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(Journal of Leukocyte Biology. 2001;70:491-509.)
© 2001 by Society for Leukocyte Biology

Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement

Peter Friedl, Stefan Borgmann and Eva-B. Bröcker

Cell Migration Laboratory, Department of Dermatology, University of Würzburg, Würzburg, Germany

Correspondence: Peter Friedl, Cell Migration Laboratory, Department of Dermatology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany. E-mail: peter.fr{at}mail.uni-wuerzburg.de


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ABSTRACT
 
Cell movement within three-dimensional tissues is a cycling multistep process that requires the integration of complex biochemical and biophysical cell functions. Different cells solve this challenge differently, which leads to differences in migration strategies. Migration principles established for leukocytes share many characteristics with those described for ameba of the lower eukaryote Dictyostelium discoideum. The hallmarks of amoeboid movement include a simple polarized shape, dynamic pseudopod protrusion and retraction, flexible oscillatory shape changes, and rapid low-affinity crawling. Amoeboid crawling includes haptokinetic adhesion-dependent as well as biophysical migration mechanisms on or within many structurally and functionally different substrates. We describe central aspects of amoeboid movement in leukocytes and the implications for leukocyte crawling and positioning strategies within interstitial tissues.

Key Words: T lymphocytes • collagen matrix • cytoskeletal dynamics • migration strategies


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INTRODUCTION
 
Leukocytes are highly migratory cells that can develop a spectrum of versatile migration strategies to enter and transmigrate through various tissues and organs [reviewed in ref. 1 2 3 ]. In the body, migrating leukocytes adapt their cell bodies to preexisting structures, yet appear simultaneously able to structurally modify matrix barriers. Putatively nondestructive T cell migration occurs within the loose connective tissue of lymphatic organs and interstitial compartments [2 ]. These compartments comprise the lymph node cortex, the mesentery, perivascular fibrillar trails along blood vessels, or the dermal papillae [2 , 4 ]. T lymphocytes are further capable of crawling along the surface of other cells, such as vessel endothelium before transmigration, fibroblastic reticulum cells in lymph nodes [5 ], and antigen-presenting cells [6 ]. The penetration of basement membranes requires attachment coupled to proteolytic mechanisms [7 ] and profound shape changes. In chronic inflammation, the interaction of T cells with resident tissue cells can result in severe proteolytic remodeling of tissue architecture, integrity, and function [8 ]. For all of these conditions, migratory traction occurs in conjunction with recognition of additional activation signals from the counterpart cell or matrix interphase, implicating overlapping pathways of cytoskeletal dynamics and signal transduction [9 ].

Such a broad spectrum of migratory capabilities through or across biophysically and biochemically very different substrates implies a great degree of flexibility and adaptability of leukocyte migration and positioning strategies. Such diversity may set leukocytes apart from other more specialized migratory cell types. As examples, fibroblasts, keratinocytes, and neurons migrate only under circumstances highly restricted in location and time, i.e., on morphogenesis or wound healing within a specific tissue context.

Cell migration within the tissues is a complex mechanochemical process that requires the integration of key events in signaling, cytoskeletal, membrane, and adhesion systems [reviewed in ref. 10 11 12 ]. Based on cell type-specific morphological and functional criteria of migration within an extracellular matrix (ECM) environment, i.e., polarization and shape change, migration velocity, cytoskeletal organization, and integrin and protease expression and function, at least three migratory prototypes can be classified: (1) amoeboid crawling can be distinguished from (2) fibroblast-like, mesenchymal migration and (3) collective cell movement, as seen in multicellular strands, sheets, or clusters [12 14 ].

The concept of amoeboid motion is most clearly established by studies using the single-cell stage of the lower eukaryotic ameba Dictyostelium discoideum [15 ]. Amoeboid movement results from alternating cycles of morphological expansion and contraction driven by cytoskeletal dynamics, shape change, and low adhesivity. These migration characteristics allow ameba to rapidly adapt to a given environment, develop high migration velocities, and contact other cells in a dynamic yet reversible manner. The Dictyostelium paradigm of movement has important implications for the understanding of cell migration strategies in higher eukaryotes, most notably for neutrophils, lymphocytes, and some tumor cells [15 , 16 ].

Fibroblast-like mesenchymal migration, as detected in fibroblasts, myoblasts, neural crest cells, and many cells from solid tumors, depends on relatively slow, adhesion receptor- and integrin-dependent cell-substrate interactions that fulfill the criteria of adhesion-mediated ("haptokinetic") migration [11 , 17 , 18 ]. These cells express high levels of integrins and matrix-degrading proteases and, while migrating, can create substantial remodeling of the ECM [19 20 21 ; reviewed in ref. 12 ].

Collective cell movement, in extension of haptokinetic migration of single cells, is dependent on highly adhesive substrate interactions and remodeling of the extracellular matrix. Multicellular migration requires the maintenance of stringent cell-cell adhesion and communication mechanisms [13 , 22 23 ; Y. Hegerfeldt, M. Tusch, E. B. Brocker, P. Friedl, unpublished results], as detected in migrating cell clusters from epidermal keratinocyte sheets, angiogenic sprouts, and tumors.

Because form follows function and vice versa, the shapes adopted by single cells or cells within tissue frequently correspond to functional specificities of those cells. We here summarize cellular and molecular strategies involved in amoeboid cell movement and use the paradigm of amoeboid crawling for understanding the locomotor mechanisms of leukocytes, particularly by T lymphocytes and neutrophils migrating on 2-D and within 3-D tissue substrates.


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THE LIFE CYCLE OF D. DISCOIDEUM
 
The amoeboid cells of the slime mold D. discoideum live as separate, independent cells in soil, leaf mold, or litter stratum, phagocytozing bacteria and yeasts. Under favorable nutrient conditions, amebas grow as individual cells that divide every few hours by individual cell division (cytokinesis). Under these conditions, Dictyostelium amebae develop chemotaxis towards the source of their food, i.e., bacterial products, such as folic acid. The single cell phenotype is commonly referred to as preaggregation stage. On challenge by adverse events, such as starvation by exhaustion of food and nutrients, amebae stop dividing and initiate a developmental program that leads to the two-step formation of a multicellular organism, the spore. First, about 104-105 cells chemotactically gather together and aggregate to form a tipped mound. The mound is surrounded by extracellular matrix and develops a spore head. The spore head elongates and falls onto the substrate to generate a multicellular slug or pseudoplasmodium, i.e., a worm-like migratory cell cluster that behaves chemotactically and phototactically. Proper slug development is dependent on differentiation and sorting into two different cell types, prestalk and prespore, that migrate and position themselves differently. Prestalk cells form the faster moving front of the slug whereas prespore cells are located in slower rear parts [25 ]. The slug is surrounded by a sheet composed of extracellular matrix (ECM) proteins and cellulose. Attracted by light and heat, multicellular migration of slugs can persist for up to 20 days. Culmination is initiated if growth conditions such as light, humidity, and external ion strength are appropriate. The slug then maturates into a differentiated fruiting body consisting of a vacuolated stalk supporting a mass of spores (spore head), stalk tube, and a basal disk. Finally, the spore germinates to form a new generation of ameba and thereby completes the life cycle [26 ] (highly instructive Dictyostelium information is available on the World Wide Web at http://dicty.cmb.nwu.edu/dicty/dicty.html).


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PARADIGM OF AMOEBOID MOTION, INCLUDING SHAPE CHANGE AND MIGRATION DYNAMICS
 
One of the earliest responses to starvation by amebae is that they start to secrete cAMP. cAMP leads to convergent chemotactic migration of the cells for aggregation and differentiation towards the mound and slug stage [15 ]. This phase of early single-cell movement has been used for extensive quantitative studies on the cell biology and molecular mechanisms of amoeboid crawling. Based on findings from the preaggregation stage of Dictyostelium, a five-step model of cell crawling was established and has been extended over the years [15 , 27 ]. These five phases form a cycling process that is initiated by pseudopod extension as a starting point [28 ]: (1) In initial actin nucleation, extrinsic or intrinsic signals are integrated by G-proteins and phosphoinosites (PIPs) leading to local actin polymerization at the leading edge; (2) during filament growth and pseudopod protrusion, as a result of actin polymerization, a pseudopod is formed and protruded; the development of a pseudopod results from elongation and cross-linking of polymerized actin to a viscous gel and unilateral swelling, prompting the outward pushing of the plasma membrane, extension of one or several leading pseudopods, and acquisition of a polarized cell shape; (3) during attachment, the pseudopod establishes an interaction towards the underlying substrate by low-adhesion mechanisms that, in the case of Dictyostelium, remains to be defined on a molecular level; (4) contraction by filament sliding occurs after attachment of the leading edge to the substrate and anterior elongation of the cell body; this contraction provides the force for translocation, and contractile force is putatively provided by myosin motors and additional mechanisms; (5) finally retraction and detachment of the cell rear occurs, during which localized release of adhesive bonds at the trailing edge allows the detachment and retraction of the rear end into the advancing cell body.

An important aspect of cell movement is how biochemical signals are integrated into characteristic and cell type-specific morphological dynamics. In resting Dictyostelium, the size of the cell body is 8–12 µm and the shape is spherical. After polarization and on crawling, the length axis averages 12–16 µm and can extend to 30 µm and more. In principle, the amoeboid morphology consists of an elliptoid core body with a broader leading edge and a narrowing trailing edge, termed the uropod. The overall morphology is highly reactive to external stimuli and can fundamentally change within seconds. Shortly before and on crawling, multiple pseudopods form around the leading edge and lateral portions of the cell body, while the uropod appears to remain a largely passive contractile zone. While the cell body advances, the bottom surface has been shown to maintain knobby foot-like actin-rich interaction zones ("eupodia") of 1 µm in diameter towards the underlying substrate [29 ], which, together with lateral adhesion sites, are thought to act as anchoring points. The average velocity varies from 4 to 12 µm/min, whereas accelerations in pseudopod extension result in peak velocities up to 25 µm/min [30 ]. If external chemotactic triggers are lacking, autochthonous pseudopodal oscillations and shape changes lead to short-lived forward movement alternating with rapid turns. Consequently, the directional persistence of nonchemotactically crawling ameba on nonaligned substrates is low, in agreement with the concept of persistent random walk [31 , 32 ].

In the natural environment, Dictyostelium cells migrate on or within three-dimensional (3D) complex substrates such as soil particles, fragmented leaves, and debris of very different physicochemical properties. The cells are able to move on humid as well as on dry substrates. Consequently, amoeboid migration must be a very robust process that is resistant to many adverse events. On the other hand, conventional laboratory substrates represent relatively smooth surfaces, such as agar, nitrocellulose filters, or cover slips, lacking biophysical constraints. As discussed below, these substrates might not mimic the natural environment closely enough, recently prompting investigations on reconstructed soil substrate [33 , 34 ].


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CYTOSKELETAL ORGANIZATION
 
The structure and dynamics of the cytoskeleton in Dictyostelium have been the subjects of intense investigations using both functional and genetic strategies [see reviews in ref 10 , 15 , 35 36 37 ]. It is likely that several molecular events converge in cytoskeletal dynamics and force generation.

Mechanisms of cell motility
Three different yet putatively synergistic mechanisms have been proposed and further studied that might contribute to cell motility: actin polymerization to filaments, myosin-mediated stiffening and contraction, and osmotic or hydrostatic force.

Actin polymerization
Actin polymerization occurs closely juxtaposed to the inner leaflet of the plasma membrane, possibly favored by random membrane fluctuations to be filled by expanding actin filaments. Actin, the main component of the microfilamentous cytoskeleton, exists as soluble, monomeric globular actin (G-actin) which homoaggregates to form filamentous actin polymers (F-actin). Actin polymerization is a directional process. Rapid aggregation of monomers occurs at one side of the filament (barbed end), while depolymerization is present at the other end (pointed end). Growing actin filaments elongate, branch, and form a viscous actin-rich gel ("gel" phase). This actin gel is characterized by a certain degree of stiffness and rigidity, allowing rapid membrane protrusion and pseudopod growth. From scanning electron microscopy studies detecting labeled actin monomers newly incorporated into the filaments, it appears that most of these barbed ends are directed towards the plasma membrane and, while growing, might generate an outward movement of the membrane [27 ].

Myosin sliding
The myosin sliding concept predicts that, also in nonmuscle cells, actin filaments can be cross-linked and contracted through myosin, generating both cortical actin stabilization and cell contraction. Myosin II (conventional myosin) forms bipolar filaments that cross-link actin filaments and stiffen cell protrusions [38 ]. Myosin II was further shown to mediate posterior contraction enabling the rear of the cell to detach from the substrate and move in concert with the leading edge [39 ]. Although myosin II-deficient cells are principally able to generate pseudopods and undergo locomotion on surfaces, the migration velocity as well as mechanical force generated at the leading edge of myosin II-deficient cells is reduced as a consequence of reduced cortical stiffness, delayed posterior release of adhesive bonds, and uropod retraction [40 , 41 ].

Cortical expansion
The cortical expansion model predicts that local osmotic pressure favors focal cytoplasmic swelling and pseudopod extension [35 ]. Osmotic pressure might be generated by ion influx at the leading edge mediated by ion channels [42 ] or the cytoplasmic release of osmotically active molecules. In addition, some hydrostatic pressure might be generated by cell contraction or uropod retraction that pumps cytoplasmic fluid into regions of increased elasticity. Net pressure of the aqueous phase could then lead to localized swelling and passive outward extension of a nascent pseudopod.

Role of cytoplasmic proteins in actin function
It is quite likely, that the above concepts synergize and can be integrated into one common scheme. In Dictyostelium, the molecular control of actin filament assembly and disassembly is provided by an ever increasing number of known cytoplasmic proteins, which bind to actin and control actin function. The following paragraphs highlight selected proteins, most of which share structural and functional homology to mammalian cells.

Capping proteins
Capping proteins bind to, protect, and sequester actin filaments. At the onset of filament growth, capping proteins dissociate from preexisting filaments and increase the number of barbed ends [43 ]. Capping proteins appear to deliver actin monomers to privileged actin nucleation centers, e.g., at the leading edge [44 ], thereby putatively confining the addition of actin monomers to growing filaments at special sites in time and space [45 ]. On the other hand, while reversibly capping older filaments, capping proteins also terminate polymerization. Capping proteins include gelsolin, protovillin, profilin, and Cap32/34. Actin uncapping and initial polymerization are rapid processes. After chemotactic stimulation, increased barbed ends are observed in Dicytostelium with 1-s delay [46 ], indicating that uncapping is a rapid process that initiates filament growth and remodeling. The importance of capping proteins in actin dynamics and remodeling is underlined by deletion mutants that develop exaggerated levels of polymerized actin while motility is impaired, putatively as a consequence of decreased actin turnover [47 ].

Severing proteins
Severing proteins bind to and intercalate between actin filaments causing filament breakage or actin monomer dissociation at the contact point. It is thought that repetitive filament severing keeps growing actin filaments at a certain length for network formation and also increases the number of freely accessible barbed ends for filament growth [summarized in ref. 48 ]. However, if G-actin levels are rate-limiting, severing will lead to depolymerization, whereas at high G-actin concentrations, nucleation and filament growth are supported [48 ]. Important severing proteins are members of the cofilin family (previously called actin-depolymerizing factors). In vitro and in vivo analyses have shown that cofilin severs filamentous actin thereby generating free barbed ends for G-actin binding and nucleation. Cofilin increases actin turnover rates [49 ], and, consequently, overexpression of cofilin leads to enhanced ruffling, pseudopod formation, and cell movement [50 , 51 ]. It was proposed that cofilin could recycle actin monomers to regions of nucleation by depolymerizing old filaments, thereby contributing to the remodeling of the actin cytoskeleton [44 , 52 ]. Other proteins exerting severing function are gelsolin, severin, and villin [53 ].

Branching and cross-linking proteins
Branching and cross-linking proteins bind to and intercalate between individual actin filaments and contribute to the formation of actin meshwork [54 ]. Cross-linking is important for the generation of a 3-D actin filament structure of increased mechanical strength and stiffness. Branching proteins include Arp2, Arp3, members of the filamin family [e.g., the 120-kDa gelation factor actin-binding protein (ABP)-120], and coronins. The actin-related proteins Arp2 and Arp3 are part of a seven-protein cross-linking and signaling complex localized in actin-rich spots within pseudopodia. The Arp2/3 complex was shown to be essential in actin network formation [55 ]. ABP-120 and coronin become localized in pseudopods of the leading edge and are thought to contribute to cross-linking and remodeling of cortical filaments in pseudopod extensions [56–59]. Other branching proteins are ABP-180 and spectrin. Cross-linking proteins, such as {alpha}-actinin, fascin, cortexillins, and calpactin cross-link actin filaments to form thicker bundles or meshwork of higher mechanical strength. Myosin II also cross-links actin filaments and contributes to cortical stiffness and pushing force in extending pseudopods [40 , 41 ].

Anchoring proteins
Anchoring proteins are candidate proteins connecting growing filaments to the inner leaflet of the plasma membrane. Anchoring proteins include Scar1, myosin I, and talin homologues. Scar1 is an important multifunctional adapter protein related to the Wiskott-Aldrich syndrome protein (WASP) family in mammals (see below). Scar1/WASP binds to membrane-inserted phosphoinositides and membrane-anchored activated G-proteins, then recruiting actin binding and branching proteins (e.g., Arp2/3) to the membrane [60 ]. Overexpression of a nonfunctional, dominant-negative Scar1/WASP fragment uncouples the actin cytoskeleton from the membrane and leads to complete loss of actin nucleation and lamellipodial extension. Myosin I ("unconventional myosin") is a candidate protein to anchor actin filaments to the plasma membrane predominantly at the leading edge. Myosin-I-deficient cells extend an increased number of pseudopods, indicating a role for myosin I in focusing and polarizing pseudopodal action [61 ]. Talin-like proteins are homologous to mammalian talin (see below), a protein that cross-links actin to transmembrane adhesion receptors [62 , 63 ]. Talin homologues localize to the tips of the filopodia, cross-link actin filaments, and are also involved in actin nucleation and assembly [62 , 63 ]. Other anchoring proteins are ponticulin, hisactophilin, synexins, and intercaptin [64 66 ].

There is a consensus that actin polymerization involves simultaneous and synergistic mechanisms including severing of preexisting actin filaments (cofilin), uncapping of preexisting nuclei by dissociation of capping proteins, and de novo nucleation (Arp2/3, Scar1/WASP) [28 , 48 , 55 , 67 , 68 ]. From a viewpoint of physical chemistry, the formation and turnover of actin-rich gels are sufficient to explain membrane protrusion and the generation of a certain degree of mechanical stiffness. In vivo evidence for this concept comes from observations on the intracellular propulsion of Listeria monocytogenes in infected eukaryotic cells. Listeria rods are pushed forward by a filamentous "tail" of endogenous host actin that forms at one end of the bacterium [reviewed in ref. 69 , 70 ]. A minimum of four components required for this actin-driven force generation were recently identified using purified proteins in cell-free extracts: actin, activated Arp2/3 complex, cofilin, and capping protein [71 , 72 ].

Several models have been proposed illustrating how these components might act together. Activated Arp2/3 induces branched nucleation and filament growth. Two actin sites have been discussed as targets of Arp2/3, the sides of older filaments (dendritic nucleation model) [68 , 73 ] and the barbed ends of actin filaments after uncapping (barbed-end nucleation model) [74 , 75 ]. Cofilin has been proposed to depolymerize pointed ends and to further accelerate actin incorporation into barbed ends, increasing the speed of filament growth [51 , 76 ]. The function of capping protein in this context is less clear. Capping protein could contribute to spatially limit and focus the polymerization process (funneling concept), e.g., by masking older filaments while sparing adjacent rapidly growing filaments [67 ]. Based on these concepts together with other in vitro as well as in vivo findings, actin polymerization alone might generate mechanical pushing force for pseudopod growth and protrusion.

It remains to be determined how the mechanical strength generated by such restricted actin assembly in vitro can be translated to the biomechanical requirements of entire cells. In vivo generation of actin filaments must be robust enough for higher-order shape change and mechanical resistance towards extracellular constraints. It is therefore likely that the biomechanics in complete cells require additional action of ABPs with overlapping function. In Dictyostelium, a single cell crawling across 2-D surfaces is a robust process that can be quite resistant to genetic ablation of structural and signaling proteins. A number of Dictyostelium deletion mutants for single ABPs (e.g., coronin, profilin, myosin IB, 120-kDa gelation factor, cortexillin, severin, cap32/34, {alpha}-actinin, or talin) have produced strikingly minor alterations in single-cell locomotion, whereas other processes such as phagocytosis, cytokinesis, and morphogenesis have been more frequently impaired [77 ]. One obvious explanation is that deleting a single or a limited number of proteins might not sufficiently impair the overall stability of the cytoskeleton, because functional compensation of the deleted proteins might be provided by remaining ABPs as a part of a redundant network. More frequently, loss of function in mutants with deletion of cytoskeletal proteins is obtained at the slug or spore stage, indicating that cell-cell and cell-substrate interactions involved in sorting and positioning at 3-D multicellular stages demand more stringent cytoskeletal action and control than biomechanically less demanding single-cell crawling [25 ].

A further critical parameter determining the molecular biomechanics of amoeboid crawling and positioning could come from the physical 3-D architecture and composition of the extracellular environment. Ponte et al. [34 ] have reported experiments on Dictyostelium gene deletion mutants (deletions of genes encoding, e.g., {alpha}-actinin, synexin, ABP-34 and ABP-120) using an artificial soil substrate that reconstructs an arguably more complex and natural in vitro environment. On reconstructed soil, these mutants display more profound locomotor and morphogenetic defects than were initially described for the same mutants using conventional laboratory substrates [34 ]. Therefore, it might be useful to investigate complex cell functions such as migration also in the context of biomechanically demanding "nature-like" 3-D environments.


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SIGNALING CONTROL OF ACTIN POLYMERIZATION
 
The best-defined mechanism leading to the initiation of cell polarization and induction of migration is outside-in signaling via chemoattractant receptors. Chemotaxis is defined as the directional cell movement towards an increasing concentration of a soluble factor, the chemoattractant or chemotaxin. Dictyostelium shows chemotaxis towards a number of compounds, and the best-characterized response is that to cAMP [78 ; reviewed in ref 26 , 79 ]. The cAMP receptors (cARs) belong to the family of serpentine/G-protein-coupled receptors with homology to serpentine chemoattractant receptors in higher eukaryotes. Cloning and deletion of four cARs in Dictyostelium (cAR1–4) shows that high-affinity cAR1 and cAR3 are essential for chemotaxis and aggregation at the single-cell stage [80 ]. Cells lacking cAR1 and cAR3 fail to develop cAMP-induced chemotaxis and aggregation [81 ]. cAMP is secreted by the cells in a paracrine fashion to neighboring cells at nanomolar oscillatory pulses with a periodicity of several minutes [36 ]. After receptor binding, an activation peak within the first minute is followed by a few minutes of unresponsiveness. cAMP then becomes rapidly degraded by a membrane-bound phosphodiesterase allowing resensitization and ensuing chemotactic cycles.

For the integration of an external chemotactic stimulus towards cell polarization and directional crawling, triggering should occur in a polarized manner, e.g., by preferential signaling and cytoskeletal activation in the cell compartment proximal to the highest chemoattractant concentration [79 ]. Preferential chemoattractant-induced signaling at the leading edge would require the polar redistribution and/or clustering of chemoattractant receptors or, alternatively, G-proteins or downstream effectors. Whereas in polarized Dictyostelium cells, cARs remain uniformly distributed, the signal transducing G-protein Gß{gamma} shows preferential accumulation at the leading edge [82 ]. Polarized G-protein activation is thought to generate downstream signaling towards different effectors, most notably to phosphatidyl-inositol-kinases (PIKs) and small GTPases [79 ]. In principle, PIKs and small GTPases synergize for downstream signaling by recruiting multiple other signaling and cytoskeletal proteins to the inner leaflet of the plasma membrane, leading to actin nucleation, polymerization, and turnover.

Accumulating evidence suggests that PIPs form a major structural link between the plasma membrane and the actin cytoskeleton. PIPs are generated by PIKs from membrane phospholipids inserted in the inner leaflet of the plasma membrane. Important PIKs include phosphatidyl-inositol-3-kinase (PI3K) and type I phosphatidyl-inositol-5-kinase (PI5KI). PI3K has been shown to be directly activated by Gß{gamma} [83 ]. Activated PI3K, in turn, phosphorylates membrane PIPs at the 3-OH position as well as other effectors at serine/threonine residues [84 , 85 ]. The predominant PIP phosphorylation products phosphatidylinositol-3,4-bisphosphate (PIP2) and phosphoinosite-3,4,5-monophosphate (PIP3) are thought to form multimeric aggregates inserted in the inner leaflet of the membrane bilayer [86 87 88 ]. Clustered PIP2 and PIP3 can be directly bound by proteins that contain either a pleckstrin homology (PH) domain, Src homology (SH) domains (e.g., SH2, SH3), or other PIP-binding domains [89 ]. Several ABPs, including Scar/WASP, gelsolin, phdA (PH domain containing protein I), and akt/PKB (also known as Rac protein kinase or protein kinase B), as well as the signaling molecules guanine nucleotide exchange factors (GEFs) [90 ] and phospholipase C{gamma} [45 ], have been shown either to contain such PIP-binding domains or to directly bind PIPs [89 ]. By interacting with PIPs, these proteins link the plasma membrane to the cortical cytoskeleton and cytoplasmic-signaling apparatus. Within seconds after stimulation by cAMP, akt/PKB [87 ], phdA [85 ], Scar1, and other proteins accumulate at the leading edge, and it has been predicted that this recruitment is PIP dependent [79 , 85 , 86 , 89 , 91 ]. The amount of cortical actin polymerization is proportional to the generation of PIPs, suggesting that PIPs favor actin polymerization [92 , 93 ]. In reverse, as PIP levels fall, the reaction of filament growth is reversibly terminated [92 ], which is consistent with findings on Dictyostelium mutants that lack PI3K or cells treated with PI3K inhibitor. These cells, presumably because of insufficient PIP generation, fail to recruit pkB, phdA, and other adapters to the leading edge [85 ]. As a consequence, PI3K-deficient cells fail to rapidly assemble F-actin at the leading edge and to polarize, produce fewer pseudopods, and do not undergo chemotaxis normally [85 ]. Taken together, local generation of PIPs and consecutive recruitment of actin binding and regulatory factors initiate and maintain uncapping, actin anchoring, and branching for spatially regulated filament growth.

PIP generation is further controlled by other signaling pathways that include low-molecular-weight GTPases of the Ras and Rac family, src-related tyrosine kinases, and other kinases. Besides initiating actin polymerization, PIPs have been shown to induce downstream signaling towards mitogen-activated protein kinase/extracellular regulated kinase pathways, Ras and Rac pathways, and protein kinase A- and cGMP-dependent effectors [26 , 94 ]. This suggests that the generation of PIPs and membrane-proximal actin polymerization accompany quite different stimuli and signal transduction pathways.

Another important PI3K substrate in Dictyostelium is Akt, the homologue to mammalian protein kinase B (akt/PKB). After phosphorylation, Akt/PKB becomes localized to the leading edge [95 , 96 ]. In turn, Akt/PKB can phosphorylate multiple effector proteins, regulating not only chemotactic response but also endocytosis, gene expression, and apoptosis [84 ]. Akt/PKB-deficient Dictyostelium cells, similar to cells lacking PI3K function, exhibit a polarization defect, aberrant chemotaxis, and reduced aggregation capacity [87 ].

The Ras and Rac family of small GTPases is involved in various pathways controlling the actin cytoskeleton. Dictyostelium cells express >40 different members of the Ras and Rac subfamily of GTPases [for complete list, see ref. 97 ] and the related GEFs. Ras and Rac GTPases interact with multiple cytoskeletal and adapter proteins that control cytoskeletal remodeling and cell motility, including ruffling, polarization, cortical stiffness, and cytokinesis [97 ]. Rac GTPases contain a PH domain for membrane localization and putative PIP binding on chemotactic response [98 , 99 ], although this feature remains to be confirmed for Dictyostelium. More than 20 downstream targets of Rac GTPases have been reported so far, among them Rho-dependent serine/threonine kinase (ROCK), myosin light-chain phosphatase, and gelsolin [100 , 101 ]. Many of the GTPases have been genetically ablated in Dictyostelium resulting in defined loss-of-function phenotypes. As examples, deletion of the small GTPase RasG, Rac1A, or Rac1b leads to severe impairment of cell polarity and single-cell movement [97 , 102 ]. On the other hand, deletion of RasS or its related exchange factor, RasGEFB results in a threefold increase in migration velocity compared with that of wild-type cells [103 ]. How downstream effectors mediate these different effects remains to be determined. Many mutations of GTPases show no apparent defects in single-cell crawling; however, they severely affect the sorting and differentiation process at the multicellular stages [97 ]. It is therefore likely that each Ras and Rac GTPase controls quite specific cytoskeletal events at a given developmental stage.

Besides its occurrence on chemotaxis, pseudopod extension has been suggested to occur as a spontaneous process involving constitutive oscillation of signaling molecules and cyclic baseline actin polymerization and depolymerization. High-resolution morphometric analysis of shape changes demonstrates that Dictyostelium cells are intrinsically vibrating bodies, transited by "self-organized, superpositioned, harmonic modes of rotating oscillatory waves" [104 ]. These morphological oscillations appear to be associated with intrinsic physicochemical oscillations of actin polarization leading to pseudopodal extensions and retractions [104 ]. In other words, the contractile apparatus of Dictyostelium exhibits a certain degree of autochthonous rhythmicity. In the absence of external triggers, this inherent oscillatory apparatus can lead to constitutive, nondirectional crawling that, as mentioned above, follows a model of persistent random walking [31 ].


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ADHESION AND DETACHMENT MECHANISMS IN METAZOAN CELLS AND COMPARISON TO DICTYOSTELIUM
 
An important issue in exploring cell movement is the control of cycling adhesion and de-adhesion events that couple the cell body to the extracellular environment. Although an array of adhesion mechanisms and intracellular effectors have been identified in metazoan cells over the past decade [105 , 106 ], the molecular basis of adhesion to extracellular structures is largely elusive in Dictyostelium. Because the integration of molecular adhesion and de-adhesion events required for migration has been predominantly established for fibroblasts and keratinocytes [11 , 101 , 107 , 108 ], we here summarize these metazoan migration models first and then discuss their implications for crawling Dictyostelium ameba.

Haptokinetic migration
In the model of haptokinetic (i.e., adhesion-dependent) migration of fibroblasts across ligand-coated surfaces [11 , 17 , 109 ], migration efficiency follows an inverse-U function relative to the adhesive force between the cell and the underlying substrate (Fig. 1A ). Maximum migration rates are achieved at intermediate strength of adhesion to the substrate [17 ], allowing the formation of new interactions at the leading edge, while preexisting bonds are sufficiently released at the trailing edge (Fig. 1A , ). Increasing adhesion, e.g., by increasing ligand density or by up-regulating adhesion receptor density, leads to locomotor reduction (Fig. 1A , ), as a consequence of impeded or delayed detachment form the rear [18 , 110 , 111 ]. On the other hand, reducing substrate adhesion to a minimum, e.g., by decreasing ligand density or by adhesion-perturbing antibodies, causes a loss of contact to the substrate [Fig. 1A , ). As net interaction and traction forces are further lowered, partial detachment and rounding up of the cells are accompanied by nonproductive oscillatory membrane dynamics and impaired migration (Fig. 1A , ) [101 , 112 ]. Besides fibroblasts and keratinocytes, the haptokinetic migration model on 2-D substrate has been confirmed for myoblasts migrating across a ligand-coated surface [113 ], as well as for fibroblast-like cells (e.g., melanoma cells) penetrating 3-D collagen matrices [21 ].



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Figure 1. Migration of fibroblasts (A) and leukocytes (B) on 2-D and within 3-D environments as a function of adhesive strength to the substrate (C). (A) Haptokinetic migration model as established from fibroblasts [see ref. 11 17 ]. In migrating fibroblasts and cells utilizing fibroblast-like migration strategies, migration efficiency in both 2-D and 3-D environments is similarly dependent on adhesive interaction to the substrate. Three prototypic migratory states are indicated by numbers. (B) Four different migratory states observed for leukocyte migration on 2-D and within 3-D substrates. As a major difference, leukocyte crawling might persist in 3-D environments after adhesion blocking (B, ), whereas fibroblast-like migration is dependent on adhesion in both 2-D and 3-D migration models (A, ). (C) Leukocyte adhesion conditions and resulting morphology, as deduced for each functional state. References correspond to migrating leukocytes only.

In mammalian cells, adhesion and de-adhesion events are represented by the assembly and disassembly of transmembranous junctions containing adhesion receptors, extracellular ligands, and cytoskeletal elements, termed either focal adhesions or focal contacts. These junctions control cytoskeletal assembly and turnover, adhesion and migration, and related signaling.

Focal adhesion
Focal adhesions contain adhesion receptors (in mammals, integrins) and cytoskeletal and signaling molecules in multimolecular complexes of 0.5–>2 µm in diameter [114 ]. Integrins bound to extracellular ligands become linked to the actin cytoskeleton via several adapter and signaling proteins, such as talin, {alpha}-actinin, filamin, focal adhesion kinase, and others [114 , 115 ]. Fully matured focal adhesions represent relatively stable cell-substrate interactions that persist for longer periods. Long-lasting contact leads to integrin-ligand strengthening and bundling of preexisting actin filaments in parallel to longitudinal thick actin bundles, termed stress fibers, inserting into these attachment zones. The formation of fully matured focal adhesions and stress fibers can be induced by activated low-molecular-weight GTPase Rho via downstream effector ROCK [101 , 116 ]. Stress fiber formation is counteracted by actin-severing proteins such as cofilin or gelsolin [117 ], as shown by inhibitory targeting of severing activity. In fibroblasts, Rac has been shown to mediate cortical actin turnover and filopodal ruffling via activation of gelsolin, thereby preventing stress fiber formation [100 ].

Focal adhesions and stress fibers appear to maintain a critical basal adhesion strength and traction. In slowly spreading and/or migrating cells, focal adhesions develop within 10 to 20 min, which is a time-consuming process [114 ] resulting in relatively stable and long-lived interactions towards the substrate that are inversely proportional to pseudopodal dynamics [117 ] and migration speed [11 , 18 , 101 ]. This does not exclude that slow turnover of focal adhesions and stress fibers can contribute to slow motion by gradual translocation. On promigratory stimulation, however, fibroblasts and keratinocytes disassemble preformed stress fibers and focal adhesions, decrease adhesiveness, and, consequently, increase the turnover of adhesion receptors for migration [118 ]. In mammals, the control of focal adhesion and stress fiber disassembly has been shown for the Rho family member Rnd1 [112 ], counteracting stress fiber assembly by the Rho and ROCK pathways [101 ]. Rnd1 appears to favor cell detachment from the rear, increasing migration speed; however, if Rnd1 is constitutively active, substrate adhesion becomes disrupted, and the cells round up, detach, and stop migration [112 ].

Focal contact
The focal contact is a smaller, less developed, and more transient relative to focal adhesions [114 ]. Focal contacts contain smaller clusters of adhesion receptors and a reduced array of cytoskeletal and signaling elements, which are not linked to stress fibers but rather to a more diffuse cortical F-actin [114 ]. Focal contacts are considered to represent more dynamic junctions predominantly under the control of Rac and Cdc42 [101 , 119 ].

Highly dynamic surface structures, such as microvilli, filopodia, and pseudopodia, frequently display no apparent structural-compartment formation of proteins in the plasma membrane. Here, F-actin, adhesion receptors, and signaling molecules show a rather diffuse distribution, indicating high molecular dynamics, lateral mobility, and turnover of protein-protein interactions.

Detachment mechanisms
Membrane flow models for locomotion predict that membrane extension at the leading edge requires the insertion of new membrane from intracellular sites, such as intracellular vesicles [120 ]. Endosomes containing internalized adhesion receptor and plasma membrane are formed at the trailing edge and traffic towards the leading edge for receptor and membrane recycling at pseudopod protrusions [120 ]. For crawling Dictyostelium cells, endocytosis and turnover rates of fluorescently tagged surface membranes have been established in the range of one cell surface equivalent every 4 to 8 min [121 ], indicating high membrane turnover rates.

If high substrate adhesivity counteracts receptor release from the substrate, as seen in migrating fibroblasts and tumor cells, detachment can be further achieved by the deposition ("shedding") of adhesion receptors, such as integrins and CD44 [19 , 110 , 122 ]. One mechanism of receptor shedding is proteolytic cleavage of cytoskeletal anchorage points by the protease calpain as well as the cytoplasmic portion of integrin adhesion receptors . Calpain cleaves the adapter proteins talin and paxillin [125 , 126 ]. By degrading cytoskeleton-membrane junctions, calpain is considered an important regulator of focal adhesion disassembly and cell detachment in fibroblasts [18 ]. Other cells, however, such as crawling leukocytes, appear to detach independently of calpain-mediated cleavage, suggesting that rapidly moving amoeboid leukocytes could use different mechanisms to regulate adhesive release at the cell rear than more adhesive, slowly moving cells [18 ].

Adhesion receptors in Dictyostelium
Although the generation of pseudopodal extension is well understood in Dictyostelium, candidate adhesion receptors in regions that contact the underlying substrate remain to be identified [127 ]. So far, neither tyrosine kinase receptors nor integrin homologues have been identified in Dictyostelium. Known transmembranous adhesion receptors include gp80/contact site A (csA) [33 , 128 ], gp24/DdCAD-1 [129 ], gp150 [130 ], and DTFA (defective in tip formation) [131 ]. gp80/csA mediates EDTA-resistant homotypic end-to-end adhesion in aggregating cells, which can be blocked by antibody [128 ]. In addition, gp80/csA-defective cells develop increased substrate adhesiveness and delayed migration (compare state , Fig. 1A ), suggesting a function of gp80/csA in the regulation of de-adhesion [33 ]. gp24/DdCAD-1 is a secreted nontransmembrane protein with sequence similarities to mammalian cadherins which binds to the extracellular surface and mediates calcium-dependent cell-cell adhesion in early morphogenesis [132 ]. gp150 is a candidate receptor involved in the sorting of prestalk and prespore cells at the mound stage [130 ]. DTFA is involved in cell-cell interaction at early aggregation as well as a later sorting stage [131 ]. So far, none of these receptors have been implicated in binding to the underlying substrate and in single-cell crawling or chemotaxis. Because specific adhesion receptors remain to be identified in Dictyostelium, it is difficult to relate the mechanism of amoeboid substrate interactions with principles of haptokinetic migration to mammalian cells. In nature, Dictyostelium is confronted with substrates of very diverse, nonuniform physicochemical properties, such as leaves and soil. It therefore remains subject to speculation whether engagement of specific adhesion receptors would be a useful strategy for interaction with such diverse environmental substrates after all.

Lack of focal adhesions/contacts in Dicytostelium
The focal-adhesion model has been mainly established using cells from higher eukaryotes, whereas in Dictyostelium amebae, adhesion receptors that mediate contact to substrates other than cells are unknown. In migrating Dictyostelium cells, on-off rates of cell substrate interactions are high (in the range of seconds to minutes), whereas long-lasting stable attachment to the underlying substrate is lacking. Cell propulsion and traction appear to result from highly volatile, low-affinity interactions rather than stable substrate binding. Consequently, focal adhesion- or focal contact-like structures are not formed, which is consistent with the concept that migration dynamics prevent focal adhesion formation and vice versa. Genetically modified ponticulin-deficient Dictyostelium cells lack approximately 90% of the normal number of high-affinity interactions between actin filaments and the plasma membrane, but migration and phagocytosis exhibit no functional alterations [65 ]. Together, these morphological and functional characteristics indicate a low degree of cytoskeletal-compartment formation coupled to low-affinity interactions between cytoskeleton, plasma membrane, and extracellular substrate. Besides in Dictyostelium, such diffuse membrane and cytoskeletal organization appears as a general feature present in fast moving leukocytes and other cells in the absence of focal contacts and stress fibers [4 , 14 , 104 , 133 ]. One conclusion of the above findings is that the cellular and molecular migration mechanisms diverge between amoeboid crawling and more adhesive haptokinetic migration strategies in fibroblasts and other cells [14 ].


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GENERAL LOCOMOTOR CHARACTERISTICS IN CRAWLING LEUKOCYTES
 
Many aspects of morphodynamics, such as cell movement, pseudopodal extension and retraction, cytoskeletal organization, phagocytosis, cell sorting, pattern formation, and the response to external stimuli, are common among Dictyostelium and higher eukaryotic cells. Leukocytes, similarly to Dictyostelium at preaggregation stage, spend most of their life cycle as amoeboid cells. Leukocytes chemotactically respond to external stimuli, perform phagocytosis, and contact other cells present within peripheral tissues. After passively circulating in the blood, leukocytes become rapidly recruited into the tissues by activated endothelium of microvessels. Leukocyte-endothelial interactions involve multiple specific adhesion molecules, chemokine signals, and biophysical events that lead to leukocyte spreading on the surface of the endothelium [reviewed in ref. 1 , 134 ]. The ensuing transmigration requires the widening of endothelial cell-cell junctions, profound morphological dynamics (squeezing), and penetration of the basement membrane followed by a long-lasting migratory response within the adjacent tissue [135 ]. Although this initial transendothelial step is a special feature of circulating bone marrow-derived cells in higher eukaryotes and lacks a clear equivalent in ameba, the crawling of leukocytes within tissues or across cells shares many characteristics of migrating Dictyostelium cells.

T cell and neutrophil polarization and migration follow a stereotypic intrinsic program in response to different external stimuli. These include chemokines, chemical or mechanical triggers, increase in temperature, contact with ECM ligands, or physical contact with other cells (e.g., endothelium and antigen-presenting cells) [2 , 6 , 136 , 137 ]. Similar to Dictyostelium, migrating leukocytes adopt a polarized morphology (Fig. 2A ) consisting of a leading edge (Fig. 2A and 2B , asterisk) that generates one or several anterior lamellipodia, an elliptoid cell body containing the nucleus, and a tail-like uropod (Fig. 2A , black arrowhead). Both in vitro and in vivo, this prototypic morphology is conserved among different leukocytes, including T cells [4 ], neutrophils [138 ], and dendritic cells [139 , 140 ]. Leukocytes display an extraordinary deformability suitable for flexible adaptation to biophysical constraints of the substrate, such as pores and spaces [141 ]. While the cytoplasm in locomoting leukocytes is flowing forward, lateral protrusions ("footholds") move backwards relative to the advancing cell body (Fig. 2B) .



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Figure 2. Amoeboid motion of a T lymphocyte within a 3-D collagen matrix. Polarized morphology, squeezing, shape change, and directional alterations of a CD4+ T cell in spontaneous locomotion after staining with calcein-AM, as assessed by confocal time-lapse reflection and fluorescence microscopy (movie can be viewed at http://www.xxx.html). Frames were taken at 20-s time intervals and reconstructed. Numbers represent individual time points (seconds). The collagen lattice was reconstructed from confocal reflection contrast (6 individual sections of 1.5 µm in depth) [230 ]. The central section of the cell body was integrated into the reconstructed fiber network at the actual cell position in z direction and displayed by false color imaging. Symbols in frame A indicate leading edge (*) and uropod (black arrowhead). In frames C and D; cell trapping within a region of narrow fibers leads to temporary stopping by obstruction (C–E, white arrowheads), whereas oscillating shape change is continued for ~200 s. From frames E to F, pseudopod extension (white arrow) at a lateral portion of the cell body initiates the process of circum-migration across a horizontal fiber in the absence of structural remodeling of the matrix structure. Variations of overall fiber architecture from frame to frame result from manual focussing of the microscopic stage in depth while the cell is crawling. Scale bar, 10 µm. _art;1>

As in Dictyostelium, stress fibers and focal contacts are lacking in crawling neutrophils [142 ] and T cells [4 ], indicating high turnover of individual filaments. To measure physical forces generated by migrating cells, spherical micropipette systems have been used to apply pressure to different cell compartments, as an estimate for stiffness generated by the cell [143 ]. The rigidity of the cell body in migrating leukocytes is relatively high, albeit the traction forces are 10- to 30-fold smaller than those detected in fibroblasts. The deformability or stiffness of the plasma membrane in nonmotile B cells is in the range of 0.1–0.2 µdyn, which is increased after cross-linking of surface receptors by lectin to 0.4–0.6 µdyn [144 ]. In comparison, forces generated by individual fibroblast ruffles are in the range of 15–30 µdyn [143 ]. The deformability of polarized neutrophils is in the range of 1 µdyn, as measured from the retention force of membrane-bound magnetic beads at single locations [145 ], whereas the total force generated by a complete migrating neutrophil has been estimated in the range of 3 mdyn [146 ]. High elasticity at the leading edge coupled to cortical stiffness and low-traction forces allows migration velocities in leukocytes similar to those in Dictyostelium, which are 10- to 30-fold higher than in fibroblasts and other cells. Average migration velocities are 6–8 µm (peak velocity, 22 µm/min) for T cells [147 ], 2–6 µm (peak velocity, 10 µm/min) for dendritic cells [148 ] and 8–16 µm (peak velocity, 28 µm/min) for neutrophils [149 ]. The velocities as deduced from low-adhesion surfaces or 3-D in vitro matrices are quite similar to the values obtained from in vivo observations, e.g., an average of 8–12 µm/min for neutrophils crawling through the loose connective tissue of the rat mesentery [149 ] or the rabbit ear [140 ]. In summary, simple shape, polarized morphology, rapid oscillatory pseudopod dynamics, and high migration velocities are common features among leukocytes and Dictyostelium ameba.


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STRUCTURE AND FUNCTION OF THE CYTOSKELETON IN LEUKOCYTES
 
The organization of the actin cytoskeleton in crawling leukocytes shares many similarities with the cytoskeleton in Dictyostelium. Most extensive information is available for intact neutrophils or neutrophil cell extracts, and some results have been confirmed for T lymphocytes. In principle, the five-step process of pseudopod extension and attachment to substrate, cell polarization, contraction of the cell body, the posterior release of attachment sites, and uropod retraction have been demonstrated in neutrophils [16 ] and to some extent in T cells [150 ]. Different sets of structural and regulatory actin binding proteins have been identified to provide filament growth, severing, branching, and cross-linking in mammalian cells, most of which are homologous to actin-binding proteins in Dictyostelium.

Capping protein homologues controlling initial filament unmasking include gelsolin, villin, radixin, capZ, adseverin, and {alpha}-actinin. Filament severing is achieved by cofilin, depactin, and actophorin [151 ]. Similar to Dictyostelium, Arp2/3 acts as a key nucleator of branched actin polymerization in neutrophils [68 , 152 ]. Other homologous branching proteins, such as filamins (homologues to ABP-120 and ABP-280 in Dictyostelium) and spectrin also contribute to branching and formation of a filament meshwork [151 ]. Filament cross-linking to parallel bundles is provided by fimbrin, {alpha}-actinin, and calpactin. Anchoring to membrane receptors occurs through filamin, talin, and {alpha}-actinin. Filamin, talin, and {alpha}-actinin have been shown to directly interact with the cytoplasmic portion of ß1 and ß3 integrins and, to some extent, ß2 integrins, representing major structural links between the actin cytoskeleton and integrins [115 , 153 154 155 ].

The kinetics and spatial distribution of actin assembly and disassembly are very similar in leukocytes and Dictyostelium. In nonmigrating spherical neutrophils, F-actin comprises approximately 30% of total actin and is diffusely distributed throughout the cytoplasm [156 ]. On chemokine triggering, initial actin polymerization occurs within <15 s [156 ]. After 30 s, the content of F-actin has doubled, and most newly assembled actin is detected in the proximity to the emerging leading lamella [156 , 157 ], whereas the cell body and uropod contain a relatively stable cortical rim of F-actin for polarity and stability [158 , 159 ]. In electron microscopic studies from neutrophil cytoplasmic lysates, individual actin filaments elongate rapidly to reach a maximum length after 15–30 s of 0.5–2 µm [158 , 160 ]. Actin filaments at the leading lamella are rapidly remodeled based on polymerization and depolymerization [158 ]. After removal of chemoattractant, actin depolymerization to basal levels is reached within 3–10 s, preferentially at the leading edge [158 ]. This time scale is consistent with in vitro estimation of actin turnover in the range of 10–30 s [161 ].

Similar to neutrophils, in T cells, chemoattractant-induced onset of migration is complete after 1–2 min [136 ]; however, more detailed information on actin filament structure and regulators of polymerization in T cells is sparse. In nonmigrating T cells residing within a 3-D collagen matrix, some focal nucleation of actin appears at contacts to collagen fibers [162 ]. With the onset of T cell locomotion, cortical F-actin is present along the rim of the cell body and is enriched in the uropod [162 ], whereas F-actin distribution is highly variable at the leading edge [4 , 159 , 162 ]. Taken together, in crawling leukocytes polymerized actin forms a highly dynamic and flexible leading edge in conjunction with a relatively stiff polarized core body and uropod.


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SIGNALING CONTROL OF CYTOSKELETAL DYNAMICS IN LEUKOCYTES
 
The molecular components controlling the actin cytoskeleton in leukocytes are largely homologous to those of Dictyostelium [15 , 163 ]. Although cAMP as a compound is not a common chemoattractant in higher eukaryotes, the molecular mechanisms of cAMP-induced signaling and induction of migration are prototypic for most other chemoattractants in mammalian cells. In neutrophils, numerous factors can initiate actin filament nucleation and growth. Examples include chemoattractants, growth factors, extracellular matrix, and multimeric or patterned molecule complexes, such as lectins, phagocytic particles, the surfaces of other cells, and other hydrophilic solid-phase substrates (e.g., nitrocellulose filters). Similar to mechanisms in Dictyostelium, the signals generated by chemokine receptors and associated heterotrimeric G-proteins lead to the activation of PI3K and/or PI5KI. Besides chemokine receptors, PIKs can be activated by Fc receptors, complement receptors, integrins, or, in T cells, triggering of the T cell receptor (TCR) [164 ]. PIKs generate PIP2 and PIP3 inserted in the inner leaflet of the membrane, thereby recruiting cytoplasmic actin-binding and -signaling molecules to the plasma membrane [165 , 166 ]. In neutrophils, an ever-increasing number of ABPs have been described that are recruited to the membrane by PIPs. These include gelsolin, profilin, ezrin, moesin, phospholipase C-{gamma} or -{delta}3, akt/PKB, Vav, and WASP [reviewed in ref. 167 ]. The importance of PIKs in actin polymerization and leukocyte motility has been shown using PI3K inhibitors. The PI3K inhibitor wortmanin leads to severe impairment of polarization and chemotaxis in neutrophils [168 , 169 ] and T lymphocytes [170 ], indicating that, similar to that in Dictyostelium, PI3K is a key regulator of actin dynamics and motility in leukocytes. Conversely, exaggerated PIP formation might overstate action polymerization and delay turnover. PI5KI overexpression in migrating fibroblasts greatly delays detachment and migration rates, suggesting that balanced amounts of PIPs are important for cytoskeletal assembly, force generation, and filament turnover [117 ].

WASP and N-WASP, homologues to Scar1 in Dictyostelium, are important for the induction of actin dynamics and migration in leukocytes and other cells [171 ]. (Wiskott-Aldrich syndrome is an inherited disorder in humans, caused by a deficiency of WASP protein function. The lack of WASP leads to severe defects in leukocyte and platelet function such as recurrent pyogenic and opportunistic infections, eczema, and thrombocytopenia. The life span of affected individuals is reduced.) WASP directly interacts with Cdc42, as shown by yeast two-hybrid screening, and it further has a PH domain for PIP binding [172 ]. After binding Cdc42/PIP2 complex at the inner leaflet of the membrane, WASP becomes activated [173 ] and recruits and activates Arp2/3 to form an actin nucleation complex for branched nucleation and filament growth [174 ]. Besides Cdc42 and PIP2, other factors have been proposed as candidate regulators of WASP activity, including profilin, phospholipase C-{gamma}, and proteins containing SH2 and SH3 motifs (e.g., Grp2 and src kinases) [175 ].

Similar to those in Dictyostelium, small GTPases are central regulators of actin polymerization and motility in higher eukaryotes. Mammalian Rho GTPases share structural and functional homology to Rac homologues in Dictyostelium [97 ]; however, only three family members, Rac, Cdc42, and Rho, have been identified [98 , 116 ]. Rho GTPases are activated by chemoattractant receptors, integrins, or growth factor receptors [176 , 177 ] as well as by intracellular signaling via src-related tyrosine kinases (Syc), the nuclear exchange factor Vav, or other small GTPases (Rho and Ras pathways) [119 , 178 ]. Rac and Cdc42 synergize for the induction of dynamic cytoskeletal events, i.e., the formation of ruffles and pseudopodia [116 ]. Rac forms a complex with PI5KI that is considered to increase PIP2 levels in the membrane [166 , 179 ]. Downstream effects of Rac to cytoskeletal dynamics and remodeling could be mediated by Rac-induced dissociation of capping proteins (e.g., gelsolin) from actin filaments to initiate filament growth [180 ]. Cdc42 binds PIP2 and WASP, as outlined above for the initiation of actin polymerization [181 ]. This is consistent with the finding, that, in T cell lines, dominant-negative Cdc42 abrogates chemokine-induced directional polarization and chemotaxis [182 ]. Because both contribute to actin filament growth, Rac and Cdc42 are considered candidate initiators and regulators of migration-driving membrane-cytoskeleton junctions of high turnover, such as ruffles, filopodia, and lamellipodia [98 116 ] (In contrast to Rac and Cdc42, Rho is involved in focal adhesion assembly and the maturation of actin filaments into stress fibers, putatively via downstream activation of the serine/threonine kinase ROCK [116 , 119 ]. Constitutively, active Rho exaggerates stress fiber and focal adhesion formation, thereby retarding migration in motile fibroblasts [101 ]. This phenotype is similar to the effect of PI5KI overexpression leading to exaggerated PIP production and actin polymerization [117 ]. Conversely, reduction of Rho or ROCK activity leads to the loss of stress fibers while polarized ruffling remains fully intact, thereby increasing migration rates [101 , 117 ]. This is consistent with the effects of Rho inhibition in immobilized monocytic cells, leading to greater spreading dynamics and membrane oscillations [183 ] and suggesting that reducing Rho activity favors dynamic cell-substrate interactions. Last, however, some degree of Rho activity may be essential in maintaining minimum contact strength to the substrate, because maximum inhibition of Rho can commit de-adhesion and loss of locomotion as a secondary event [101 ] (corresponding to Fig. 1 A , ).

In summary, similar to Dictyostelium, PIKs, WASP, and Rho GTPases represent important effectors controlling actin polymerization and motility. As one difference, however, mammalian cells simultaneously integrate outside-in signaling from chemoattractant receptors, ECM-binding integrins, and other receptors, whereas in Dictyostelium at single-cell stage, chemoattractant receptor-mediated signals appear predominant.


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FUNCTION OF ADHESION RECEPTORS IN LEUKOCYTE MIGRATION
 
In metazoan cells, the major ECM-binding cell surface receptors that mediate adhesion and migration are members of the superfamily of integrin receptors. Integrins are heterodimeric receptors consisting of a set of {alpha} chains noncovalently associated with a limited number of ß chains. The extracellular integrin domain binds to ECM components or to ligands on other cells, whereas the cytoplasmic portion reversibly connects to cytoskeletal and signaling proteins [reviewed in ref. 105 184 ]. On cell activation, integrins can become "activated" by intracellular signals as well as up-regulated or de novo expressed. In the absence of expression regulation, integrins may undergo conformational change leading to increased binding strength towards monomeric ligands ("affinity regulation") [185 ]. In addition, binding to multimeric ligands such as ECM macromolecules or an adjacent cell surface triggers lateral integrin aggregation and clustering in the plasma membrane, which locally increases substrate-binding avidity in the absence of affinity change ("avidity regulation") [186 ]. Integrin occupancy and clustering initiate recruitment of ABPs (e.g., talin, filamin, {alpha}-actinin, and paxillin), as well as signaling molecules (e.g., Rho GTPases, PI3K, focal adhesion kinase, and src-kinases), to the plasma membrane to form focal contacts or adhesions [105 ]. Integrin-mediated signals regulate further integrin apposition, actin assembly, cell polarity, and migration [119 ].

Nonactivated naive T cells and neutrophils express low levels of ß1, ß3, ß4, and ß7 integrins on the surface, whereas ß2 integrins are constitutively expressed at high levels [187 , 188 ]. Integrins that bind interstitial collagen, i.e., {alpha}1ß1, {alpha}2ß1, {alpha}3ß1, and {alpha}vß3, are either not detectable or expressed at very low levels in naive T cells and neutrophils [187 ]. Integrin {alpha}vß3 additionally binds to vitronectin and fibronectin [189 ]. Other ECM components, such as fibrinogen and fibrin, are bound by {alpha}Lß2 or {alpha}Mß2 integrins [190 ]. Within minutes after activation, neutrophils up-regulate the surface expression of ß1 integrins as well as {alpha}Lß2, {alpha}Mß2, and {alpha}vß3 integrins from cytoplasmic stores [191 192 193 194 ]. In contrast to neutrophils, T cells require long-term activation (>2 days) to up-regulate most adhesion receptors (e.g., ß1, ß2, ß3, and ß7 integrins), whereas others are newly expressed, such as the collagen receptors {alpha}1ß1 and {alpha}2ß1 [187 ].

Both subsets, constitutively expressed as well as up-regulated integrins, cooperate to mediate adhesion and migration in the tissue. If compared with 2-D haptokinetic migration of fibroblasts (Fig. 1A) , the interdependence of adhesion receptor-mediated attachment force shows a similar nonlinear relationship to locomotor rates in leukocytes on ECM-coated surfaces (Fig. 1B and 1C) .

Haptokinetic migration in leukocytes
Accumulating evidence suggests that the model of haptokinetic migration is valid for crawling leukocytes, most notably associated with ß1 and ß3 integrins. In different types of 2-D haptokinetic migration assays, the migration of preactivated T cells on or towards fibronectin is supported by {alpha}4ß1 and {alpha}5ß1 integrins [3 , 195 ], corresponding to an optimal balance of adhesion and de-adhesion rates (Fig. 1B , ). Activation of ß1 integrins in otherwise low-adhesion T cell lines increases adhesion to immobilized fibronectin and thereby favors traction and migration [196 ]. Maximum migration on 2-D fibronectin substrate is nearly completely blocked by adhesion-perturbing antibodies against {alpha}4, {alpha}5, or ß1 chains [195 ] or soluble monomeric fibronectin [196 ], suggesting that loss of adhesion impairs migration as a secondary effect (Fig. 1B , ). Similar to peripheral T cells, thymocytes utilize {alpha}4ß1 and {alpha}5ß1 integrins for migration across fibronectin [197 ]. Likewise, haptokinetic motility of T cells across model basement membrane (matrigel) as well as across individual basement membrane components, such as laminin or type IV collagen, is reduced by anti-laminin or anti-{alpha}6 integrin antibody [198 , 199 ].

In neutrophils, chemotaxin-induced activation and extravasation lead to the de novo expression of {alpha}2ß1 integrin both in vivo and in vitro. For neutrophils recruited into the rat mesentery in vivo, the migration velocity is reduced by blocking anti-{alpha}2 or -ß1 integrin antibody or antagonistic {alpha}2ß1-binding peptide DGEA (Asp-Gly-Gln-Ala), as assessed by intravital microscopy and tracking of cell paths [149 ]. Because ß2 integrins appear not to be required for in vivo crawling, it is likely that activated neutrophils utilize newly expressed {alpha}2ß1 integrin as a principal receptor for migration within collagenous tissue [149 ]. Similarly, {alpha}vß3 integrins support neutrophil migration across fibronectin (Fig. 1 B, ) and reduction in substrate binding by blocking anti-{alpha}vß3 antibody, which impairs neutrophil crawling as a secondary effect (Fig. 1 B, ) [200 ].

In addition to ß1 integrins, haptokinetic leukocyte migration can be supported by an intriguing number of structurally and functionally different receptors and ligands. Lymphocytes are capable of locomotion over the surface of different cell types, such as macrophages, dendritic cells, fibroblasts, glioma cells, and endothelial cells [201 , 202 ]. After contact with an antigen-presenting dendritic cell, T cells can establish a 2-D crawling migration type across the antigen-presenting cell and readily receive migration-driving as well as activation signals from the same counterpart surface [6 ]. T cell crawling across intercellular adhesion molecule (ICAM)-1-coated surface [203 ], across endothelium, or within glioma spheroids [202 ] is mediated by ß2 integrins, most importantly lymphocyte function-associated antigen (LFA)-1/{alpha}Lß2, interacting with ICAM-1 on the counterpart substrate. LFA-1 is an important and versatile integrin linking the actin cytoskeleton to promote adhesive as well as migratory cell-cell interactions [203 , 204 ]. Fc receptor-positive lymphocytes are able to move across surfaces coated with antibody complexes [205 ]. The capacity to utilize a wide range of different substrates for haptokinetic migration indicates that multiple sets of surface receptors are simultaneously available to leukocytes for dynamic connection to the actin cytoskeleton. Hence, although the receptor usage might be diverse on different 2-D substrates, the importance of maintaining minimum adhesiveness for migratory traction remains preserved among leukocytes and other cell types.

Adhesive locomotor arrest
On the highly adhesive side of leukocyte substrate interaction, integrin activation and increased substrate binding can outgrow the capacity to detach, forcing the cell to slow down and, ultimately, arrest locomotion (Fig. 1B , ). Increasing the general level of integrin-mediated attachment leads to the trapping of ruffles to the substrate followed by cell flattening and nonpolar-spreading behavior. Induction of constitutive high-affinity ß1 integrin binding in resting T cells by activating antibody leads to T-cell immobilization that can be reversed by counteracting blocking anti-ß1 antibody [4 ], suggesting that the ratio of high- to low-affinity binding determines net translocation. Similar effects of activating ß1 integrin antibody are mediated via {alpha}4 and {alpha}5 integrins trapping crawling eosinophils on fibronectin by adhesive sticking [206 ]. Along these lines, paradoxical effects of otherwise migration-driving cytokines [e.g., tumor necrosis factor (TNF-{alpha})] and chemoattractants have been described. In T cells activated by SDF-1 or regulated on activation, normal T expressed and secreted (RANTES), immobilized TNF-{alpha} can impede migration [207 ], putatively by anchoring the cell to the substrate [208 ]. Increased attachment forces can also be achieved by retarding detachment of the uropod. Intracellular calcium fluxes are required for uropod retraction and detachment in crawling neutrophils. Buffering calcium delays retraction and anchors the uropod to the substrate, rendering ruffling and pseudopodal activity at the leading edge ineffective [209 ].

Chemoattractant-induced neutrophil migration through 3-D fibrin lattices or matrigel is dependent on integrin {alpha}Mß2 yet independent of ß1 integrins [210 ]. However, after stimulation by N-formyl-methionyl-leucyl-phenytalanine, {alpha}Mß2 appears to collaborate with integrin {alpha}5ß1 and shift fibrin binding towards a high-affinity state, increasing adhesion yet reducing migration rates [210 ]. A similar adhesive mechanism is likely to mediate the stopping of T blasts crawling across ICAM-1-containing lipid bilayers via LFA-1-mediated adhesion. After TCR triggering, adhesive engagement of LFA-1 mediates T cell flattening and spreading on the substrate followed by sticking and migratory arrest, despite ongoing vigorous cytoskeletal dynamics [211 ]. Together, these observations support the concept that environment-induced increases in ligand binding above the capacity to generate traction at the leading edge favor immobilization ("sticking") of otherwise highly motile leukocytes. From a mechanistic point of view, this process has been compared with the "accumulation of flies on a fly paper" [210 ].

Biophysical migration strategies
In most of the above described migration-blocking experiments, maximum reductions of crawling velocity rates are from 50 to 70%, leaving a level of residual migration at velocities ranging from 2 to 10 µm/min [136 ]. In particular, if leukocytes are incorporated into 3-D substrates, considerable residual migration is present after adhesion blocking [4 , 149 ]. The cellular and molecular mechanisms of residual migration after receptor blocking are poorly understood. Residual substrate binding and haptokinetic migration are likely to be provided by compensatory receptors not affected in a particular assay. Furthermore, residual migration may result from nonadhesive "biophysical" mechanisms.

It has been noted that a nonadhesive 2-D substrate can support T lymphocyte attachment and migration if structural discontinuities support pseudopod anchoring and the generation of some retention force [212 ]. In 3-D substrates consisting of interstitial space, fiber strands, and cell interphases, the body of a migrating cell is bordered and three-dimensionally restricted by extracellular structures. The importance of biophysical constraints for cell crawling has been explored for neutrophils in locomotion within a flat glass capillary space. If the spacing height of two adjacent glass interphases is ~5 µm relative to an average neutrophil diameter of 6.5 to 8 µm, a slight squeezing of the cell body occurs, and neutrophil crawling and chemotaxis might not be reduced by adhesion-blocking antibodies against ß1, ß2, or ß3 integrins [213 , 214 ]. Such "adhesion-independent" locomotion, however, requires bilateral interaction to both chamber walls [213 ]. If the capillary height is increased above 14 µm (hence removing the upper contact phase), the cells are forced to unilaterally attach to the substrate via ß2 integrins. In this case, neutrophil crawling is blocked by anti-ß2 integrin antibody [213 ]. The most obvious explanation for such different adhesion and migration requirements towards an identical substrate is that, on spatial restriction and passive contact with a biophysical substrate, neutrophils can use an attachment-independent mode of locomotion (Fig. 1B , ), whereas migration on a 2-D surface is secondary to unilateral binding provided by adhesion receptors (Fig. 1B , ).

One important motility mechanism in biophysically complex environments is shape change [150 , 215 ]. In 3-D tissues, similar to the narrow glass chamber model [213 ], leukocytes are passively surrounded and trapped by collagen fibers and other ECM components [2 , 212 ], potentially bypassing active attachment for substrate contact. Early studies on T cell migration showed that nonactivated peripheral-blood T cells could not attach to a 2-D surface coated with collagen or fibronectin [150 , 212 ]. This low binding strength is best explained by low integrin expression and the maintenance of integrins in a nonadhesive state in resting T cells [216 , 217 ]. However, because such nonadhesive T cells are still capable of crawling into and within 3-D collagen or fibronectin matrices, migration-driving forces in 3-D ECM might range below the forces required for 2-D attachment, prompting concepts of adhesion-independent crawling [150 , 212 ]. If the matrix gaps, pores, and clefts are large enough relative to cell size and deformability [218 ], a 3-D environment appears to favor biophysical migration strategies of low adhesive strength (Fig.1B , ). The simplest mechanism leading to biophysical migration is based on cytoskeletal flow, cortical stiffening, and coordinated shape change, as derived from Dictyostelium pseudopod stiffening and protrusion. T cells can use stiff lateral outward pseudopod protrusions ("footholds") that remain stationary and locked between fibers for some seconds while the cell mass flows forward [212 ]. As T cells squeeze through regions of narrow spaces, constriction points or rings can act as anchors for anterograde flow of the cytoplasm and propulsion of the cell mass [212 ]. Footholds, constriction rings, and slight yet fully reversible matrix distortion on detachment have also been reported for neutrophils [219 ] and dendritic cells [139 ] on migration through 3-D ECM. The concept of migration strategies of very low adhesivity is consistent with negative results on T cells that move through 3-D collagen matrices. Here, migration occurs independently of ß1, ß2, ß3, and {alpha}v integrin-mediated adhesion, as shown by a combination of blocking anti-integrin antibodies [4 ]. In conclusion, footholds and constriction rings could represent cellular strategies to generate physical anchors that function despite low attachment forces (Fig. 1 B, ) as long as external constraints support a squeezing, pushing, and gliding mode of migration.


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DIRECTIONAL GUIDANCE OF LEUKOCYTE LOCOMOTION
 
Chemotaxis is an important mechanism by which leukocytes accumulate in the tissues both in vitro and in vivo, as comprehensively summarized elsewhere [220 221 222 223 ]. In addition to chemotactic cues, leukocytes are very responsive to the physical and chemical patterns of the substrate. Most tissues in the body are nonrandomly organized. Migratory cells exhibit a tendency to follow the 3-D geometry of the tissues as a consequence of physical and immobilized chemical guidance mechanisms. Two major concepts have been developed in the past on how cell migration and positioning are influenced by a solid-phase environment, namely contact guidance and haptotaxis.

Contact guidance
Guidance by physical structures preserved in tissues and other solid-phase substrates is termed contact guidance [224 ]. The concept of contact guidance was initially established for the extension of outgrowing neurites along the fibers of an oriented fibrin clot [225 ] and confirmed for guidance of many other cells, such as fibroblasts, leukocytes, and macrophages interacting with different substrates of nonrandom texture [reviewed in ref. 226 ]. In principle, contact guidance represents the alignment of the length axis of the cell body or parts of it in parallel to a preformed physical structure such as structural discontinuities in the form of matrix fibers (Fig. 2A) , a groove, or an elevated border. A special case of 3-D contact guidance in vitro may be represented by "chimneying," i.e., cell crawling through a narrow capillary tube [213 ]. Contact guidance results from a stereotypic guidance reaction, which can be elicited by an array of natural and synthetic substrates, e.g., matrix fibers, a groove in the cover slip, or textile or plastic fibers. This versatility and apparent substrate independence could arguably implicate mechanisms of cell-substrate interactions beyond specific receptor-ligand interaction and molecular recognition.

The cellular and molecular mechanisms involved in contact guidance remain to be determined. Physicochemically, contact guidance might be a consequence of the oriented strengthening of integrin-substrate linkages after borders of maximum rigidity [106 ]. The concept of integrin-ligand strengthening is supported by aligned focal contact-like structures preferentially developing in fibroblasts along the interface of a grooved surface but not in interactions with flat substrate regions [227 ]. The outcome is a polarized stabilization of the structural cytoskeleton along environmental discontinuities, leading to the adaptation of the cell body along these extracellular guidance cues. In 3-D tissue environments, contact guidance along edges of increased rigidity is probably combined with the flowing and gliding of the cell body along paths of least resistance, as detected for T cells crawling through a 3-D collagen fiber network (Fig. 2 ; for a video clip, see http//:www.xxx). T cells slide through open gaps along preformed pathways oriented in parallel to and guided by adjacent collagen fibers (Fig. 2A 2B 2C) . Instead of structurally remodeling collagen fibers, regions of increased fiber density and narrow texture [Fig. 2D and 2E (white arrowhead)] are readily circum-migrated leaving the preformed matrix structure fully intact [Fig. 2E 2F 2G (white arrow)]. Because T cells tend to orient along the random order of collagen fibers, the path structures obtained from T cell populations migrating within nonaligned 3-D collagen lattices follow the model of persistent random walk [2 , 212 ]. Within aligned collagen matrices, T lymphocytes show a bias for movement in parallel to the length axis of the fibers, implicating contact guidance mechanisms [212 , 228 ]. The minimum extension of a physical cue to initiate morphological cell patterning has been established in the range of 30 nm in macrophage-like cells [229 ]. The diameter of individual collagen fibers ranges from 70 to 280 nm, although bundles can reach extensions of micrometers [230 ]. Unlike chemotaxis, contact guidance in the absence of further stimuli is a bidirectional locomotion cue that, on its own, would not lead to directed cell accumulation; however, it would predefine a limited number of pathways that can be used by cells within a given structural environment.

Haptotaxis
Cell movement towards an increasing concentration of immobilized, i.e., surface- or matrix-bound adhesive or biochemically active molecules is termed "haptotaxis" [231 ]. In vitro, stable gradients of soluble chemoattractants can be generated by various diffusion techniques imposing directional cell migration. In vivo, however, it is difficult to conceive how soluble gradients might be upheld. Under in vivo conditions, physical-perturbing events such as muscular contraction, convection of extravascular fluid, and lymph flow might interfere with the graded diffusion of soluble substances, forming stable gradients. One mechanism of maintaining a concentration gradient over time is the immobilization of soluble factors to ECM via ECM-binding domains, such as TNF-{alpha} binding to laminin [208 , 232 ]. Some chemokines such as RANTES can bind to glycosaminoglycans (e.g., heparan sulfate) on cell surfaces and fibrillar structures, whereas the cell-binding domain is accessible by passenger cells [233 , 234 ], thereby potentially generating an immobilized chemokine scaffold.

It is important that cell orientation to topographical or chemical cues has a fairly large random component ("noise") for 2-D substrates [235 ] as well as 3-D fibrillar matrices [148 ]. In vivo, it is likely that nondirectional random walking, chemotaxis, haptotaxis, contact guidance, and adhesive arrest are simultaneously present to various degrees. Accumulation at a given site could then result from several converging mechanisms: (1) positive attraction, i.e., by chemotactic or haptotactic gradients of cytokines and (2) random or nonrandom walk along physical matrix structures; these promigratory events are likely combined with (3) migratory arrest provided by local stopping cues, such as trapping of migrating T cells by integrin activation [4 ] and immobilized cytokines [207 ]. As an endpoint of cell entry into tissue, adhesion-driven cell immobilization could result in efficient cell accumulation in regions of interest. Last, it is noteworthy that the leukocyte crawling efficiency may be determined by the physical width of preformed matrix gaps and trails. On vasodilatation or inflammation, the degree of tissue hydration and swelling might significantly contribute to location and speed of leukocyte infiltration by reversible widening of preexisting matrix pores and gaps for passenger cells, representing an adaptive navigation mechanism beyond single-cell function.


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CONCLUSIONS
 
Leukocyte migration is now understood as a plastic and adaptive process rather than a uniform stereotypic event. In the course of evolution, essential basic migration mechanisms appear to have arisen in primitive cells, as represented by Dictyostelium ameba. These migration strategies might have been conserved in some if not most cell types throughout further development of metazoan organisms. With the development of complex tissue structures as well as the rise of the specific immune system, ancient basic migration strategies appear to have become more sophisticated and diverse, i.e., including diverse sets of adhesion receptors, signaling cascades, and matrix-remodeling strategies. These additional migration strategies might have specifically evolved on the basis of more intimate cell-tissue interactions, whereas basic migration instruments such as cytoskeletal turnover or pseudopod dynamics were retained. Consequently, for the understanding of differences in leukocyte migration and positioning strategies, it might be helpful to operationally distinguish basic, i.e., phylogenetically old, crawling mechanisms present after constitutive trafficking through tissues and across resident tissue cells, from more specialized adhesion and migration events occurring in the context of specific environmental conditions. For leukocytes, such more specific events might include transendothelial migration, T cell crawling across antigen-presenting cells, or cytotoxic conjugation and killing, which then are further determined by highly specific sets of receptors and signaling cascades. On the other hand, highly specific recognition strategies, e.g., on triggering TCR by major histocompatibility complex-peptide complexes, are likely to retain basic events in cytoskeletal dynamics and turnover [9 ].

The paradigm of amoeboid movement will continue as a source of insights and probes into the study of cell motility in mammalian cells. Amoeboid single-cell movement appears to represent an efficient migration strategy optimized for speed and flexibility for cells that appear to function in very different environments under both beneficial and adverse conditions. The finding that Dictyostelium cells can apparently crawl across many different sorts of substrates, either living or nonliving, might stimulate further efforts to dissect robust, versatile shape-driven migration strategies from specific adhesion receptor-dependent haptokinetic processes. In light of these findings in Dictyolstelium, one of the most overlooked principles of migration and positioning in leukocytes might depend on morphological stiffness and shape change generated by a highly dynamic actin cytoskeleton in response to adjacent extracellular patterns.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Fr-1155/2-1) and the Wilhelm-Sander Foundation (96.030.2). We acknowledge Katarina Wolf for critical reading of the manuscript and Bong-Seok Song for confocal time-lapse microscopy.

Received February 15, 2001; revised July 9, 2001; accepted July 30, 2001.


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