Responding to attraction: chemotaxis and chemotropism in Dictyostelium and yeast

reviews

Responding to attraction: chemotaxis and chemotropism in Dictyostelium and yeast

directed growth and movement, respectively.

movement and growth. For an effective response, cells must be able to detect small differences in attractant concentration, and detection must convey directional information to the cell. It is likely that the intracellular transmission of an external signal requires amplification to increase sensitivity and adaptation (an adjustment in sensitivity) to disregard constant signals. To generate a response to a chemoattractant requires polarization of the cytoskeleton and subsequent growth or movement towards the chemoattractant source. For growth or movement up a chemoattractant gradient, a cell must be able to desensitize its response either by adaptation to increased levels of signal or by degradation of the chemoattractant. In many instances, it is necessary that cells be able to detect and respond rapidly to subtle differences in attractant concentration. Furthermore, a response to an external signal must override or reprogram default cellular growth processes. Dictyostelium are able to sense a chemoattractant gradient by a G-protein-coupled receptor and respond by pseudopod extension up a gradient3. Similarly, budding yeast respond to a gradient of mating pheromone via a G-protein-coupled receptor by localized growth directed towards the source of the gradient, resulting in a pear-shaped cell called a shmoo4 (Figs 1 and 2). Although the rapid movement of Dictyostelium amoebae towards a chemoattractant source, compared with the slow growth of a yeast cell towards its mating partner, appears superficially different, it is likely that, in these two organisms, the underlying mechanisms for signal detection and transmission of directional information are similar. The molecular genetics of both Dictyostelium and yeast have made it possible to identify and examine the components required for chemotaxis and chemotropism. Recent studies suggest that the basic mechanisms for these cellular processes are conserved in different systems.

Robert Arkowitz is in the Division of Cell Biology, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH. E-mail: ra2@ mclmb.cam.ac.uk

Chemotaxis, the ability to migrate towards an attractant, and chemotropism, the ability to grow towards an attractant, are crucial for responding to a complex environment. Chemotaxis is essential for leukocyte and macrophage migration to sites of tissue damage and infection1. In addition, axonal outgrowth to target cells is crucial for development of the nervous system2. Nerve cells can grow either towards an attractant or away from a repellent, and the molecular mechanisms of attraction and repulsion are likely to be similar. Aggregation of the social amoeba Dictyostelium discoideum to form a multicellular structure involves movement towards a cAMP gradient3. The budding yeast Saccharomyces cerevisiae grows towards a gradient of mating pheromone produced by a cell of the opposite mating type4. Dictyostelium and budding yeast therefore provide paradigms for chemotaxis and chemotropism, respectively. Chemotaxis and chemotropism require the ability to detect a signal, transmit this signal intracellularly and respond to the signal by oriented

Models for detection of directional information The ability of a variety of organisms to detect and respond to an attractant has been studied extensively (for comprehensive reviews, see Refs 1, 3 and 4), but how the binding of a ligand by a receptor translates into directional movement or growth remains a mystery. Several models have been proposed to explain how a cell is able to derive directional information from a chemoattractant gradient3, including spatial comparison, temporal comparison or a combination of both. Sensing by the spatial mechanism requires cells to be able to detect differences in signal concentration across the cell, a mechanism that is likely to occur in stationary cells such as nerve axons and yeast. In a temporal mechanism, analogous to the process of chemotaxis seen in bacteria, it is the change in concentration as a function of time that is important. For example, in an amoeba such as Dictyostelium, a temporal mechanism could involve the extension of pilot pseudopods in all directions, with reinforcement or further extension of pseudopods directed up a gradient. A combination of spatial and temporal mechanisms would require temporal changes in

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0962-8924/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.

Robert A. Arkowitz Polarized growth in response to external signals is essential for both the internal organization of cells and generation of complex multicellular structures during development. Oriented growth or movement requires specific detection of an external cue, reorganization of the cytoskeleton and subsequent growth or movement. Genetic approaches in both the budding yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum have shed light on the molecular and cellular aspects of growth or movement towards an external signal. This review discusses the mechanisms and signalling pathways that enable yeast and Dictyostelium cells to translate external signals into

PII: S0962-8924(98)01412-3

trends in CELL BIOLOGY (Vol. 9) January 1999

reviews (a)

Mating pheromone

(b)

Mating pheromone

(c)

Mating pheromone

(d)

Mating pheromone

detection of attractant concentration, with additional activation at the end of the cell proximal to the attractant source (or inhibition at the distal side). The signal-to-noise hypothesis postulates that fluctuations in ligand receptor binding occur across the cell Actin cortical patches Actin filaments such that the direction of polarization in uniform attractant concentrations is FIGURE 1 determined by chance, and, in a gradi- Steps required for oriented growth. As an example, budding yeast is shown with a black arrow ent of attractant, the probability of po- denoting the direction of polarized growth during vegetative growth. The graded arrow represents a larization is highest in the direction of concentration gradient of mating pheromone. (a) A vegetatively growing haploid cell comes in contact the signal5. This hypothesis incorpo- with a pheromone gradient. (b) The pheromone gradient results in activation of receptors, dissociation rates aspects of both temporal and spa- of Ga from Gbg and spatial activation of signalling molecules (denoted by X). (c) This internal tial mechanisms because fluctuations landmark (X) results in reorganization of the cytoskeleton towards the pheromone gradient, leading to in receptor occupancy across a cell can oriented growth towards the pheromone source (d). occur as a function of time. In such a stochastic mechanism, determination of the directo read out mean receptor occupancy, yet the subtion of the gradient could be accomplished by marktraction of this signal might involve some negative ing the location on the cell where the first stimulus or counteracting signal occurring uniformly across is received (or the first stimulus above a certain the cell. A number of cellular processes are likely to threshold)6. It is hard to imagine how a cell could contribute to this adjustment in sensitivity (adaptation), including degradation of the signal and redetect the first stimulus during exposure to a movceptor, internalization of the signal and receptor, ing gradient (a wave) or a steep gradient in which regulation of receptor synthesis and desensitization ligand binding would be fast7. It has been difficult of receptor signalling. to rule out any of the models for signal detection, As well as detecting chemoattractants, receptors but new results from Dictyostelium (see below) are also need to convey positional information about most consistent with the spatial mechanism, which the source of the attractant. In yeast lacking is also applicable to chemotropism in nonmotile pheromone receptors, downstream activation of the cells. For simplicity, the detection of chemoattracpheromone-response pathway does not restore tant gradients will be discussed further assuming a chemotropism12,13, suggesting that the receptor spatial model. transmits spatial information to the cell. In the absence of mating pheromone, the pheromone recepReceptors tor is localized uniformly over the surface of the Seven-transmembrane-domain G-protein-coupled cell13 (Fig. 2). In shmoos, the pheromone receptor receptors are involved in detecting many chemoattractants. Direct evidence of receptor function localizes to the mating projection, and, even in cells in chemotaxis and chemotropism has been shown without a discernible mating projection, a patch in a number of different organisms by receptor of receptor is evident on the plasma membrane13. expression in a nonresponsive cell line or deletion Is receptor localization important for yeast of the gene encoding the receptor. Yeast8, chemotropism? Polarized receptor localization occurs irrespective of whether cells are treated with a Dictyostelium9, neutrophils10 and nerve growth uniform concentration or a gradient of pheromone, cones2 can detect and orient to concentration differences of approximately 1–10% over their width, which varies from ~4 mm for yeast to 10 mm for (a) (b) (c) neuronal growth cones. Optimal chemoattractant detection occurs when the concentration at the cell midpoint is in the range of the Kd for the respective receptor. This means that cells can detect very small differences in receptor occupancy. For example, in a yeast cell 50 mm from a point source of 70 nM alphafactor mating pheromone, the half of the cell closest to the source would have 2610 occupied receptors and the half furthest from the source would have 2540 (assuming a Kd of 8 nM and 8000 receptors cell–1)8. It appears that orientation is related to FIGURE 2 the difference in receptor occupancy across a cell and not to the absolute number of occupied recepDistribution of receptors for mating pheromone in budding yeast cells treated with tors11. Hence, cells are able to subtract out the mean mating pheromone. The alpha-factor receptor Ste2p–GFP is shown by confocal receptor occupancy such that, in the above exfluorescence microscopy. Images prior to alpha-factor addition (a), after 1 h with ample, the yeast cell would read out a 135 signal on 110 nM alpha-factor (b) and after 2 h with alpha-factor (c). The brightly fluorescent the side of the cell closest to the source (2610–2575) structures within the cells are the vacuoles, which accumulate receptor and green and a –35 signal on the half furthest from the source fluorescent protein (GFP). Note the fluorescence at the cell periphery in (a) and at the shmoo tips in (b) and (c). Bar, 5 mm. (2540–2575). It remains to be seen how cells are able trends in CELL BIOLOGY (Vol. 9) January 1999

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reviews (a)

(c)

(b)

(d)

FIGURE 3 Distribution of receptors for cAMP during chemotaxis by Dictyostelium. The sequence shows a cAR1–GFP fusion in cells turning in response to movement of a cAMP source. The position of a micropipette generating the cAMP gradient is indicated by ‘1’. In (a) and (b), the micropipette is positioned above the cells, and, in (c) and (d), the micropipette has been moved to below the cells. Note the uniform distribution of receptor around the circumference of the cells. Bar, 10 mm. GFP, green fluorescent protein.

suggesting that receptor polarization is a consequence of growth directed towards the mating projection. In mammalian neutrophils and lymphocytes, chemoattractant receptors exhibit a nonuniform cell-surface distribution. Activation of neutrophils with the chemoattractant N-formylmethionylleucylphenylalanine (fMLP) does not result in a redistribution of chemoattractant receptor14. In the cases where the distribution of chemoattractant receptors is polarized15,16, polarization appears to require cell migration, raising the possibility that the receptor localization reflects directed secretion to this leading edge. Recent studies in Dictyostelium and macrophages demonstrate that cells with a uniform distribution of receptor can nonetheless undergo chemotaxis. Dictyostelium cells exposed to gradients of cAMP show no change in the uniform distribution of a cAMP receptor fused to green fluorescent protein (GFP) on the cell surface17 (Fig. 3). In addition, the receptor tyrosine kinase for colony-stimulating factor 1 (CSF-1) on macrophages challenged with a CSF-1 concentration gradient is endocytosed and found in vesicles throughout the cell upon stimulation18. Thus, chemotaxis is possible without a polarized distribution of the chemoattractant receptor. It might indeed be that cells, such as Dictyostelium, that need to respond very quickly to changes in chemoattractant are aided by the uniform distribution of receptor on the cell surface. Nevertheless, receptor activation must transmit spatial information from the chemoattractant gradient to the inside of the cell, and it is likely that local activation of a receptor is sufficient for communicating this spatial information to the cell. Receptor posttranslational modification After a receptor binds to a ligand, a number of different processes occur at the level of the receptor,

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including desensitization and internalization. Desensitization refers to a decrease in the sensitivity of a cell to chemoattractant, generally either by direct modification or deactivation of the downstream signalling. The sensitivity of a cell to a chemoattractant can also be modulated by receptor internalization via ligand-induced endocytosis and proteolytic degradation of the receptor. Posttranslational phosphorylation of receptors is essential for both desensitization and internalization. In mammalian, Dictyostelium and yeast cells, phosphorylation of the receptor cytoplasmic C-terminus both at serine and threonine residues occurs following ligand binding. Studies of Dictyostelium cAMP receptors19, human chemoattractant protein 1 receptor (CCR2B)20 and human myeloid fMLP receptor21 all indicate that removal of these phosphorylation sites affects receptor internalization, recycling and desensitization but has little effect on chemotaxis. By contrast, removal of the majority of the C-terminus of the Gprotein-coupled CCR2B receptor20, the interleukin 8 receptor22 or the yeast alpha-mating pheromone receptor (L. Vallier, J. Segall and M. Snyder, pers. commun.) causes defects in chemotaxis/chemotropism. However, these effects could be due to the defects in G-protein activation resulting from receptor truncation. Overall, posttranslational modification of G-protein-coupled receptors does not appear to be crucial for chemotaxis or chemotropism but might be important for the cellular processes requiring chemotaxis or chemotropism. Regulation of downstream signalling and chemoattractant degradation Other forms of desensitization include regulation of receptor downstream signalling and chemoattractant degradation. Regulation of downstream signalling is accomplished in part by a ubiquitous class of proteins called RGS (for ‘regulator of G-protein signalling’). Mutations in the yeast RGS SST2 cause cells to be supersensitive to mating pheromone. Orientation assays, which measure the orientation of the mating projection in a pheromone gradient, show that sst2 – cells are able to orient in gradients with very low concentrations of mating pheromone, whereas, at the higher concentrations of mating pheromone that are required for orientation of wildtype cells, orientation is substantially reduced8. In addition, Dictyostelium and yeast degrade their chemoattractants. In both organisms, chemotaxis/chemotropism occurs even when degradation of attractant is prevented8,23, indicating that degradation is not necessary for movement or growth towards a chemoattractant source. While undoubtedly both these means of desensitization are physiologically important for cellular processes requiring chemotaxis and chemotropism, they are not essential for orientation of movement or growth in an attractant gradient. Transmitting signals to the internal signaltransduction machinery Cells undergoing chemotaxis and chemotropism polarize their actin cytoskeleton towards the source of the chemoattractant. How does ligand activation trends in CELL BIOLOGY (Vol. 9) January 1999

reviews TABLE 1 – GENES AFFECTING CHEMOTAXIS/CHEMOTROPISM IN DICTYOSTELIUM AND BUDDING YEAST Gene

Functions

Dictyostelium cAR1-4 cAMP receptor CRAC Cytosolic regulator of adenylyl cyclase Requires activated Gbg subunits for membrane translocation Ga-2 Heterotrimeric G-protein a-subunit Ga-4 Heterotrimeric G-protein a-subunit Gb Heterotrimeric G-protein b-subunit Erk1 Mek1 AleA

MAP kinase necessary for cell aggregation MAP kinase kinase RasGEF necessary for cell aggregation

PiaA

Necessary for cell aggregation

Yeast STE2 STE3 GPA1 STE4 STE18 SST2 FAR1 CDC24 CDC42

a-Mating pheromone receptor essential for mating a-Mating pheromone receptor essential for mating Heterotrimeric G-protein a-subunit essential for mating Heterotrimeric G-protein b-subunit essential for mating Heterotrimeric G-protein g-subunit essential for mating Regulator of heterotrimeric G-protein signalling Cyclin-dependent kinase inhibitor required for mating-pheromone arrest GEF for Cdc42 necessary for polarized growth Small G-protein necessary for polarized growth

Role in chemotaxis

Refs

Required for cAMP chemotaxis Not required for chemotaxis

Reviewed in Ref. 30 31,32

Required for cAMP chemotaxis Required for folic acid chemotaxis Required for chemotaxis to all known chemoattractants Required for chemotaxis to high but not low cAMP concentrations Required for cAMP chemotaxis Required for cAMP chemotaxis, but mutants show weak chemotactic response Required for cAMP chemotaxis, but mutants show weak chemotactic response

Reviewed in Ref. 30 Reviewed in Ref. 30 25,26

Required for a-mating pheromone chemotropisma By analogy to STE2, assumed to be required for a-mating pheromone chemotropism Required for chemotropisma

12,13

12,13

Required for chemotropisma

13

Required for chemotropisma

13

Required for chemotropism to high but not low a-mating pheromone concentrationsa,b Required for chemotropismb

8,39 39,41

Required for chemotropismb

43

Likely to have a role in chemotropism

48

34,35 36 37 38

12,13

aDemonstrated

by mating-partner selection experiments. by measurement of growth orientation to a pheromone gradient. Abbreviations: GEF, GDP–GTP exchange factor; MAP, mitogen-activated protein. bDemonstrated

of a chemoattractant receptor direct reorganization of the actin cytoskeleton? This question has begun to be addressed using genetic approaches in both Dictyostelium and budding yeast, which have identified a number of genes involved in chemotaxis and chemotropism (Table 1). Although chemotaxis in Dictyostelium and chemotropism in yeast have different functions, both require activation of heterotrimeric G-proteins and downstream signalling that is in part mediated by small G-proteins and their exchange factors (GEFs). In many organisms, chemoattractant binding to G-protein-coupled receptors results in dissociation of Ga from Gbg (Fig. 5) and it appears that Gbg activates downstream chemotactic/chemotropic responses12,22,24–26. Dictyostelium Dictyostelium cells respond chemotactically to cAMP and folic acid27. The chemoattractant cAMP trends in CELL BIOLOGY (Vol. 9) January 1999

activates both adenylyl and guanylyl cyclase, and folic acid activates guanylyl and perhaps adenylyl cyclase. Cells lacking adenylyl cyclase activity are unable to form a multicellular aggregate because cAMP is necessary for intercellular communication. Such mutants do, however, exhibit chemotaxis towards a source of cAMP, showing that adenylyl cyclase activation is not absolutely required for chemotaxis28. By contrast, guanylyl cyclase activation appears to be essential for chemotaxis29, indicating that cGMP functions as a second messenger for chemotaxis. The same Gb subunit (present as Gbg) is required for response to both cAMP and folic acid25,26. A screen for mutants that were unable to respond to either cAMP or folic acid identified nine complementation groups29. Of particular interest are two mutants called KI-8 and KI-10; the former has little to no intracellular cGMP and the latter does not show characteristic increases in cGMP

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reviews

FIGURE 4 Distribution of CRAC–GFP during Dictyostelium chemotaxis. The sequence shows the time-course (0, 10, 20 and 30 s) of CRAC–GFP localization in cells exposed to a cAMP gradient generated by a micropipette. Note the polarized distribution of CRAC–GFP on the edge of cells adjacent to the cAMP source. Bar, 5 mm. CRAC, cytosolic regulator of adenylyl cyclase; GFP, green fluorescent protein.

levels upon stimulation with chemoattractant. In response to chemoattractants, both mutants increase intracellular cAMP and actin polymerization, suggesting that they are able to respond yet are specifically defective in the directional aspect of the response. A number of other mutants such as synag7/dagA, erk2, aleA and piaA are defective in cell aggregation30 (Table 1). Each of these mutants is defective in adenylyl cyclase activation, and collectively they indicate that the adenylyl cyclase pathway is necessary for intercellular communication, whereas the guanylyl cyclase pathway is required for chemotaxis, perhaps eliciting a directional response. A cytosolic regulator of adenylyl cyclase, referred to as CRAC, was identified biochemically from wildtype cells and this cytosolic activity was able to reconstitute adenylyl cyclase activity from synag7 and dagA mutants. Although synag7/dagA mutants have an attenuated chemotactic response, they do respond to a gradient of cAMP31. CRAC translocates from cytosolic to membrane fractions upon chemoattractant stimulation32. Binding of CRAC to membrane fractions depends on the activation of the heterotrimeric G-protein and requires Gb. The temporal and spatial dynamics of this membrane translocation have been examined recently using a CRAC–GFP fusion protein33. Addition of uniform

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concentrations of cAMP to cells results in rapid translocation of CRAC to the cell periphery, whereas exposure of cells to a cAMP gradient causes the selective and transient recruitment of cytosolic CRAC to the stimulated edge of cells (Fig. 4), providing evidence for the local activation of the G-protein signalling system even when the chemoattractant receptor is distributed uniformly. Actin polymerization is necessary for cell movement and pseudopod extension. Strikingly, the dynamic and polarized localization of CRAC is unaffected in cells treated with the actin-depolymerizing drug latrunculin A. These nonmotile cells are still able to polarize their chemotactic signal-transduction machinery, arguing against a temporal model for gradient sensing and suggesting that these cells can detect small differences in receptor occupancy across their length – similar to yeast cells. A number of chemoattractants also stimulate mitogen-activated protein (MAP) kinase cascades, but the role of these pathways in chemotaxis is not clear. erk2, which encodes a MAP kinase, was identified as a mutant that is unable to aggregate, and erk2 – cells are defective in chemotaxis to folate and cAMP34,35. However, erk2 – cells respond chemotactically to low concentrations of cAMP34, indicating that chemotaxis is not dependent on erk2 activation. Instead, an inability of erk2 – cells to repolarize in high chemoattractant concentrations implies that this MAP kinase is important for adaptation to high cAMP concentrations. Mek1 is a MAP kinase kinase, and mek1– cells form small aggregates yet continue to differentiate normally36. mek1– cells are defective in cAMP-dependent chemotaxis and activation of guanylyl cyclase36, suggesting that the Mek1 MAP kinase cascade is involved in regulation of intracellular cGMP. Erk2 activation is independent of Mek1, indicating that these kinases lie in different pathways36. Thus, although MAP kinase cascades can affect chemotaxis, this effect is probably indirect, perhaps owing to feedback between directed movement and MAP kinase signalling. The Dictyostelium gene aleA (aimless) encodes a RasGEF that is required for chemotaxis37. AleA– cells are specifically defective in the activation of adenylyl cyclase either by cAMP receptors or by GTPgS, but they do, however, show a weak chemotactic response. piaA (pianissimo) encodes another Dictyostelium protein required for adenylyl cyclase activation38. Pianissimo is a 130-kDa cytosolic protein necessary for chemotaxis and has homologues both in S. cerevisiae and Schizosaccharomyces pombe. While erk2 –, piaA– and aleA– cells are all defective in chemotaxis, these mutants still exhibit a very weak chemotactic response in contrast to Gb–, cells which are completely unable to perform chemotaxis25, implying that this single Gb transmits information to both cAMP- and cGMP-dependent pathways. Yeast The molecular basis of chemotropism in budding yeast has become more accessible owing to its relatively simple life cycle and the completion of its genome sequence. In yeast, genetic techniques have trends in CELL BIOLOGY (Vol. 9) January 1999

reviews enabled the identification of genes essential for chemotropism. One approach used to screen for such genes (Table 1) is to test the ability of cells to select a partner between pheromone-producing and pheromoneless cells of the opposite mating type. Such studies have shown that the G-protein-coupled mating pheromone receptors (STE2 and STE3), Ga (GPA1), Gb (STE4) and Gg (STE18) are crucial for chemotropism12,13. None of the pheromonedependent MAP kinase cascade components is required for mating-partner selection12. FAR1, encoding a cyclin-dependent kinase inhibitor necessary for pheromone-dependent cell-cycle arrest, is also essential for oriented growth in response to a pheromone gradient39–41. far1-s mutants were isolated in a screen for cells that had enhanced mating defects when mated to an enfeebled mating partner42. Molecular characterization of far1-s alleles showed that most of the mutations are clustered in the C-terminus of the protein, in contrast to the far1 alleles with defects in cell-cycle arrest, which were clustered in the N-terminus41. far1-s mutant cells form normal shmoos, arrest growth, induce matingspecific genes and polarize their actin cytoskeleton in response to mating pheromone, in contrast to Dfar1 cells, which are unable to arrest growth in response to mating pheromone. Careful analysis of the point mutants showed that they are specifically defective in their ability to grow towards a mating partner and not in their ability to detect mating pheromone and indicate that the cell-cycle-arrest function and growth-orientation function of FAR1 are two separable activities. These studies provide the first demonstration of an intracellular protein functioning specifically to transmit directional information. As yet, there are no known Far1p homologues in higher eukaryotes. Far1p probably functions to couple cell-cycle arrest to growth orientation during mating. A screen for mating-specific mutants in the Cdc42p GDP–GTP exchange factor (GEF) Cdc24p has revealed a role for this exchange factor in chemotropism43. Cdc42p is a small GTPase involved in organizing the actin cytoskeleton (see below). Both CDC42 and CDC24 are essential yeast genes; conditional mutants grow in an unpolarized fashion and have defects in bud-site selection44,45. The mating-specific cdc24-m mutants are normal for vegetative growth and polarization of the actin cytoskeleton. These mutants have normal pheromone-dependent responses, including polarization of the cytoskeleton, indicating that they can detect mating pheromone. However, they are unable to orient growth to a gradient of mating pheromone. At a molecular level, the mutant Cdc24p proteins are unable to interact with the yeast Gb protein Ste4p. These results suggest that binding or activation of Cdc24p by yeast Gb is essential for chemotropism and link external signalling via the pheromone receptor to regulation of the organization of the actin cytoskeleton by Cdc42p. The similar phenotypes of far1-s and cdc24-m mutant cells suggest that CDC24 and FAR1 function in the same chemotropism pathway. An attractive trends in CELL BIOLOGY (Vol. 9) January 1999

possibility is that these two proteins mark a site in the vicinity of activated pheromone receptors, thereby providing a link between the directional information from the chemoattractant gradient and the inside of the cell. Such a model would require local activation or localization of these proteins to the side of the cell where the pheromone receptors are activated (adjacent to the pheromone source). This internal landmark could then serve as a point of nucleation for the actin cytoskeleton and other proteins necessary for directional formation of the mating projection (Fig. 1b). Small GTPases Signalling from chemoattractant receptors must at some level reach small GTP-binding proteins, such as Rho, Rac and Cdc42, which control organization of the actin cytoskeleton. These small GTPases regulate the assembly of actin stress fibres, focal adhesion sites, membrane ruffles, protrusive lamellipodia and filopodia46. Neuroblastoma cells induce formation of filopodia and lamellipodia in response to a concentration gradient of acetylcholine, and preinjection of dominant–negative Cdc42 and Rac1, respectively, inhibits this response47. By contrast, lysophosphatidic acid (LPA)induced neurite retraction appears to be mediated by RhoA. Cdc42p plays a role in yeast mating, but the level at which this occurs is unknown48. A recent study of the role of Cdc42, Rac1 and RhoA in CSF-1induced macrophage chemotaxis found that microinjection of a constitutively active version of each of these G-proteins results in a reduction of chemoattractant-induced cell motility18. Similarly, microinjection of dominant–negative Rac1 or inhibition of Rho using C3 transferase, an exoenzyme that ribosylates and inhibits Rho function, block CSF-1-mediated cell migration. Interestingly, microinjection of dominant–negative Cdc42 increased the migration speed of CSF-1-treated cells, yet these cells show no bias in migration direction with respect to a CSF-1 concentration gradient, indicating a complete lack of chemotactic response. However, they show a somewhat polarized morphology, suggesting that they are able to respond to CSF-1 but cannot transmit directional information. In leukocyte lysates49, Dictyostelium lysates49 and permeabilized yeast cells50, Cdc42 specifically induces actin polymerization, suggesting that locally activated Cdc42 can lead to oriented growth or movement. Presently, no CDC42 gene has been identified in Dictyostelium, raising the possibility that a related small GTPase such as one of the Rac proteins might fulfil this function. The requirement for Cdc42 in chemotaxis and the role of the Cdc42p GEF Cdc24p in yeast chemotropism together imply that these two proteins are likely to play fundamental roles in chemotaxis/chemotropism in a number of organisms. Overriding internal growth processes To respond to an external chemoattractant gradient requires that cells be able to override internal growth and organization programmes and direct

25

reviews (a)

Yeast Pheromone-response pathways

as far1 and cdc24 that have defects in growth orientation of mating projections41,43, the projection forms adjacent to the previous bud site. Conversely, when a gradient of pheromone is applied and then removed, wild-type cells form buds at their shmoo tips8. Together, these results imply that there is relocalization of the machinery for polarized growth to different growth sites in response to internal signals and external signals. A simple mechanism to accomplish such a switch would be to make at least one component common to both growth processes. Cdc24p is a candidate for such a switch as it is necessary both for bud-site selection and positioning of the mating projection.

Mating pheromone Receptor Ga Gbg MAP kinase cascade

Far1p Cdc24p Cdc42p? Actin polymerization

Cell-cycle Gene arrest induction

Chemotropism (b)

Dictyostelium cAMP/folate-response pathways cAMP

Folate

Receptor

Receptor

Ga Gbg

Ga Gbg

Aimless Pianissimo

CRAC Erk2

?

Adenylyl cyclase

Guanylyl cyclase

cAMP

cGMP

Actin polymerization Intercellular signalling

Chemotaxis FIGURE 5

Chemotropic and chemotactic signalling pathways in yeast and Dictyostelium. (a) Budding yeast pheromone-response pathways. The role of Cdc42p in chemotropism is speculative. (b) Dictyostelium cAMP/folate-response pathways. An unknown aspect of the Dictyostelium response is represented by a question mark. The chemotactic mutants KI-8 and KI-10 are likely to function between the heterotrimeric G-protein and guanylyl cyclase. Aimless, Pianissimo and Erk2 are shown next to adenylyl cyclase because they reduce adenylyl cyclase activation. Mek1 is not shown as it is not clear where it fits in.

growth towards the source of attractant. How this switching is achieved is unclear. In budding yeast, cells must be able to switch between bud-site selection (polarized growth mediated by internal signals) and positioning of the mating projection adjacent to the pheromone source. In the presence of a uniform concentration of pheromone39,51 or in mutants such

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Maintaining directional growth or movement Although initiation of directional growth or movement is crucial for chemotactic or chemotropic processes, maintenance of growth or movement towards a chemoattractant source is equally important. The ability to follow a gradient requires that cells be able continuously to sense a gradient. Nerve cells are able to read out gradients over long distances. For such a readout process to occur, it is likely that the chemoattractant receptor would need to be present over the entire surface of the growing projection in order to detect subtle changes in the chemoattractant gradient. Growth up a gradient probably requires desensitization of chemoattractant receptors in order to maintain maximum sensitivity. The behaviour of cells in multiple chemoattractant gradients is more complex. For example, leukocytes express several different chemoattractant receptors and in vivo are likely to encounter different signals that will ultimately direct their path. Navigation through complex mixtures of chemoattractants can be accomplished by a step-by-step process of response to one chemoattractant after another52. This combinatorial effect of different chemoattractants is thought to be essential for selective and efficient cell guidance and targeting of leukocytes and neurons. Concluding remarks Many different motile and nonmotile cells have the ability to sense and respond to chemoattractants in their environment. The process of translating small differences in chemoattractant concentration across the length of a cell into directional movement or growth involves communication between G-protein-coupled receptors, heterotrimeric Gproteins, GEFs and small GTP-binding proteins such as Cdc42. Dictyostelium and yeast use G-proteincoupled receptors to detect differences in chemoattractant concentration across their cell length. While the mechanistic basis for chemotaxis in Dictyostelium and chemotropism in yeast are similar (Fig. 5), it is premature to compare the intracellular molecules necessary for transmitting spatial information in the two organisms. The time constraints placed on chemotaxis in Dictyostelium and chemotropism in yeast are very different, and it is therefore likely that response time is optimized in Dictyostelium, whereas choice of site for mating trends in CELL BIOLOGY (Vol. 9) January 1999

reviews projection growth is emphasized in yeast. By contrast, the commitment of a yeast cell to extend a mating projection in a particular direction is probably much higher than the orientation of Dictyostelium. In both chemotaxis and chemotropism, a number of outstanding questions remain. • How are cells able to determine the difference in receptor occupancy over their length? • What mechanisms provide the amplification of such small differences in receptor occupancy? • Are there negative or counteracting signals that enable a cell to subtract out receptor occupancy across its surface? • How do different processes such as attractant internalization and degradation, receptor internalization and degradation, regulation of receptor expression, oriented secretion and sensitization or desensitization of G-protein signalling result in an efficient temporal and spatial response to a chemoattractant gradient? Yeast and Dictyostelium provide ideal model systems in which to address these questions and elucidate the molecular processes essential for oriented cell growth and movement. References 1 Bokoch, G. M. (1995) Blood 86, 1649–1660 2 Lohof, A. M. et al. (1992) J. Neurosci. 14, 1253–1261 3 Devreotes, P. N. and Zigmond, S. H. (1988) Annu. Rev. Cell Biol. 4, 649–686 4 Chenevert, J. (1994) Mol. Biol. Cell 5, 1169–1175 5 Tranquillo, R. T., Lauffenburger, D. A. and Zigmond, S. H. (1988) J. Cell Biol. 106, 303–309 6 McKay, D. A., Kusel, J. R. and Wilkinson, P. C. (1991) J. Cell Sci. 100, 473–479 7 Tomchik, K. J. and Devreotes, P. N. (1981) Science 212, 443–446 8 Segall, J. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8332–8336 9 Mato, J. M. et al. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4991–4993 10 Zigmond, S. H. (1977) J. Cell Biol. 75, 606–616 11 Zigmond, S. H. (1981) J. Cell Biol. 88, 644–647 12 Schrick, K., Garvik, B. and Hartwell, L. H. (1997) Genetics 147, 19–32 13 Jackson, C. L., Konopka, J. B. and Hartwell, L. H. (1991) Cell 67, 389–402 14 Gray, G. D. et al. (1997) J. Histochem. Cytochem. 45, 1461–1467 15 Sullivan, S. J., Daukas, G. and Zigmond, S. H. (1984) J. Cell Biol. 99, 1461–1467 16 Nieto, M. et al. (1997) J. Exp. Med. 186, 153–158 17 Xiao, Z. et al. (1997) J. Cell Biol. 139, 365–374

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Acknowledgements I thank M. Bassilana, R. Kay, N. Lowe, A. Nern and S. Munro for comments on the manuscript and P. Devreotes for the images in Figs 3 and 4.

GFP movies The CRAC–GFP movie described in this article (see Fig. 4) is one of the many included in the trends in CELL BIOLOGY GFP movies CD, to be distributed with the journal next month. Other Dictyostelium movies include migrating slugs, cytoskeletal organization and cell division. There are over 150 movies in total, from a wide range of cell types and organisms. The CD is being compiled and edited by Beat Ludin and Andrew Matus of the Friedrich Miescher Institute, Basel, Switzerland, and is made possible through generous sponsorship from Applied Imaging, Chroma Technology Corp., Clontech, Leica Microsystems, Life Science Resources (LSR) and Universal Imaging.

trends in CELL BIOLOGY (Vol. 9) January 1999

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