibiting thisresponse alone does not always block chemo-taxis (16, 33, 40, 63, 105). Dictyostelium cells orneutrophils lacking PI3Ks still carry out essen-tially normal chemotaxis in steep gradients orunder specific adhesive conditions. In contrast,pten- cells have high basal PIP3 levels and ex-aggerated PIP3 increases with chemoattractantstimulation, causing defects in cell morphology,

directed migration, and cell-cell signaling (45).Taken together, these results suggest that par-tially redundant pathways act in parallel withPIP3 signaling to mediate chemotaxis. One suchpathway appears to involve Phospholipase A2

(PLA2), because the simultaneous loss or in-hibition of PI3K and PLA2 activities causesa stronger chemotactic defect than does theloss of either activity alone (15, 112). Anotherparallel pathway described in detail below in-volves TorC2- and PKB-mediated phosphory-lation events.

PKB and PKB Substrate Modules

Activated Ras proteins and PIP3 accumula-tions are involved in the regulation of thechemoattractant-induced activities of two ma-jor kinases, PKBA and PKBR1, and the phos-phorylation of PKB substrates (SupplementalTable 1). Like mammalian PKBs, each kinaseis phosphorylated within a hydrophobic mo-tif (HM) by TorC2 and within an activationloop (AL), presumably by phosphoinositide-dependent protein kinases (PDKs). In cellslacking putative TorC2 subunits PiaA or Ras-interacting protein 3 (Rip3), chemoattractantsfail to elicit phosphorylation of the HM of ei-ther PKB (62). The absence of HM phospho-rylation prevents phosphorylation of the AL ofPKBR1 and substantially decreases phospho-rylation of the AL of PKBA. Unlike typicalPKBs, including PKBA, PKBR1 lacks a PIP3-sensitive PH domain and is tethered consti-tutively to the membrane by myristoylation,and therefore its activation is independent ofPIP3.

The two PKBs act somewhat redundantlyto phosphorylate a series of substrates, includ-ing TalinB, the RacGAP GacQ, the RasGEFsGEFS and GEFN, PakA, and a putativephosphoinositide 5-kinase (PI5K) (21, 62).The bulk of chemoattractant-mediated PKB-specific phosphorylation events are insensitiveto inhibition or disruption of PI3Ks, indicatingthat PKBR1 is the predominate kinase. Thephosphorylation of substrates is substantiallyreduced in pkbR1- cells and nearly abolished


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in piaA- cells. Consistently, pkbR1- and piaA-cells have impaired chemotaxis (18, 62). Fur-thermore, phosphorylation of specific PKBsubstrates is exaggerated and prolonged inpten- cells, which have extraneous projectionsthat interfere with chemotaxis. These defectsare suppressed by simultaneous disruption ofPKBA, indicating that the effects of PIP3 aremediated by this protein (M. Tang, M. Iijima,Y. Kamimura & P. Devreotes, manuscript inpreparation). Thus, in this series of mutants,the level of PKB substrate phosphorylation cor-relates well with the observed behavior of thecells. Consistently, inhibition of PKBs in mam-malian cells has been reported to interfere withchemotaxis (35, 130). The functions of specificPKB substrates in Dictyostelium are currentlybeing investigated; it is expected that each hasa distinct role in mediating chemotaxis.

cAMP Module

The oscillatory production and secretion ofcAMP by ACA mediate cell-cell signal relay,and, although not necessary for chemotaxis indifferentiated cells, the regulation of this re-sponse provides important insights into thechemotactic signaling networks (93; see alsoSupplemental Sidebar 2). Activation of ACArequires PIP3 accumulation and PKB activa-tion. An early study showed that, in vitro, ad-dition of supernatants from piaA- or crac- cellsrestored nonhydrolyzable GTP (GTPγS) stim-ulation of ACA to extracts from crac- or piaA-cells, respectively, but only wild-type super-natants restored activation to extracts of crac-/piaA- cells (18). For ACA activation, Cracrequires an intact PH domain, which medi-ates the recruitment of Crac to PIP3 at theleading edge of the cell (90). Consistently, in-hibitors of PI3Ks block activation of ACA invitro, and pten- cells display excessive ACA ac-tivation (24, 45). The involvement of TorC2strongly suggests that activation of ACA alsorequires PKB activity and probably phosphory-lation of one or more PKB substrates. Prelim-inary evidence shows that ACA is not activatedin pkbR1-/pkba- cells (H. Cai, Y. Kamimura,

C. Parent, F. Comer, S. Das & P. Devreotes,manuscript in preparation).

cAMP is synthesized by ACA and degradedboth intracellularly by the phosphodiesteraseRegA and extracellularly by membrane-boundand secreted phosphodiesterases (2). A circuitinvolving Erk2, RegA, ACA, and PKA hasbeen proposed to contribute to the spontaneousoscillations of cAMP levels that occur dur-ing development (80; see also SupplementalSidebar 2). In addition to activating ACA, a re-ceptor also activates Erk2, which inhibits RegA,thus preventing the degradation of cAMP. Theresulting elevated levels of cAMP activate PKA,which in turn deactivates ACA. In this model,the oscillations arise from a combination of thisinhibition and a positive feedback loop in whichcAMP stimulates the receptor.

cGMP/Myosin II Module

Activation of the receptor and heterotrimericG-protein triggers a transient burst of cGMPthat is closely associated with the chemotac-tic response in Dictyostelium (reviewed in Ref-erence 113). The cGMP increase is mediatedby membrane-bound and soluble guanylyl cy-clases (GCA and sGC, respectively) (98). Curi-ously, GCA has 12 transmembrane domains, asdo adenylyl cyclases; both GCs have catalyticdomains that are similar to those of the ACs.Unlike mammalian GCs, neither DictyosteliumGC contains a heme group. Simultaneous dis-ruption of GCA and sGC prevents cGMP ac-cumulation and leads to a defect in chemotaxis.Mutations in the complementation group re-ferred to as streamer F, which maps to the genefor a cGMP-specific phosphodiesterase, PdeD,were some of the earliest described chemotacticdefects (26). Streamer F mutants have exces-sive and prolonged stimulus-mediated cGMPaccumulation, have excessive Myosin II associ-ation with the cortex, and are hyperpolarized(reviewed in Reference 88). These findings ledto the concept that cGMP positively regulatescell polarity.

The Roco protein kinase family membercGMP-binding protein C (GbpC) appears to


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bind to and mediate the effects of cGMP (9).GbpC contains Leucine-rich repeat (LRR),Ras, and MAPKKK domains, like other Rocofamily members, and also a unique C-terminalextension with cyclic nucleotide binding andGEF domains (reviewed in Reference 81). Invitro, cGMP activates the GbpC GEF domain,which in turn activates the Ras domain and leadsto the subsequent activation of the MAPKKKdomain (109). GbpC activity mediates the re-cruitment of Myosin II to the cortex at the rearof the cell, which is important for generating thetension and contraction that facilitate chemo-taxis (6, 9). Disruption of the gbpC gene causesdefects in polarity and chemotaxis that resem-ble the loss of GCs or Myosin II (6, 9, 98, 126).Recent evidence suggests that PIP3 may also beinvolved in restricting Myosin II to the laggingedge, because Myosin II localization is abnor-mal in pten- cells during cytokinesis and othergrowth stages (53, 95, 125).

RasB/Rap1/MHCK Module

The activation of receptor and G-proteintriggers the activation of RasB and Rap1,which regulate Myosin Heavy Chain Kinases(MHCKs) and Myosin II. The phosphoryla-tion of Myosin II Heavy Chain (MHCA) byMHCKs promotes the disassembly and releaseof Myosin II from the cortex and opposescGMP-mediated Myosin II assembly (reviewedin Reference 7). RasB and Rap1 are thought tomediate the chemoattractant-induced recruit-ment and activation of MHCKA at the front ofcells, resulting in the restriction of Myosin II tothe rear (31, 79). Furthermore, it has been pro-posed that PakA, which is localized at the lag-ging edge, inhibits MHCK activity at the rear(19).

Several studies support the involvement ofRasB or Rap1 in the activation of MHCKs (56,57, 72, 87). Excessive activation of RasB, causedby the overexpression of the GEF domain fromGEFQ, recruits MHCKA to the membrane andphenocopies mhcA- cells (87, 126). Conversely,gefQ- cells underphosphorylate and overassem-ble Myosin II, which also impairs chemotaxis

(87). Rap1 and its putative effector, tyrosinekinase-like protein Phg2, are also speculatedto activate MHCK at the leading edge of thecell (56, 57, 72). Rap1 is activated by the GEFdomain of cGMP-binding protein D (GbpD),and although GbpD contains cyclic nucleotide-binding domains, it is not stimulated by cGMPor cAMP (6, 72). Disruption of GbpD or ex-pression of dominant-negative Rap1 leads toexcessive polarization and reduced lateral pseu-dopodia extension, whereas overexpression ofGbpD, expression of constitutive-active Rap1,or disruption of RapGAP1 leads to enhancedadhesion, many protrusions, and inhibition ofchemotaxis (56, 57, 72). This phenotype is par-tially suppressed when GbpD is expressed inphg2- cells, suggesting that Phg2 mediates someof the effects of GbpD and Rap1 (72). As ex-pected from the restriction of MHCK to theleading edge, Phg2, RapGAP1, and markersthat should be specific for Rap1-GTP are alllocalized to the front of cells (56, 57). Recentevidence suggests that Rap1 activity is regulatedby RasG, which is consistent with the slowerchemoattractant-mediated activation of Rap1compared with the other Ras proteins (5, 57).However, other reports state that prolonged ac-tivation of RasG does not appear to increaseRap1 activation (134).

Linking the Signaling Networkto the Cytoskeleton

Chemotaxis depends on both myosin-mediatedcontraction at the rear of the cell and actinpolymerization at the front. It is thought thatsignaling through the Rac proteins mediatesactin polymerization in Dictyostelium as in neu-trophils and other cell types. Disruption ofsome of the individual Dictyostelium Rac pro-teins or putative Rac exchange factors and ef-fectors results in impaired chemotaxis (20, 38,52, 76, 89, 92, 103). However, a definitiverole for the Racs has been elusive owing toredundancy between the many Rac proteins.Indeed, although there has been intense inves-tigation of the cytoskeletal events involved inactin polymerization, little is known about the


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mechanisms by which signaling events regu-late the actin cytoskeleton, but there are someclues. First, excessive or prolonged levels ofPIP3, as found in pten- and nf1- cells, elevatethe actin polymerization response and inter-fere with chemotaxis (16, 45, 134). The ef-fects of elevated PIP3 in these mutants maybe a result of excessive PKB substrate phos-phorylation, because disruption of PKBA inpten- cells restores polarity, suppresses ex-traneous pseudopodia, and enhances chemo-taxis (M. Tang, M. Iijima, Y. Kamimura &P. Devreotes, manuscript in preparation).Therefore, phosphorylation of certain PKB

substrates may be an important link between re-ceptor signaling and actin polymerization. Sec-ond, signals mediated by contractive forces atthe rear may regulate actin polymerization aspart of a negative feedback loop. For exam-ple, mhcA- cells have an increased number oflateral pseudopodia and chemotactic defects,similar to pten- cells (45, 126). By reading ex-tracellular gradients and integrating the reg-ulation of actin polymerization and myosin-based contraction, the signaling network caninterpret the intracellular compass and trans-late inputs from chemical stimuli into directedmigration.

SUMMARY POINTS

1. A conserved process referred to as chemotaxis guides the migration of cells, such asneurons, leukocytes, stem cells, and simple amoeba, along chemical gradients.

2. Chemotaxis can be conceptually divided into the processes of motility, directional sensing,and polarity.

3. Eukaryotic cells can compare and react to the small concentration differences acrosstheir dimensions. In some cells, adaptation to constant chemotactic stimulation allowssubtraction of ambient chemoattractant concentrations and greatly increases the accuracyof gradient sensing.

4. Genetic analysis of Dictyostelium is revealing that chemotaxis depends on a network ofoverlapping interactions among receptors, G-proteins, Ras proteins, PI3Ks, protein ki-nases, and phosphatases.

FUTURE ISSUES

1. What are the components involved in the generation and propagation of waves of cy-toskeletal proteins that are seen on the basal cortex of migrating cells? What is thecausal relationship between these waves and cell motility, and how are they affected bychemotactic stimuli and gradients?

2. What is the molecular mechanism of adaptation? Evidence suggests that it occurs betweenG-proteins and Ras proteins. How are the Ras proteins activated and inactivated by G-protein signaling?

3. What is the nature of the positive feedback mechanisms that drive polarity, and howare proteins restricted to the front or back of polarized cells? How is it that differentcomponents of the same signaling pathway sometimes show widely different localizationpatterns?

4. What is the relative importance of the parallel pathways in the chemotactic signalingnetwork, and what is the purpose of the apparent redundancy?


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5. How is actin polymerization activated and regulated by the chemotactic signaling net-work?

6. To what extent is the chemotactic signaling network described in Dictyostelium applicableto other systems, such as leukocytes, fibroblasts, and cancer cells?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Kristen F. Swaney and Chuan-Hsiang Huang contributed equally to this review. The authors wishto thank Pablo Iglesias and Yulia Artemenko for critical reading of the manuscript and Peter vanHaastert for scientific suggestions. KFS is supported by the American Heart Association. CHHis a Harold L. Plotnick Fellow of the Damon Runyon Cancer Research Foundation. This workwas supported by NIH grants GM28007 and GM34933 to PND.

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Annual Review ofBiophysics

Volume 39, 2010Contents

Adventures in Physical ChemistryHarden McConnell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Global Dynamics of Proteins: Bridging Between Structureand FunctionIvet Bahar, Timothy R. Lezon, Lee-Wei Yang, and Eran Eyal � � � � � � � � � � � � � � � � � � � � � � � � � � � � �23

Simplified Models of Biological NetworksKim Sneppen, Sandeep Krishna, and Szabolcs Semsey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �43

Compact Intermediates in RNA FoldingSarah A. Woodson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �61

Nanopore Analysis of Nucleic Acids Bound to Exonucleasesand PolymerasesDavid Deamer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �79

Actin Dynamics: From Nanoscale to MicroscaleAnders E. Carlsson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �91

Eukaryotic Mechanosensitive ChannelsJohanna Arnadottir and Martin Chalfie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 111

Protein Crystallization Using Microfluidic Technologies Based onValves, Droplets, and SlipChipLiang Li and Rustem F. Ismagilov � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 139

Theoretical Perspectives on Protein FoldingD. Thirumalai, Edward P. O’Brien, Greg Morrison, and Changbong Hyeon � � � � � � � � � � � 159

Bacterial Microcompartment Organelles: Protein Shell Structureand EvolutionTodd O. Yeates, Christopher S. Crowley, and Shiho Tanaka � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Phase Separation in Biological Membranes: Integration of Theoryand ExperimentElliot L. Elson, Eliot Fried, John E. Dolbow, and Guy M. Genin � � � � � � � � � � � � � � � � � � � � � � � � 207

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Ribosome Structure and Dynamics During Translocationand TerminationJack A. Dunkle and Jamie H.D. Cate � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 227

Expanding Roles for Diverse Physical Phenomena During the Originof LifeItay Budin and Jack W. Szostak � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 245

Eukaryotic Chemotaxis: A Network of Signaling Pathways ControlsMotility, Directional Sensing, and PolarityKristen F. Swaney, Chuan-Hsiang Huang, and Peter N. Devreotes � � � � � � � � � � � � � � � � � � � � � 265

Protein Quantitation Using Isotope-Assisted Mass SpectrometryKelli G. Kline and Michael R. Sussman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

Structure and Activation of the Visual Pigment RhodopsinSteven O. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 309

Optical Control of Neuronal ActivityStephanie Szobota and Ehud Y. Isacoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

Biophysics of KnottingDario Meluzzi, Douglas E. Smith, and Gaurav Arya � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 349

Lessons Learned from UvrD Helicase: Mechanism forDirectional MovementWei Yang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 367

Protein NMR Using Paramagnetic IonsGottfried Otting � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

The Distribution and Function of Phosphatidylserinein Cellular MembranesPeter A. Leventis and Sergio Grinstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Single-Molecule Studies of the ReplisomeAntoine M. van Oijen and Joseph J. Loparo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 429

Control of Actin Filament Treadmilling in Cell MotilityBeata Bugyi and Marie-France Carlier � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 449

Chromatin DynamicsMichael R. Hubner and David L. Spector � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 471

Single Ribosome Dynamics and the Mechanism of TranslationColin Echeverrıa Aitken, Alexey Petrov, and Joseph D. Puglisi � � � � � � � � � � � � � � � � � � � � � � � � � � 491

Rewiring Cells: Synthetic Biology as a Tool to Interrogate theOrganizational Principles of Living SystemsCaleb J. Bashor, Andrew A. Horwitz, Sergio G. Peisajovich, and Wendell A. Lim � � � � � 515

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Structural and Functional Insights into the Myosin Motor MechanismH. Lee Sweeney and Anne Houdusse � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Lipids and Cholesterol as Regulators of Traffic in theEndomembrane SystemJennifer Lippincott-Schwartz and Robert D. Phair � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 559

Index

Cumulative Index of Contributing Authors, Volumes 35–39 � � � � � � � � � � � � � � � � � � � � � � � � � � � 579

Errata

An online log of corrections to Annual Review of Biophysics articles may be found athttp://biophys.annualreviews.org/errata.shtml

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eukaryotic chemotaxis: a network of signaling...

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