Changing directions in the study of chemotaxis - Nature

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Changing directions in the study of chemotaxis Robert R. Kay*, Paul Langridge*‡, David Traynor* and Oliver Hoeller*

Abstract | Chemotaxis — the guided movement of cells in chemical gradients — probably first emerged in our single-celled ancestors and even today is recognizably similar in neutrophils and amoebae. Chemotaxis enables immune cells to reach sites of infection, allows wounds to heal and is crucial for forming embryonic patterns. Furthermore, the manipulation of chemotaxis may help to alleviate disease states, including the metastasis of cancer cells. This review discusses recent results concerning how cells orientate in chemotactic gradients and the role of phosphatidylinositol-3,4,5-trisphosphate, what produces the force for projecting pseudopodia and a new role for the endocytic cycle in movement. Pseudopod An organelle-free projection that is thicker than other types of cell projection. Pseudopodia contain filamentous actin and are formed by cells such as amoebae.

Substratum The surface on which cells move. The nature of the substratum can change the behaviour of cells.

Lamellipodium A thin, organelle-free projection that contains filamentous actin. Lamellipodia are formed by cells such as fibroblasts or keratocytes.

*MRC Laboratory of Molecular Biology, Hill Road, Cambridge CB2 0QH, UK. ‡ Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, New York 10032, USA. Correspondence to R.R.K. e-mail: [email protected] doi:10.1038/nrm2419

Chemotaxis — the guided movement of cells in chemical gradients — probably emerged early in eukaryotic evolution, and even today striking similarities are apparent between the movement of amoebae and neutrophils. Chemotaxis of these crawling cells is an extremely slow form of movement. Fibroblasts move at up to 1 µm per minute, and even fast-moving amoebae, neutrophils and keratocytes only attain 10–40 µm per minute, which is 100 times slower than swimming bacteria. A neutrophil that moves at 10 µm per minute covers its own body length in approximately 1 minute, which is equivalent to a human taking an hour to cover 100 metres. Chemotaxis allows neutrophils or Dictyostelium amoebae to find their bacterial prey and also allows amoebae, when starved, to aggregate into multicellular masses using cyclic AMP (cAMP) signals. Chemotaxis is a key mechanism by which tissues and organs become organized during development. For example, the chick body plan is laid down partly by the invagination of primitive streak cells, guided by positive and negative chemotaxis to fibroblast growth factors1, neurons interconnect using chemotactic growth cones and the lateral line organ of the zebrafish forms by the chemotactic migration of a mass of precursor cells2. Chemotactic ability can lie dormant in fibroblasts, but is evoked when a wound needs to be healed. In the metastasis of cancer cells, it would be desirable if chemotaxis could be completely suppressed, as it is the escape and spread of cells from the primary tumour that makes cancer so intractable3. In the classical movement cycle, a cell projects forward an organelle-free pseudopod and makes new adhesions to the substratum. The cell body then advances,

nature reviews | molecular cell biology

and the back detaches and retracts, before the whole cycle is repeated. However, this simple picture belies a more complex reality, as crawling cells can vary greatly in morphology. Fish keratocytes move using a delicate lamellipodium and an unchanging body shape, whereas amoebae and neutrophils have much thicker pseudo podia and constantly pull themselves into uncomfortable shapes. But this variety might represent no more than variations on a theme; Dictyostelium cells can migrate with a typical amoeboid morphology, but can also move like keratocytes4, as highly elongated snake-like cells during aggregation or as ‘paddle’ cells with smooth, bleb-like pseudopodia5. In this review, we focus on two major issues for which recent results have shed new light: how cells orientate in chemotactic gradients and the mechanism of pseudopod projection from the cell body. We also touch on a less explored topic — the ‘surface-area problem’ — that links movement and the endocytic cycle. We focus in particular on fast-moving cells, such as Dictyostelium cells, which undergo chemotaxis towards cAMP, and on neutrophils and their relatives.

Steering the cell Cell behaviour in chemotactic gradients. For most cells, unguided movement is of little use, as cells need to go to specific places or find particular things. Accordingly, although some cells, such as Dictyostelium amoebae, are intrinsic ally motile, many others need at least global stimulation with a chemoattractant to initiate movement. Much is revealed by observing cells as they move in chemotactic gradients. Their paths depend greatly on the strengths of the gradients and on whether volume 9 | june 2008 | 455

© 2008 Nature Publishing Group

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Figure 1 | Behaviour of Dictyostelium cells in gradients. a | A rounded cell thatNature is attracted by| aMolecular strong gradient Reviews Cell Biology of cyclic AMP (cAMP) that has been released from a micropipette. The experiment shows the ability of cells in strong gradients to orientate accurately — they move directly up the gradient with little deviation. Coloured outlines represent the position of the cell at successive time points and the asterisks mark the micropipette positions. The cell was chilled so that it became rounded and lost polarity, and was then warmed and stimulated. b | A cell re-orientating in a cAMP gradient of intermediate strength produced by a micropipette that has moved to a new position. The cell does not move directly up the gradient, but re-orientates more gradually. New pseudopods are produced by splitting existing ones and the cell orientates by favouring the more accurate of the daughter pseudopods. Panel b reproduced, with permission, from Nature Cell Biology Ref. 10  (2007) Macmillan Publishers Ltd.

Leading edge The front of the cell.

PtdIns(3,4,5)P3

A signalling phospholipid and minor component of the plasma membrane that serves as a binding site for proteins that contain specific pleckstrinhomology domains.

or not they are polarized (FIG. 1). In steep gradients, and especially if the cells are unpolarized, neutrophils and Dictyostelium amoebae can project pseudopodia directly up the gradient and move, with little deviation, towards the chemoattractant source6–8. Evidently, these cells can directly perceive the direction of the gradient. A second behaviour is apparent when polarized cells change direction in response to an altered gradient 9: these cells often turn like a car to match the new gradient, constantly maintaining their original front end. In weaker gradients, several different cell types use a different strategy10. These cells do not orientate pseudopodia directly up the chemotactic gradient, but instead produce new pseudopodia by splitting existing ones with little reference to the gradient (Supplementary information S1 (movie)). Cells then steer by favouring the daughter pseudopod that is up-gradient, which then

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becomes dominant, and the unsuccessful daughter is retracted. In this way, the chemotactic gradient can act over the whole life of a pseudopod, allowing time averaging for the directional signal to be picked up over background noise. At extreme sensitivities, neutrophils and Dictyostelium cells can navigate in gradients that differ by only a few percent in chemoattractant concentration down their length11,12, which for Dictyostelium cells translates into a difference of just 5–10 occupied receptors13,14 out of perhaps 50,000 total receptors. These cells take a more uncertain track that is best described as a biased random walk, presumably because noise from fluctuating receptor occupancy and internal sources of asymmetry nearly obscure the gradient15,16. This range of behaviours can be viewed as the outcome of competition between three processes that affect pseudopod production: the propensity of cells to spontaneously produce pseudopodia in random directions; the influence of the chemotactic gradient on the positioning of pseudopodia formation and on pseudopodia lifetime; and, in polarized cells, an increased stability of the leading edge. In weak gradients, spontaneous pseudopod formation predominates; in strong gradients, pseudopod formation is mostly controlled by the gradient; and in polarized cells there is a tendency to maintain the existing pseudopod, even in adverse gradients. Signal transduction and directional sensing. A key property of the signal-transduction pathway is its ability to convert a continuous gradient into a local response to trigger pseudopod formation. This is most apparent with unpolarized cells in steep gradients, and implies that cells can compare receptor occupancy over their surface and determine where the concentration is the highest: this is called directional sensing17. A key hypothesis is that receptor occupancy produces at least two antagonistic signals: a short range and possibly autocatalytic signal that promotes pseudopod formation and a longer range signal that acts globally as an inhibitor17,18. Second messengers can be categorized by their potential range: membrane lipids, such as phosphatidylinositol‑3,4,5-trisphosphate (PtdIns(3,4,5)P3), have a short range and are best suited to convey local information, whereas faster diffusing, cytoplasmic mole cules, such as cyclic GMP (cGMP), are more suited to a global role19. Where does directional sensing reading occur in the chemotactic signal-transduction pathway? In Dictyostelium cells, cAMP is detected by seven‑ transmembrane domain receptors, of which cAMP receptor‑1 (cAR1) is the most important. cAR1 couples to the heterotrimeric G protein Gα2βγ, and both the receptor and the G protein are genetically essential for chemotaxis20 (FIG. 2): they are uniformly distributed over the cell surface and seem to faithfully transmit the external chemotactic gradient into the cell21. Lower in the pathway, PtdIns(3,4,5)P3 behaves differently22,23: it forms sharp gradients in the membrane that are steeper than the external chemotactic gradient24 and can be visualized by its binding to a pleckstrin-homology (PH) domain–green fluorescent protein (GFP) reporter. www.nature.com/reviews/molcellbio


REVIEWS cAMP receptor

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Figure 2 | A simplified version of the upper part of the Nature Reviews | Molecular Biology Dictyostelium chemotactic pathway for cyclicCell AMP. After activation of a cyclic AMP (cAMP) receptor, gradient sensing is proposed to occur in the section between the activated heterotrimeric G protein and the activation of RasC and RasG. It is proposed that the pathway then splits into at least two redundant pathways that are mediated by various phosphatidylinositol 3-kinases (PI3Ks) or by target of rapamycin complex-2 (TORC2). Two important components are not shown: phospholipase A2, which might form part of another redundant pathway56, and the guanylyl cyclases60, the activation of which lies genetically downstream of RasC and RasG53. GEF, guanine nucleotideexchange factor.

Dictyostelium type‑I PI3Ks The domain organization of these proteins resembles mammalian type‑I phosphatidylinositol 3‑kinases, especially because they have a Ras-binding domain.

Chemotactic index A measure of the accuracy of chemotaxis that is calculated by taking the cosine of the angle between a line directly up the gradient and one that connects a cell’s start point to its end point. A value of 1 is directly up the gradient.

Neutrophils also produce steepened PtdIns(3,4,5)P3 gradients25,26, but those of fibroblasts are not steepened, which is consistent with the limited chemotactic performance of fibroblasts27. PtdIns(3,4,5)P3 gradients in Dictyostelium cells are produced by the activation of phosphatidylinositol 3‑kinases (PI3Ks) in the up-gradient region of the cell and by the loss of the opposing phosphatase and tensin homologue (PTEN) phosphatase from the membrane in the same region28,29. PtdIns(3,4,5)P3 gradients are more than just intermediates in a signalling cascade — they have a life of their own. Intense PtdIns(3,4,5)P3 gradients form in randomly moving Dictyostelium Gβ-null cells, in which all signalling through the heterotrimeric G proteins is disrupted30. They seem to be sustained by a positive feedback loop that requires activation of the small GTPase Ras and that is broken when actin poly merization is inhibited with latrunculin. PtdIns(3,4,5)P3 gradients or ‘patches’ have self-organizing properties. When patch formation is triggered by uniform stim ulation with cAMP, the number of patches that form, but not their size or lifetime, depends on the stimulus concentration; and following local cAMP stimulation, PtdIns(3,4,5)P 3 patches behave as if regulated by a long-lived local inhibitor of unknown nature31,32. Thus, Dictyostelium cells seem to have a basal excitable system that produces PtdIns(3,4,5)P3 gradients without receptor input, but can be stimulated and orientated by a chemotactic gradient.


The role of PtdIns(3,4,5)P3 gradients in chemotaxis. As PtdIns(3,4,5)P3 gradients often signify the site where a pseudopod will form, it has been suggested that they might specify this site and in doing so act as ‘chemotactic compasses’ that guide the cells33. However, after growing suspicions34,35, recent results have demonstrated that PtdIns(3,4,5)P3 signalling cannot be the sole compass and that it is probably only one of several semi-redundant ways of guiding cells. Early work that supported the PtdIns(3,4,5)P3 compass hypothesis reported that chemotaxis is severely impaired in Dictyostelium cells that have been treated with the PI3K inhibitor LY294002 or in Dictyostelium mutants that lack two of the five Dictyostelium type‑I PI3Ks (the mutant cell chemotactic index was 0.29 compared with 0.86 for the wild type)28,36. Subsequently, it was found that cells could recover from PI3K-inhibitor treatment and resume chemotaxis with an accuracy that is statistically indistinguishable from control conditions37. Reinvestigation of the PI3K1–/PI3K2–-double-knockout mutant showed that, in suitable conditions, mutant cells could undergo chemotaxis with an efficiency that was near to that of wild-type cells37,38. Although PTEN-null mutants produce broader and longer-lived PtdIns(3,4,5)P3 gradients than wild-type cells, which causes the excessive formation of pseudopodia29, they too can undergo chemotaxis with high efficiency39. Furthermore, mutant cells in which all five type‑I PI3Ks and PTEN have been eliminated can still undergo efficient chemotaxis in steep cAMP gradients, even though they have lost the ability to produce PtdIns(3,4,5)P3 gradients39 (mutant cells have a chemotactic index of 0.95, compared with a wildtype chemotactic index of 0.96) (FIG. 3; Supplementary information S2,S3 (movies)). However, movement of the PI3K mutants is not normal — they are slower, orientate poorly in weak gradients and their random movement in the absence of a chemotactic gradient is slower38,39. A similar pattern of results has emerged in neutrophil chemotaxis. PI3K inhibitors were initially reported to reduce chemotaxis40–42, but more detailed examination showed that at least some inhibited cells can undergo efficient chemotaxis towards the chemotactic peptide fMLP43,44. Contrary to initial indications45–48, detailed examination also showed that mouse PI3Kγ‑–/– neutrophils undergo chemotaxis towards fMLP as efficiently as wildtype cells, even if additional PI3K inhibitors are added to target PI3Kβ and PI3Kδ. Likewise, ablation of SH2domain-containing inositol phosphatase (SHIP), which is the major PtdIns(3,4,5)P3 phosphatase in these cells, still allows efficient chemotaxis26 (FIG. 3). The varying results of neutrophil chemotaxis assays might be due to confounding variables, such as the ability of drug-inhibited cells to recover after a short delay44, reduced proportions of motile cells in mutant or drug-treated populations (resulting in reduced chemotaxis in transwell assays), differences in the substratum and differences in cell priming43. An exception is the PI3K-dependent chemotaxis of neutrophils towards attractants, such as interleukin‑8 (IL‑8). These cells reach their final destinations in an animal by navigating along gradients of endogenously produced chemoattractants, such as IL‑8, and towards volume 9 | june 2008 | 457


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Figure 3 | PtdIns(3,4,5)P3 gradients are not required for chemotaxis. a | Dictyostelium wild-type (WT)Cell cells and Nature Reviews | Molecular Biology knockout (KO) mutant cells that lack all ‘type‑I’ phosphatidylinositol 3-kinases (PI3Ks), or the phosphatase and tensin homologue (PTEN) phosphatase for phosphatidylinositol3,4,5-trisphosphate (PtdIns(3,4,5)P3), are shown moving towards cyclic AMP39. b | Mouse neutrophils that lack PI3Kγ or the SH2-domain-containing inositol phosphatase-1 (SHIP1) that acts on PtdIns(3,4,5)P3 are shown moving towards the chemotactic peptide fMLP26. The tracks of cells that are moving towards a source of chemoattractant (at the top of each panel) are shown. It is evident that cells can undergo chemotaxis efficiently, even if their ability to form PtdIns(3,4,5)P3 gradients is genetically impaired or abolished. These and other experiments disprove the once popular hypothesis that PtdIns(3,4,5)P3 gradients are essential for chemotaxis. However, PI3K activity is required for efficient random movement and for efficient chemotaxis in shallow gradients. Panel a modified, with permission, from Ref. 39  (2007) Cell Press. Panel b reproduced, with permission, from Nature Cell Biology Ref. 26  (2007) Macmillan Publishers Ltd.

the chemoattractants that are released from their end targets, such as the bacterially produced fMLP. Faced with two conflicting gradients, these cells follow fMLP rather than IL‑8, which seems to be reflected in their diff ering signal-transduction pathways: IL‑8 chemotaxis is completely blocked by PI3K inhibitors, whereas fMLP chemotaxis is not44,49. If not PtdIns(3,4,5)P 3 , then what? The working assumption of several groups is that PtdIns(3,4,5)P3 gradients act redundantly with at least one other unidentified guidance pathway. At what stage might 458 | june 2008 | volume 9

this pathway branch off from the signal-transduction pathway and what is its nature? Clearly, the branch must lie between the essential upstream components of the pathway — the cAMP receptor and Gα2βγ — and PI3K (FIG. 2). Type‑I PI3Ks of Dictyostelium cells are characterized by the possession of a Ras-binding domain, which binds to activated Ras proteins. Ras is rapidly activated by cAMP and is localized to the leading edge, similar to PtdIns(3,4,5)P3 (Refs 50,51). Knockout of any single Ras protein leaves cells with substantial chemotactic ability, but combining expression of a dominant-negative RasG protein with a null allele of aimless (aleA), a Ras guanine nucleotide-exchange factor, or knocking out both RasC and RasG almost abolishes chemotaxis towards cAMP51–53. Therefore, it seems that Ras function lies in the common direction-sensing pathway, which thereafter splits, with one branch going through PI3K and another going through an unknown pathway. By this logic, the other branch might connect through a protein with a Ras-binding domain. A strong candidate is Rip3, a component of the target of rapamycin complex-2 (TORC2), because it has a Ras-binding domain and null mutations of TORC2 components impair chemotaxis54,55. An unbiased genetic screen for the components of the alternative pathway in Dictyostelium revealed an additional candidate: phospholipase A2 (PLA2)56. Chemotaxis mutants were sought that were only defective when PI3K was inhibited using LY294002. The identification of PLA2 as a candidate is supported by drug experiments in which cocktails of inhibitors block chemotaxis only when both PI3K and PLA2 are inhibited57. PLA2 releases free fatty acids and lysophospho lipids from phospholipids and is stimulated by cAMP, which is consistent with a direct role in chemotaxis. However, a triple mutant of PLA2 and two PI3Ks can still undergo chemotaxis towards cAMP56,58, showing that a further guidance pathway remains undisrupted (the triple mutant has a chemotactic index of 0.55 compared with a wild-type chemotactic index of 0.96). Of the second messengers that are controlled by the cAMP signalling pathway, both calcium influx 59 and cAMP production can be genetically abolished without much disruption to chemotaxis. Abolition of cGMP production, by the deletion of the two known guanylyl cyclases, severely impairs chemotaxis. This occurs, at least in part, because cGMP is required for myosin II filament formation at the back of the cell19,60. The production of cGMP occurs downstream of RasC and RasG53, and it is probably not a suitable signal to specify where a pseudopod will form because it spreads too widely within the cell19. In summary, PtdIns(3,4,5)P3 lies in an excitable circuit that is important for random movement and, although not essential for cell guidance, it might act redundantly with a guidance pathway that has not yet been fully identified. Direction sensing in Dictyostelium cells occurs somewhere between the cAMP receptorcoupled G protein and RasC and RasG, and the guidance pathway might bifurcate after the Ras proteins. www.nature.com/reviews/molcellbio


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Figure 4 | F‑actin waves. Waves of actin polymerization that move along the plasma Nature Reviewscells | Molecular membrane are visualized in different cell types. a | Dictyostelium that areCell Biology expressing a filamentous (F)‑actin-binding protein that is fused to green fluorescent protein (GFP). b | HL60 cells that are expressing the HEM1 component of the SCAR– WAVE complex that is fused to GFP. The HEM1 waves are assumed to be associated with F‑actin waves and to be similar to the Dictyostelium waves, but this has not yet been confirmed. c | The hypothetical structure of a wave. It is proposed that waves leave behind a refractory zone that does not allow another wave to pass until it has recovered. Actin polymerization might be initiated in the leading edge of the wave by the actinrelated protein-2/3 (ARP2/3) complex and F‑actin that has been depolymerized in the trailing edge; see Ref. 88 for a heuristic model of wave propagation. The scale bar represents 5 µm. Panel a courtesy of T. Bretschneider and G. Gerisch, University of Warwick, UK, and the Max Planck Institute, Germany. Panel b reproduced from Ref. 88.

Hydrostatic pressure A force that is applied by fluid to any surface it is in contact with. Hydrostatic pressure allows forces that are produced by the contraction of the back of a cell to be transmitted to the front.

Photobleaching The bleaching of fluorophores, such as green fluorescent protein, by light of sufficient intensity. Photobleaching can be used to create small bleached spots on the structure of interest, the movement of which can then be monitored.

The motor for pseudopod extension Pseudopods can be projected entirely off the substratum, only attaching later in their life, and so do not need local adhesions to extend61. The dominant view is that the leading edge of a cell is driven forward by dendritic actin polymerization that occurs adjacent to the membrane62,63. Of the competing ideas, membrane flow64 has received little experimental support65,66, whereas hydrostatic pressure is emerging as an important driver of movement. Actin polymerization at the leading edge. A dense meshwork of actin filaments lies directly beneath the leading edge of many motile cells. The fast-growing, barbed ends of these filaments point towards the membrane. Both photobleaching and speckle microscopy show that actin filaments are extended at the membrane and then flow back and are disassembled deeper in the cell, thereby releasing actin monomers for another cycle67,68. The actin meshwork contains long and short filaments and


is dendritically branched69,70, although this is contested71. Similar mechanisms of actin polymerization drive the intracellular motility of various pathogens72 and could apply force to the plasma membrane by a thermal-ratchet mechanism73, in which the filament flexes away from the membrane owing to thermal motion, is extended and then flexes back again. It is generally thought that the major nucleator of actin polymerization at the leading edge is the actinrelated protein-2/3 (Arp2/3) complex, which nucleates from the side of pre-existing filaments, thus producing a dendritic meshwork62,74. The contribution of the Arp2/3 complex to movement is difficult to test genetically, as it has many pleiotropic effects75. Depletion of an essential subunit of Arp2/3 using RNA interference (RNAi) produces cells with greatly reduced lamellae75,76, although partial loss-of-function mutants in Dictyostelium have only mild movement defects77. The Arp2/3 complex must be activated by Wiskott–Aldrich syndrome protein (WASP) or suppressor of cAMP receptor (SCAR; also known as WAVE) proteins, which might transmit signals from the chemotactic receptors78. Null mutants of SCAR and its regulatory complex have only moderate motility defects in Dictyostelium79. WASP seems to be essential, and WASP mutants with reduced activity show reduced actin polymerization in response to a chemoattractant and a severe movement defect80. Together, these results indicate that the Arp2/3 complex has a substantial role in the motility of a cell, and that both SCAR and WASP are involved, although other modes of motility have not been excluded. Two other actin-nucleation methods might also be involved. Formins nucleate linear actin filaments and protect them during growth by capping their fast-growing barbed ends. Formins are important for filopodium formation81, but recent experiments indicate that the formin mammalian Dia2 can also be important for lamellipodium formation, as RNAi knockdown of Dia2 expression substantially reduces both the lamellipodial area and the rate of expansion in melanoma cells70. In a hybrid scheme derived from studies on carcinoma cells, actin polymerization is initially triggered by cofilin that has been activated at the leading edge82,83. Cofilin severs the actin filaments and creates barbed ends, thereby triggering rapid actin polymerization. Because the ARP2/3 complex binds preferentially to the newly polymerized, ATP-rich filaments, it is recruited to the newly extended filaments and amplifies the initial effect. F-actin waves. Current models for actin polymerization at the leading edge62 do not readily explain the spontaneous, excitable properties that are characteristic of pseudopod formation in many freely moving cells. These properties are more easily explainable if motility uses the ‘waves’ of actin polymerization that have been observed travelling on the plasma membrane of Dictyostelium cells (FIG. 4; Supplementary information S4 (movie)). Waves of actin polymerization were first reported several years ago in confocal microscopy studies84, and are clearly revealed by total internal reflection fluorescence (TIRF) microscopy, which illuminates only a narrow zone of volume 9 | june 2008 | 459


REVIEWS the cell adjacent to the substratum85–87. Similar waves in the HEM1 component of the SCAR/WAVE complex (an activator of the ARP2/3 complex and hence of actin polymerization) have also been discovered in HL60 cells88 (Supplementary information S5 (movie)). These waves move at speeds of several micrometres per minute, which is comparable to the movement speed of the cells that host them and, although tied to the membrane, the waves can extend some way into the cytoplasm. Waves that travel in opposite directions can exist in the same cell, and when these collide they mutually annihilate each other. This indicates that they propagate in an excitable medium, leaving behind a refractory zone that does not immediately support the propagation of another wave. The composition and mechanism of the propagation of these waves is largely unknown. As waves can form without chemotactic stimulation and can initiate pseudo pod formation following a collision with the plasma membrane at the side of the cell, they could be a basic feature of cell motility that is used for chemotaxis. However, the existence of waves raises more questions than it answers. For example, how does actin polymerization in the waves relate to actin polymerization at the leading edge? What distinguishes permissive membrane from refractory membrane? Can a chemoattractant initiate or guide the waves?

Speckle microscopy A technique in which nearly all of the chromophores in a structrure, such as actin–green fluorescent protein that has been polymerized into filamentous actin, are photobleached such that those that remain can be tracked individually as speckles.

HL60 cells Human leukaemia cells that can be induced to differentiate into motile neutrophil-like cells.

Hydrostatic pressure, blebs and myosin II. Hydrostatic pressure has long been suspected to provide an alternative force for the extension of pseudopodia. It can produce blebs — unusual rounded extensions of the plasma membrane. Blebs are thought to form at sites where the actin cortex detaches from the plasma membrane, allowing the bleb to expand by a rapid influx of cytosol that is driven by hydrostatic pressure. This occurs without an increase in the volume of the cell and depends on myosin II. Actin is not enriched at the membrane during the expansion phase, which confirms that expansion is not driven by actin polymerization. Rather, actin only accumulates under the membrane bleb as expansion ceases and is likely to help stabilize or retract the bleb89–94. In many cells, blebbing occurs around the perimeter and does not result in directional movement. As early as 1973, however, it was proposed that blebbing drives the movement of Fundulus blastoderm cells95. Some of the best examples of the bleb mode of movement come from cells that move on more challenging terrain than a glass slide. The primordial germ cells of zebrafish migrate from the embryo periphery through the developing tissue to the gonads, extending large, classical blebs that are devoid of an actin cortex during their expansion phase96 (Supplementary information S6,S7 (movies)). Both bleb formation and movement depend on myosin II and can be inhibited using blebbistatin, which targets myosin II. Tumour cells are often characterized by copious blebbing89. As a model for metastasis, they can be induced to invade a three-dimensional matrix that mimics the extracellular environment that is found in tumours. Many do this with a rounded morphology and numerous membrane blebs97.

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Blebbing is not immediately apparent in time-lapse films of Dictyostelium cells that are undergoing chemotaxis, but a detailed study of real-time filamentous (F)‑actin distribution reveals that small blebs period ically form at the leading edge of these cells92. Genetically, bleb formation in Dictyostelium cells depends on myosin II93. Myosin II-null mutants move slower than the wild type, their pseudopodia are more flaccid98 and, crucially, they project pseudopodia at half the speed99. These defects are not readily explained by models in which the leading edge is projected solely by the force of actin polymerization. These defects are to be expected if myosin II contributes to hydrostatic pressure, either directly (through its contractility) or indirectly (by its contribution to cortical strength)100. Therefore, it seems that the contractile forces that produce blebbing make a real contribution to the extension of the leading edge, even when blebbing is not as apparent. Little is known about how blebbing is regulated during chemotaxis. Local disassembly of actin in the cell cortex might weaken the attachment between the cytoskeleton and the membrane, such that detachment occurs under hydrostatic pressure. Consistent with this, drug-induced depolymerization of the cortex can induce blebs101 and mutants in the actin cytoskeleton can show increased blebbing89,93. Alternatively, hydrostatic pressure might increase locally at the position on the cell perimeter where a bleb will form91, possibly because of an influx of calcium ions at the region of protrusion96. Classical blebbing is stimulated by a chemoattractant in Dictyostelium cells, which suggests that there is a link between the chemotaxis machinery and the forces that produce blebbing93. Whatever the connection, blebbing in cells that are undergoing chemotaxis orientates with the gradient92, implying that the chemotactic steering machinery connects to a distinct set of targets from those that orientate dendritic actin polymerization and pseudopod survival. Modes of motility: a hybrid engine? It is becoming apparent that cells have an extraordinarily dynamic engine for chemotaxis that is not solely composed of the mechanisms that have been presented in the dendritic nucleation model62. Cofilin and formins might also stimulate actin polymerization in pseudopodia, and hydrostatic pressure might make an important contribution to pseudopod expansion. It could be that many cell types operate a hybrid engine, in which the balance between moving by actin polymerization and hydrostatic pressure is weighted differently and can be changed according to the circumstances.

The surface-area problem The importance of the endocytic cycle for recycling the ‘feet’ from the back of a moving cell to the front is well known102. Another role for the endocytic cycle might be in surface-area regulation. Moving neutrophils or amoebae periodically become rounded and then flatten out again61,66,103,104, and they will often project a pseudo pod from the front of the cell before they retract the back of the cell105 (FIG. 5; Supplementary information S8 (movie)). These changes in shape probably produce www.nature.com/reviews/molcellbio


Relative surface area

REVIEWS the SNARE complexes that are formed when a vesicle docks to a membrane, and in the tsNSF mutant the endocytic cycle is almost completely blocked at the restrictive temperature. Under these conditions, the cells become rounded and reduce their surface area, but they can still polarize to cAMP and polymerize actin in an attempt to form a pseudopod. Without sufficient membrane, however, the pseudopod cannot expand. How might the endocytic cycle be used to regulate surface area in cells that are undergoing chemotaxis? One possibility is that the balance between exocytosis and endocytosis is regulated by membrane tension. This can be sensed by stretch-activated channels that open under tension to allow calcium into the cell and are periodically activated in moving cells110.

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Figure 5 | Three-dimensional reconstructions of a Dictyostelium cell that is undergoing chemotaxis towards cAMP. When viewed in three-dimensions, cells such Nature Reviews | Molecular Cell Biology as Dictyostelium amoebae and neutrophils change radically in shape as they move, often alternating between rounded and flattened morphologies. From simple geometry, their surface area would be expected to fluctuate as they move, which has been confirmed by direct measurement. These increases in surface-area are considerably greater than the amount allowed by membrane stretching, and therefore these increases are proposed to be due to regulated changes in endocytosis and exocytosis. Reconstructions are from confocal sections of cells that express a fluorescent plasma-membrane marker, with filopodia truncated; see Ref. 66 for further information.

SNARE complexes (soluble N-ethylmaleimidesensitive fusion protein attachment protein receptor complexes). Proteins that are required for the fusion of membrane vesicles. They form a complex during fusion that has to be resolved before they can be reused.

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changes in surface area, and indeed changes of 20–30% over a few minutes can be measured from confocal reconstructions of moving Dictyostelium cells66. Such changes greatly exceed the amount by which membranes can stretch without rupture106, thus raising the surfacearea problem: how do moving cells adjust their surface areas to match their changes in shape? As neutrophils and Dictyostelium amoebae do not have obvious plasma-membrane foldings to act as reserves for surface expansion, the only way that extra surface components can be supplied is through the exocytosis of membrane. Dictyostelium cells maintain an active endocytic cycle in which their entire surface is taken up in around 10 minutes66,107, and regulation of this cycle would provide a way of regulating surface area. Indeed, when the endocytic cycle is perturbed, motility is strongly affected. Clathrin-null mutants of Dictyostelium move more slowly and irregularly than the wild type108, and a temperature-sensitive (ts) mutant in the essential NSF (N-ethylmaleimide-sensitive fusion) protein is almost completely paralyzed within a few minutes of incubation at the restrictive temperature66,109 (Supplementary information S9 (movie)). NSF is required for resolving

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Conclusions Cells are basically excitable — as shown by the spontaneous formation of PtdIns(3,4,5)P3 gradients, F‑actin waves and pseudopodia — and this leads to a basal motility that is harnessed in chemotaxis. Chemotaxis is almost certainly regulated by redundant signalling pathways, not only at the level of redundant components, such as various PI3K proteins or Ras proteins, but also at the whole circuit level. At a minimum, PtdIns(3,4,5)P3 reports directional sensing by the cell and stimulates basal motility, but it is not by itself essential for cell guidance: at least one further steering pathway awaits discovery. Possible components of further pathways include the TORC2 complex and PLA2. Current models of pseudopod extension that are based solely on dendritic actin polymerization at the leading edge seem to be incomplete. They do not account for the F-actin waves, which have been observed travelling along the membrane, and there is strong evidence that hydrostatic pressure makes an important contri bution to pseudopod expansion. The endocytic cycle has an under-appreciated role in cell motility, particularly in surface-area regulation. Cell motility is a fantastically complicated process and it is clear that major discoveries remain to be made. What steering routes exist and what are their targets? Do these targets regulate pseudopod initiation, pseudopod survival, bleb initiation or all of the above? What are the relative contributions of actin polymerization and myosin II-dependent bleb formation in driving pseudo pod formation? How does membrane trafficking relate to movement, specifically in surface-area regulation? This profusion of questions makes chemotaxis an exciting field, in which further twists and turns are to be expected, no doubt mimicking a cell proceeding, with some uncertainty, towards its target by chemotaxis.

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Acknowledgements

We would like to thank M. Bretscher for stimulating our inter‑ est in cell motility and the anonymous reviewers and many others for discussion.

DATABASES UniProtKB: http://ca.expasy.org/sprot cAR1 | cofilin | IL‑8 | NSF | PLA2 | RasG | Rip3 | SCAR | SHIP | WASP

FURTHER INFORMATION Robert Kay’s homepage: http://www2.mrc-lmb.cam.ac.uk/CB/Kay_R/group

SUPPLEMENTARY INFORMATION See online article: S1 (movie) | S2 (movie) | S3 (movie) | S4 (movie) | S5 (movie) | S6 (movie) | S7 (movie) | S8 (movie) | S9 (movie) All links are active in the online pdf

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