Cell Motility and the Cytoskeleton 18:245-257 (1991)

Review Cell Locomotion: New Research Tests Old Ideas on Membrane and Cytoskeletal Flow Julian P. Heath and Bruce F. Holifield

Department of Cell Biology, Baylor College of Medicine, Houston, Texas Recent studies on the mobility of membrane markers on crawling cells indicate that there is no long-range centripetal flow of membrane proteins or lipids during cell locomotion. In this article we reflect on the history of ideas about membrane flow in cells, and we discuss how these new findings will shift the focus of research in cell locomotion away from the cell surface to the molecular interactions and dynamics of the actin cytoskeleton. Key words: lipid flow, cytoskeleton, actin, microscopy

INTRODUCTION

In this article we put forward our view of how ideas about the mechanisms of cell locomotion, in particular of the relative contributions of the cell surface and the actin cytoskeleton, have developed over the past three decades or so. One feature that seems to be common to all motile cells, and which has generated an unusual amount of debate and controversy, is the phenomenon of centripetal flow. Centripetal flow refers to the rearward redistribution of cell structures and cross-linked membrane proteins and external cell surface markers on substratumattached cells and to the capping of surface markers on lymphocytes. It was the widespread curiosity as to the nature of this characteristic of flow that gave rise in the 1970s to two models of tissue cell locomotion that are the subject of the first part of this review: membrane flow and lipid flow. These models were similar in that both postulated a flow of membrane components, but were different in the importance given to membrane flow and to the cytoskeleton as the prime mechanism for motility. But now, new data on surface flow collected with stateof-the-art video microscopical techniques challenge these older models. So we close this review with a detailed look at the dynamic structural events occurring in the lamellipodium and the leading lamella of a fibroblast during locomotion. The body of literature published over the last 30 years on cell locomotion and membrane flow is so large 0 1991 Wiley-Liss, Inc.

and the contributors so many that we cannot hope to do justice to all aspects of this subject. Furthermore we are mindful of the fact that the record of published papers does not necessarily accurately reflect the evolution of ideas or the total volume of experimental work on the topic. So in this review we focus on the crawling locomotion of metazoan fibroblasts, for that is the cell type with which we are most familiar, but the behavior and motile machinery of epithelial cells, neurons, neutrophils, and even some invertebrate amoeboid cells is proving to be sufficiently similar to give us confidence that there is a single set of locomotory mechanisms employed by all cell types. Two symposia on cell locomotion held in 1960 [Harris, 19611 and in 1972 [Porter and Fitzsimmons, 19731, and a monograph by Trinkaus [1969], give an excellent bird’s eye view of the state of the field in the 1960s. For more recent and broader reviews of cell movement see Abercrombie [1980], Trinkaus [ 19851, Lackie [ 19861, Bershadsky and Vasiliev [1988], and Bray and White [ 19881. FIBROBLAST LOCOMOTORY BEHAVIOR

Before we review various models of cell locomotion, it is useful to set the scene and define some terms by Accepted December 13, 1990. Address reprint requests to Dr. Julian P. Heath, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.

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Fig. 1. Phase-contrast light micrograph of a C3H mouse fibroblast showing the fan-shaped leading lamella and the ruffling lamellipodia typical of a crawling cell. Scale bar = 10 )*.m. Fig. 2. Scanning electron micrograph of a human fibroblast showing ruffling larnellipodia. Scale bar = 10 Fm.

giving a brief account of the structure and locomotory behavior of fibroblasts in culture. Most moving fibroblasts are approximately fan shaped with a broad flattened anterior region called the lamella that extends in front of the thicker and central perinuclear region where most of the major organelles are found (Fig. 1). During locomotion, new contacts with the substratum are made at the lamellar margin [Izzard and Lochner, 19761 where thin sheets of cytoplasm, called lamellipodia, are protruded (Figs. 1, 2). The formation of focal cell-substratum contacts is mediated by precursor structures lying within the lamellipodia variously called microspikes, filopodia, or actin ribs, all of which describe the short rib-like bundles of actin filaments commonly found in lamellipodia [DePasquale and Izzard, 19871. Newly formed focal adhesions in turn

initiate, through mechanisms poorly understood, the development of actin stress fibers that pass backward and obliquely through the lamella, terminating near the nucleus. Adhesions remain stationary as the cell passes over them and they and their associated stress fibers usually disassemble at the rear of the lamella [Heath and Dunn, 19781. Some adhesions persist and eventually wind up at the rear of the cell if the cell changes direction and forms a new leading lamella. Stress fibers are absent or less prominent in some fibroblasts as well as in neutrophils and macrophages, and yet these cells move quite efficiently, so the role of stress fibers in locomotion is not clear; whether stress fibers are present or not there could be an additional tractive force developed in the actin networks that constitute a ventral cortical layer in all cells. The central regions of the cell are generally not adherent to the substratum, but the trailing region of the cell, or tail, is anchored to the substratum at its distal portion. Translocation appears to involve competition between the two sets of adhesions and as a result the advancement of the cell is sometimes jerky, with the tail often being rapidly detached and withdrawn into the perinuclear region. Tail detachment is often accompanied by a phase of rapid protrusion at the front end showing that the cellular mechanisms of retraction and protrusion are somehow intimately linked [Chen, 1979; Dunn, 1980; Heath, 1982, 1983al. Lamellipodia extend from the anterior margin of the lamella, taking the form of thin veils of cytoplasm less than 200 nm thick, and up to 5 bm long (Figs. 1, 2). They are made up almost exclusively of an axially oriented network of actin filaments [Hoglund et al., 1980; Small, 19811 (Fig. 3). The most obvious behavioral feature of lamellipodia is ruffling, which describes the way in which lamellipodia cyclically extend forward, lift up away from the substratum, undulate, and then move rearward, in some cases for several micrometers, before dissolving into the dorsal surface of the lamella (Figs. 1 , 2). Ruffling occurs to varying degrees in most cells in culture, and is also seen in cells in vivo [Trinkaus, 19731. Although we now recognize that lamellipodia need not upturn as ruffles in order for tissue cells to migrate efficiently, the presence of ruffles does help in distinguishing active cells from quiescent ones. CELL LOCOMOTION 30 YEARS AGO

The behavioral aspects of cell locomotion were well characterized 30 years ago when a collection of articles on cell movement and adhesion was published [Harris, 19611. Significantly, there was a general consensus at that time that the cytoplasmic streaming and cortical contractions seen in amoebae, and the contractil-

Membrane and Actin Flow

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ity and cleavage of tissue cells, were probably brought about by contractile elements in the cytoplasm that bore a close relationship to the proteins of muscle [Wolpert, 1960; Allen, 1961; Hoffman-Berling, 19631. In the 1960s studies on the mechanism of fibroblast locomotion focused on the phenomenon of ruffling and its role in locomotion. It was already known that the cell is most adherent in regions of active ruffling as shown by microdissection [Chambers and Fell, 19311. Since ruffling ceased at sites of cellkell contact and then appeared elsewhere as cells changed their directions of migration, it was theorised that the ruffling membrane tends to control the direction of the mechanism that produces cell locomotion [Abercrombie and Ambrose, 19.581. Indeed, Abercrombie [ 19611 more assertively declared that “the main locomotory organ of the fibroblast is its ruffled (undulating) membrane.” And an early model of fibroblast locomotion [Ambrose, 19611 postulated that ruffles were symptomatic of a train of peristaltic contractile waves passing through the cell. MEMBRANE FLOW

These initial studies raised the key question as to the source of the plasma membrane required to envelop newly protruded ruffles. Was new cell surface externalised as a lamellipodium was protruded or did the plasma membrane passively follow changes in cell shape? And if the former, was the centripetal motion seen in cells indicative of a flow of membrane through the surface? These questions concerning membrane flow were addressed in a large body of experimental and theoretical work in the late 1950s and 1960s on the locomotion of amoebae [discussed in Allen, 1961; Goldacre, 19611. Although innovative in their use of fluorescent antibodies [Wolpert and O’Neill, 19621 and particulate ligands as markers of membrane movements in amoebae and slime molds [Shaffer, 1963, 1968; Garrod and Wolpert, 1968; Hulsmann and Haberey, 19731, these studies never satisfactorily resolved the question, perhaps because of differences in cell types and experimental probes and the limitations of such approaches on free-living amoebae that have a thick cell coat distal to the true plasma membrane. In the search for evidence of membrane flow in tissue cells, an important contribution to the problem came from Marcus [ 19621 who studied the expression of Newcastle disease viral proteins in cultured HeLa cells. He found that there was a surface expression of viral haemagglutinin initially only at the extreme margin of the lamella and gradually expression extended over the whole cell surface. Marcus realised that the apparent movement of haemagglutinin across the lamella could have been due either to insertion at progressively more

Fig. 3. Correlated differential interference contrast (A) and fluorescence (B) micrographs showing the leading lamella (la) and the lamellipodial region (Ip) of a spreading IMR 90 human fibroblast fixed and stained with rhodamine phalloidin. Note the actin-containing ribs in the lamellipodium, and the texture of the intervening cytoplasm, both of which display movement in living cells. Scale bar = 20 pm.

rearward sites or to centripetal movement of proteins inserted only at the cell margin, and he designed experiments that established that the latter was indeed the case. However, whether this centripetal redistribution was due to passive diffusion or to a directed transport process was not determined. Marcus’ study was the first to show protein insertion at protrusive margins and redistribution of membrane receptors relative to the substratum; these two processes are key aspects of later membrane flow models of cell locomotion. MEMBRANE FLOW AND CELL LOCOMOTION

In 1970 Abercrombie and coworkers proposed the first general hypothesis of the mechanism of fibroblast locomotion. Their ideas arose from painstaking statistical analyses of the protrusive activity of the lamella and

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ruffles and the transport of particles across the lamella [Abercrombie et al., 1970a-cl. The centripetal transport of particles attached to the lamella had been seen earlier on chick fibroblasts by Ingram [1969] and on neuronal growth cones by Bray [1970]. And Abercrombie et al. [197Oc] also noted that there was a generalised movement of something within the lamella that they described as “a series of indefinite shadows chasing each other steadily backwards” in the lamella. This was probably the first description of actin arcs [Heath and Dunn, 1978; Heath, 1983al. Abercrombie and coworkers’ hypothesis stated that membrane was inserted at the leading edge and flowed rearward to a sink at the back of the lamella where it was internalised and recycled. They concluded that the flow was probably asymmetric, involving mainly the dorsal surface since adhesions to the substratum would tend to block the flow on the lower surface; furthermore, they thought that the flow involved only lamellipodia and the lamella, other regions of the cell

being stable. The lifting up of ruffles was explained as a sign of especially rapid surface assembly. The hypothesis explained centripetal transport since any particles attached to the membrane would be transported along with it. Significantly, and this is sometimes overlooked, they speculated that a contractile process, rather than thrust generated by the membrane flow, plays the major part in drawing the cell forward [Abercrombie et al., 19711. Abercrombie realized that the particles used in the surface marking experiments were not true membrane components. In a later paper [Abercrombie et al., 19721 he examined the redistribution of concanavalin A receptors on fibroblasts and was able to show clearance of the marker from cell margins, in accordance with the hypothesis. Further support for the membrane flow model came from Edidin and Weiss [1972] who found that the antibody-induced redistribution of H-2 antigens on mouse fibroblasts occurred on cells that were ruffling and actively locomoting. Figure 4 shows the character-

Fig. 4. Stages in the centripetal transport of patched surface receptors on human fibroblasts. Cells incubated in an antibody against the HLA surface receptor followed by a fluorescent anti-immunoglobulin have a uniform distribution of fluorescent patches of HLA-antibody complexes (A). After 10 minutes (B) the patches have moved inward from

ruffling cell margins, and by 30 minutes (C) the patches have collected over the perinuclear region. D shows the organization of actin, revealed by fluorescent phalloidin, in the fibroblast shown in B. Note the coincidence of the retreating margin of the patches (B) with actin arcs (D). Scale bar = 20 p m .

Membrane and Actin Flow

istic sequence of events during the antibody-induced redistribution of HLA receptors on the lamella of human fibroblasts in culture. Harris [1973, 19761 subsequently refined and expanded the membrane flow model. He proposed that plasma membrane components, both lipids and proteins, are dissolved into the cytoplasm throughout the cell, recycled through the cytoplasm, and inserted at sites where the membrane was under greatest tension, i.e., at the leading edge. He proposed that membrane continuously flows rearward over the lamella on both dorsal and ventral cell surface. This was based on observations that particles were transported on both upper and lower cell surface [Harris and Dunn, 19721. Like earlier thinkers, Harris postulated that a cortical actin-based contractile system exerts tension on the cell surface and causes the flow: this results in rearward movement of membrane on the upper cell surface but generates traction on the ventral which propels the cell forward. A problem for both the Abercrombie and Harris models was the difficulty of demonstrating the membrane cycling through the cytoplasm. However, the known rates of membrane turnover and synthesis measured biochemically were thought to be compatible with the rate of membrane flow during locomotion [Abercrombie et al., 19711. In the case of neurones, however, there is an obvious necessity for insertion of new membrane to allow axonal elongation; that vesicular organelles were commonly seen in the growth cone was taken as evidence for both the nature and the externalization site of the new membrane. Bray [I9731 developed a model of growth cone motility, containing some parallels to the Abercrombie model of fibroblast motility, that proposed that vesicles arriving at the growing tip of the neurite are transported by membrane-associated actin and myosin to the tip of the filopodia where they are externalised. The transport of particles on the growth cone [Bray, 19701 was taken as evidence of a flow of cell surface from the leading edge back to the base of the growth cone. ROLE OF THE CYTOSKELETON IN LOCOMOTION

In these early models of cell locomotion, the contribution of the actin-cytoskeleton in generating force was implied but was of course not clarified because few details were known. In the late 1960s and early 1970s electron microscopy had revealed actin-like filaments in stress fibers and in lamellipodia [Buckley and Porter, 1967; Spooner et al., 197 1 ; Goldman et a]. , 19731. And the discoveries that actin filament growth was correlated with changes in cell form [Behnke et a]., 197 I ; Tilney et a]., 19731 gave substance to the idea that actin polymerization can drive protrusion. Only following the intro-

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duction of probes for immunofluorescence microscopy of cytoskeletal structures in the mid-1970s did it become clear that stress fibers [Lazarides and Weber, 1974; Weber and Groeschel-Stewart, 19741 and the actin cortices of fibroblasts [Zigmond et al., 19791 contained a sarcomere-like array of muscle proteins. Furthermore, throughout the decade of the 1970s new evidence accumulated from structural and biochemical experiments that submembraneous actin and actin-binding proteins were linked to surface receptors on fibroblasts [Ash et al., 1977; Bourguignon and Singer, 1977; Toh and Hard, 19771and epithelial cells [Albertini and Anderson, 19771 and were associated with the antibody-induced caps on lymphocytes [Braun et al., 1978; Flanagan and Koch, 19781. All of this contributed to increasing acceptance of the concept that the cytoskeleton has the dual role as a modulator of the distribution of cell surface receptors and as a generator of traction for cell locomotion. But even though these data provided strong evidence for actomyosin-like force-generating systems in the cytoskeleton, as was postulated by earlier workers, the role of membrane flow in movement was not seriously questioned since it was still necessary to explain the source of the cell surface over newly extended cell processes. That the source of surface lay in the cytoplasm, as argued in the membrane flow hypothesis, was first challenged in a simple experiment by Erickson and Trinkaus [ 19761. They used scanning electron microscopy to measure the surface area of fully spread BHK21 cells and their rounded-up mitotic counterparts which were covered with blebs and microvilli. The surface areas of the two morphologically different populations was found to be closely similar, implying that cells respreading after cytokinesis could redistribute their surface membrane through shape changes rather than by externalisation of cytoplasmic membrane stores. THE LIPID FLOW HYPOTHESIS

The idea that the cell surface was flowing like a sheet over the lamellipodia and leading lamella during locomotion did not take into account the emerging concept that membranes were fluid and their protein components were capable of independent movement [Frye and Edidin, 1970; Taylor et al., 19711. The fluidity of membrane lipids and proteins formed the keystone of a new and radically different hypothesis of cell motility proposed by Bretscher in 1976. The new model stated that both the centripetal movement of surface structures and the forward locomotion of the cell were brought about not by a contractile process involving the cytoskeleton, but by a directed recycling of membrane lipids through the cell [Bretscher, 1976; and see also Bretscher 1982, 19841. The new model was a refinement of Aber-

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crombie’s, taking into account the diffusibility of proteins in the plasma membrane and the pathways of endocytosis. Bretscher’s model has attracted much attention and caused some controversy, but it is fair to say that some of the interest in the model came from the lack at that time of any concisely stated alternate model of how the cytoskeleton could drive lymphocyte capping and cell locomotion. Bretscher’s model is elegant for its simplicity. At its root lies the fact that cells constitutively internalize cell surface lipids into coated vesicles via receptor-mediated endocytosis, taking in up to the equivalent of their whole cell surface area each hour. The model proposes that the endocytosed lipids and any recycling proteins are returned to the cell surface in a directed manner at the leading edge of a motile cell. This produces a net flux of lipid, rather than bulk membrane, from the front of the cell to the rear. Cell surface proteins and receptors that are not endocytosed maintain a surface distribution determined by their individual lateral diffusion coefficients and the rate of rearward lipid flow. Membrane proteins having large diffusion coefficients (on the order of lop9 cm2/sec) can maintain a uniform distribution on the surface by lateral diffusion against the flow. A prediction of the lipid flow model is that the higher the rate of recycling of a particular protein, the greater its density will be near the sites of reinsertion into the cell surface, since it will be internalized again before it can diffuse away. So it was naturally of interest when a number of reports showed that certain types of rapidly recycling growth factor receptors, including low-density lipoprotein [Bretscher, 19831 and transferrin [Bretscher, 1983; Ekblom et al., 19831, were concentrated at cell margins, whereas two apparently slowly recycling proteins showed a more even distribution [Bretscher, 19821. Further support for a polarized insertion of proteins into the plasma membrane came from studies showing that newly synthesized vesicular stomatitis viral glycoproteins were inserted into the leading edges of infected cells [Bergmann et al., 1983; Kupfer et al., 19871. However, the large time difference between protein insertion and examination of the cells makes definitive statements about exactly where and when the protein is externalized in relationship to lamellipodial protrusion very difficult. Bretscher’s model nicely explains the capping of cross-linked receptors. Large molecular aggregates have lower diffusion coefficients and thus, unable to make progress against the flow, will be swept to the rear of the cell and capped. The fact that a wide variety of different membrane proteins can be capped indicates a degree of non-specificity in the mechanism. Bretscher argued that this favors a lipid flow mechanism since a cytoskeletal mechanism would require the unlikely promiscuous interaction between actin and almost any membrane pro-

tein. This argument was supported by the finding that glycolipids [Revesz and Greaves, 1975; Stern and Bretscher, 1979; Speigel et al., 19841, which reside in the outer leaflet of the lipid bilayer, and even exogenously applied stearoylated dextrans [Wolf et al., 19801, can both be induced to cap by cross-linking. Given that none of these lipids can have any direct association with submembranous actin or other cytoskeletal proteins, one has to postulate an alternative mechanism for the centripetal flow of these molecules. Since the flow of membrane occurs on both surfaces of a cell, Bretscher argues that there will be a rearward thrust of the ventral membrane against the substratum that would serve to propel the cell forward. Recently he has demonstrated that the extracellular matrix receptor integrin may be externalised at the leading edge where it is needed to form cell-matrix adhesions [Bretscher, 19891. These resist the rearward thrust of the lipid flow and allow forward movement. Controversially, Bretscher finds no necessity to invoke any involvement of the cytoskeleton in his general mechanism of cell locomotion.

ARGUMENTS AGAINST LIPID FLOW

A key argument against the lipid flow model is that the measured diffusion coefficients of most membrane proteins are too low to permit uniform distributions of the proteins given the hypothesized rate of lipid flow, but the normal distribution of many proteins prior to inducing capping does appear to be uniform. Bretscher counters this argument by pointing out the possibility of artifact in the most common method for measuring lateral diffusion coefficients, fluorescence recovery after photobleaching (FRAP). Second, the capping of membrane components such as glycolipids and integral membrane proteins lacking cytoplasmic polypeptide domains [Edidin and Zuniga, 19851 can occur by a cytoskeletal mechanism if these components interact with other transmembrane proteins that in turn associate with cytoskeletal elements. This type of mechanism has evolved in several forms [Bourguignon and Singer, 1977; De Petris, 1978; Dunn, 1980; Dembo and Harris, 19811, with a finding of at least one membrane protein in lymphocytes having some characteristics expected for a protein mediating the association of patched antigens with the cytoskeleton [Turner and Shotton, 19871. Finally, the fact is sometimes overlooked that the occurrence of membrane insertion near the leading edge of motile cells is not by itself a proof for the existence of a general flow of membrane components. For example, membrane insertion at the leading cell margin might oc

Membrane and Actin Flow

cur, but at a rate too slow to sustain the required rate of rearward flow or to cover the surface of new protrusions. Also, in the absence of rearward lipid flow, a density gradient of recycling receptors on the lamella could arise by simple diffusion of receptors away from the localised site of insertion. This is a reasonable explanation for the findings of Marcus [1962] and Bretscher [1983]. Clearly, whether a net rearward flow of lipid will result from insertion at the leading edge depends on the rate of insertion and we have no good quantitative data on this process.

DIRECT TESTS OF MEMBRANE FLOW

Surprisingly, since the bulk membrane and lipid flow models were put forward there have been few conclusive experimental tests of either model. Vasiliev and coworkers [1976] found that Con-A receptors were inserted into the plasma membrane randomly rather than at the leading cell margin. Middleton [ 19791 was the first to test the models of bulk membrane flow by surface marking with antibodies. He marked rat fibroblasts with a monovalent anti-Thy 1 antibody at O"C, allowed them to crawl for 30 minutes at 37"C, and then traced the Thy-1 marker with a fluorescent second antibody. He found no dilution of the anti-Thy-1 label, showing that there was no bulk flow of membrane to the rear of the cell driven by membrane externalised at the front of the cell. A similar conclusion was reached by Heath [ 1983bl who found no dilution of an antibody marker on the surface of chicken heart fibroblasts during locomotion. But neither of these studies examined the possibility that the failure of the membrane markers to redistribute was due to high diffusion coefficients or to recycling of the markers themselves. In the past year, two groups led by Ken Jacobson at the University of North Carolina, Chapel Hill, and Michael Sheetz at Washington University in St. Louis have reported on their studies of the movements of membrane receptors and lipids on crawling cells. The two groups used different but highly sensitive computer-processed-video microscopical assays for membrane flow: both groups conclude that there is no evidence for it. The Chapel Hill group used multiple fluorescent antibody labeling and low-light level digital fluorescence microscopy to test the prediction of the lipid flow hypothesis that the redistribution of non-recycling membrane markers is determined by their diffusion coefficients [Holifield et al., 19901. On mouse fibroblasts two different integral membrane proteins, Pgp-1 , also called GP80, and influenza haemagglutinin (HA), tagged with specific fluorescent antibodies, showed closely similar diffusion coefficients of about 3 x lo-"' cm* sec-I, as

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measured by the FRAP technique. When antibodies to the receptors were added to the cells, the Pgp-1 antigenantibody complex was capped while the HA was not. Care was taken to prove that the HA antigen-antibody complex was not recycled and reinserted at the leading edge during the capping of the Pgp-1 complex. This result supports the earlier findings of Middleton and of Heath and clearly shows there is no bulk membrane flow; moreover, the findings are incompatible with the lipid flow model since they show that two markers having similar diffusion coefficients can behave independently. In another test of the two models, the St. Louis group used high-resolution video microscopy of macrophages and highly motile fish keratocytes labeled with 40 nm gold particles coated with concanavalin A [Sheetz et al., 1989; Kucik et al., 19891. This technique detected motion of the particles with nanometer precision at video frame rates of 30 Hertz [Gelles et al., 19881. Due to the precision of measurement of particle displacement, a plot of mean square displacement versus time for diffusing particles provided a sensitive assay for the effects of membrane or lipid flow on diffusing particles. They found that different particles within a small surface area could simultaneously undergo random diffusional motion or directed motion both rearward and forward with respect to the cell; and furthermore any single particle could alternate between random and directed modes of motion. This behaviour is obviously incompatible with the lipid flow hypothesis. The fact that particles diffusing randomly on rapidly migrating keratocytes kept pace with the advancing cell margin suggests that plasma membrane components that are not capped might simply drift forward passively as a cell crawls [Kucik et al., 19891. In what is to date probably the most direct test of the lipid flow hypothesis, Ken Jacobson's group [Lee et al., 19901 stained neutrophils with a fluorescent lipid analog, di-I, and examined the redistribution of the probe following the photobleaching of a bar-shaped pattern on the lamella perpendicular to the direction of movement. If rearward lipid flow were occurring, the bleached area would be expected to move rearward before the fluorescence intensity recovered by lateral diffusion of unbleached dye. But they found that during the 5 seconds that it took for the fluorescence to recover, the bar moved forward at the same velocity as the cell margin. This result directly demonstrates that lipids of the plasma membrane move forward relative to the substratum as a tissue cell crawls forward, although it is not clear whether this involves only the dorsal surface, the ventral surface, or both. One is left with the conclusion that the model of a rearward flow of plasma membrane lipids in locomoting cells is incorrect.

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NEW IDEAS ABOUT FIBROBLAST LOCOMOTION

Can we finally lay to rest the bulk membrane flow model as envisaged by Abercrombie, Harris, and others? We think the answer is an unequivocal yes. And do these findings lay to rest the lipid flow hypothesis? Probably, but we think that controversy may continue. The reason is the plain fact that cells constitutively internalize and recycle some plasma membrane components creating a local flow of lipid at sites of exocytosis. Furthermore these sites can be at the cell margin. However, it seems probable that such flow will be in the submicrometer range, rather than over the long distances envisaged by Bretscher. So these new data should give us confidence that the cell surface transport and capping phenomena of motile tissue cells reflect the activities of the cytoskeleton. That this is so is indicated by a recent study in which mutant Dictyostelium amoebae, lacking conventional myosin, failed to cap Con-A-receptor patches [Pasternak et al., 19891. With this well-founded confidence in the central role of the cytoskeleton in the centripetal transport phenomenon of motile fibroblasts, we turn our attention first to the contribution of actin to lamellipodial transport and protrusion.

fined structure that is best seen in time-lapse video recordings, but it is conspicuous enough to be seen by careful direct observation of cells on the microscope. In differential interference contrast optics (DIC) the flow takes the form of a mottled pattern of densities, seen in Figure 3A, that moves rearward at 0.1-0.2 p d s e c and occurs both along actin ribs and between them from the tip of the lamellipodium to the base [Fisher et al., 19881. The flow appears to be always directly rearward, but curiously the motion of actin ribs can be orthogonal to the main direction of flow. This centripetal flow of material both beneath and on the lamellipodial surface is a characteristic feature of most if not all tissue cells, including fibroblasts [Fisher et al., 19881 and neuronal growth cones [Forscher and Smith, 19881. It is important to point out that in fibroblasts the flow seen in lamellipodia and in the leading lamella is a flow of material relative to the frame of reference of the substratum. This must not be confused with the situation in which an apparent flow is created by objects in and on the cell that are stationary with reference to the substratum and hence appear to move away from the leading edge as the cell moves forward. THE NATURE OF LAMELLIPODIAL FLOW

LAMELLIPODIAL FLOW AND ACTIN DYNAMICS

Electron microscopy shows that lamellipodia contain a network of F-actin filaments predominantly oriented with their “barbed” or fast growing ends toward the distal margin of the lamellipodium [Small et al., 1978; Hoglund et al., 1980; Small, 19811. Passing through this network are the bundles of actin filaments, called actin ribs, that can project a short distance beyond the anterior margin (Figs. 3, 4). But this static picture does not convey the high level of activity shown by lamellipodia. Not only do they continuously extend, ruffle, and fall back, but superimposed on the general cycle of protrusion and retraction are the activities of the actin ribs that develop apparently at random within the actin network and then display a complicated pattern of extension and retraction, lateral motion, and fusion with one another [Fisher et al., 1988; Izzard, 19881. These filament bundles appear to coalesce from nodes that are rich in the protein talin [Izzard, 19881, which has been shown to bind the cytoplasmic domains of certain extracellular matrix receptors [Horwitz et al., 19861. The lamellipodium contains several other cytoskeletal proteins including filamin and a-actinin that could be involved in filament bundling, but the dynamic interactions of these proteins in the actin network remains to be resolved. Concomitant with the activities of the well-defined actin ribs is a prominent rearward flux of less well-de-

What, then, is moving back in the lamellipodium? There is evidence that it is a flow of actin. Direct observation shows that actin-containing structures such as the ribs move laterally and pass rearward through the lamellipodium [Fisher et al., 19881. An important contribution to the field came from Wang [1985] who microinjected cells with fluorescent G-actin. The label was incorporated into the lamellipodia, and when a spot was photobleached at the tip of an actin rib, the fluorescence gradually recovered while the bleached area moved rearward at 0.013 p d s e c . This result is consistent with polymerization of actin filaments at the lamellipodial margin and the rearward flux, or treadmilling, of actin monomers through stationary filaments. This possibility has been strengthened by the recent EM demonstration of the addition of microinjected biotinylated actin to the anterior ends of microspikes in fibroblasts [Okabe and Hirokawa, 19891. But the rate of movement of actin structures and of particles observed by DIC microscopy is at least an order of magnitude greater than that detected by Wang in his photobleaching experiments. This difference cannot yet be explained. An alternate explanation for lamellipodial flow is that polymerization of actin occurs at the cell margin and the whole actin network is continuously transported rearward dragging any attached surface particles and membrane protein complexes with it. This model of actin flow in lamellipodia was developed by Fisher et al.

Membrane and Actin Flow [ 19881 and Forscher and Smith [ 19881 and has been discussed in detail in recent reviews by Smith [1988], Mitchison and Kirschner [ 19881, Adams and Pollard [ 19891, and Small [ 19891. A key piece of evidence for the model comes from the work of Forscher and Smith [ 19881 on cytochalasin B-treated Aplysia neuronal growth cones. The drug caused a rapid retreat of the whole actin network from the cell margin; since cytochalasin B binds to the fast-growing and anteriorly sited ends of actin filaments, depolymerization of the network would be expected to occur from the center of the cell outward if actin monomers were treadmilling through filaments. Forscher and Smith propose that retreat of the actin from the cell margin is due to a centripetal pull on the filaments by a myosin motor.

CONSEQUENCES OF ACTIN FLOW IN LAMELLIPODIA

If we accept the actin flow hypothesis of lamellipodia1 transport, what are its consequences and predictions? First, there must be continuous polymerization of actin irrespective of cell locomotion since judging by current data [Fisher et al., 1988; Forscher and Smith, 19881 the rearward movement of structure occurs in cells that have stationary lamellipodia. Given the mean flow rate of 0.2 pndsec, each actin filament requires around 66 monomers of actin per second in order to keep up with the flow. Such rapid rates of actin assembly have been seen in vivo in Thyone sperm [Tilney and Inoue, 19821. The inference to be drawn here is that regulation of the rates of actin assembly will decide whether a lamellipodium protrudes, remains stationary, or retracts [Mitchison and Kirschner, 19881. It will be of interest to determine whether rates of actin flow do actually change with rates of lamellipodial protrusion. Second, there is the question as to what happens to the filaments at the base of the lamellipodium. Two possibilities are that either the actin is rapidly broken to monomers or oligomers by depolymerization in concert with actin fragmenting proteins such as gelsolin, or it remains in a filamentous form and is redistributed elsewhere, perhaps into the lamella. Maximal rates of polymerization and depolymerization of actin measured in vitro fall far short of those required in the lamellipodium [Pollard, 19861. Third, what is the motor for the actin flow and where is it located in fibroblasts? In view of the absence of myosin I1 from lamellipodia, new models are focusing attention on the role of mini-myosin or myosin I in rearward lamellipodial transport. As proposed for growth cones [Mitchison and Kirschner, 1988; Smith, 19881, myosin I may lie in a stable submembraneous layer in the lamellipodium where it can drive actin rearward. An al-

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ternate possibility is that the motor is located either on some stable elements within the lamellipodium, such as the actin ribs, or sited within the more central regions of the cytoskeleton such as the dorsal cortical actin sheath in the lamella where myosin I1 is found [Heath, 1983al. Any free myosin I molecules could be available to bind to membrane complexes and transport them anteriorly [Small, 1988; Adams and Pollard, 19891; in this regard, the occasional anterior transport of particles, against the more obvious centripetal direction of lamellipodial flow, has been observed on highly motile fish keratocytes [Kucik et al., 19901. And finally, a model based on a general rearward actin flow also raises the problem of what types of interaction occur between the actin filaments and membrane-associated adhesion molecules. If the myosin motor driving actin flow is to do any work, it must be anchored or else the forces propelling the actin rearward would be cancelled by forward motion of the motor. The most likely way of anchoring the motor is through transmembrane linkages to the substatum. But it is difficult to conceive that stable adhesive contacts are formed if the filaments are moving backward and/or disassembling. For this reason the concept of a molecular clutch has been introduced [Mitchison and Kirschner, 19881 to allow slippage between actin filaments and any associated substratum-based transmembrane complexes along the length of the lamellipodium in a neuronal growth cone. The concept of such a clutch mechanism would be supported if it were demonstrated that there was a difference in rates of the rearward actin flow dependent on the rate of locomotion, on the degree of adhesiveness of the substratum, or on the location of the adhesions. But, in the case of fibroblasts, lamellipodia are generally adherent to the substratum only at their bases; during protrusion, the major portion of a lamellipodium projects anteriorly some distance above the substratum. This is clearly seen by interference reflection light microscopy and by electron microscopy, and is indicated by the fact that particles can attach and move back on both surfaces of the fibroblast lamellipodium [Harris and Dunn, 19721. Furthermore, fibroblasts moving through a three-dimensional lattice of gelled collagen fibers extend lamellipodia into the open spaces of the gel [Heath and Peachey, 19891. One answer to the problem of how to develop a transmembrane linkage between a motile sheet of actin filaments and a stationary substratum is to accept that some of the actin within the lamellipodium does not engage in centripetal flow. LAMELLAR TRANSPORT AND CAPPING

The centripetal flux of cytoskeletal material is not confined to lamellipodia. This fact was noted by Aber-

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crombie et al. [ 1970~1 and others many years ago. There is ample evidence for a continuous rearward flux of actin in the form of arcs (shown in Fig. 4D) in the large fanshaped lamellae of crawling cells [Heath and Dunn, 1978; Soranno and Bell, 1982; Wang, 1984; Heath, 1981, 1983a; Holifield et al., 19901. If the mechanism for generating actin flow in the lamellipodium is still unclear, then the movement of the dorsal cortical actin in the lamella may be simpler to understand. As shown in Figure 4d, in circularly spread fibroblasts the dorsal cortical actin is circumferentially arranged [Heath, 1983a; Small, 19881. Myosin I1 is located among the actin filaments in the cortex as judged by the punctate staining with antibodies to muscle myosin [Zigmond et al., 1979; Heath, 1983bl. So the sliding of actomyosin filaments would tend to draw the cortex rearward. There is a striking parallel in the behaviour of the contractile ring, a band of actin filaments that develops at the site of cleavage during cytokinesis. The association of myosin with these filaments results in a potentially contractile structure which apparently possesses some mechanical connections to the plasma membrane. Interestingly, cross-linked membrane proteins on mitotic cells are transported to the cleavage furrow as the contractile ring forms [Koppel et al., 19821, in a process strikingly similar to the transport of patches with moving arcs in fibroblasts. It has been argued that a release of cortical tension at the poles of a dividing cell could induce contractile ring formation [White and Borisy , 19831. Whether an analogous release of tension at the lamellar margin [Bray and White, 19881 is a prime mechanism in lamellar cortical flow is not known. Exactly how the flow in lamellipodia is coordinated with that in the lamella is unclear. The striking lamellar flow seen in chicken fibroblasts formed the basis of the continuous contraction model of Dunn [1980] which proposed that the whole actin network in the lamella was continuously moving rearward to a site where the network was broken down and recycled to the front of the cell. It may be that only the lamellipodium and the dorsal cortical regions of the lamella are engaging in this flow. At least a portion of the ventral cortex is engaged in cell-substratum contacts and is generating the tension necessary for translocation, and so probably is resistant to rearward movement; the posterior cortical regions of the cell are probably fairly stable. Nevertheless, the fact that stress fibers remain stationary as the cell moves forward and then decay, coupled to the fact that processes at the rear of the cell are regularly drawn in and disappear, means that there must be continuous disassembly of cortical actin in these structures which will be added to the material cycling through the lamellipodia and dorsal cell surface [Abercrombie, 19801. A similar situation must occur in the neuronal growth cone

where new filopodia extend anteriorly and then gradually become more laterally and rearwardly placed and disappear as the growth cone moves forward [Bray and Chapman, 19851. The recycled actin and actin associated proteins must then diffuse forward for reassembly in lamellipodia. The form of this recycled material, the routes taken through the lamella, and the details of its reincorporation into the cytoskeleton are still a mystery. NON-ACTIN BASED MOTORS

Finally there is a caveat. We must be cautious in placing actin at the foundation of all cell locomotory mechanisms. The highly motile amoeboid locomotory behavior of nematode spermatocytes is phenotypically similar to that of fibroblasts and many other more commonly studied cell types, except in one respect: actin is not present in detectable amounts [Nelson et al., 19821. These cells do, however, contain an abundance of a polymerizable protein that forms dynamic filament bundles which assemble at the margins of pseudopods [Roberts and King, 19891 and show a flux relative to the cell margin [Sepsenwol and Taft, 19901. These cells also transport aggregated cell surface markers rearward [Roberts and Ward, 19821and insert membrane proteins at the tips of pseudopods [Pavalko and Roberts, 19871. So, despite the lack of actin, nematode spermatocytes are able to display a repertoire of locomotory behavior and mechanisms that is similar to fibroblasts which clearly indicates these mechanisms are probably common to all cell types. ACKNOWLEDGMENTS

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Cell locomotion: new research tests old ideas on membrane and cytoskeletal flow.

Recent studies on the mobility of membrane markers on crawling cells indicate that there is no long-range centripetal flow of membrane proteins or lip...
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