Cell, Vol. 87, 601–606, November 15, 1996, Copyright 1996 by Cell Press Getting Membrane Flow and the Review Cytoskeleton to Cooperate in Moving Cells

Mark S. Bretscher of a fibroblast is a few microns per minute: this velocity MRC Laboratory of Molecular Biology decreases towards the rear of the cell so that objects Hills Road collect over the nuclear region. And finally, the capping Cambridge CB2 2QH process itself is completely inhibited in lymphocytes England (where it has been studied most) by preincubating the cells with a combination of cytochalasin B and colchi- cine, drugs which are known to interfere with the organi- zation of microfilaments and microtubules respectively. This year sees the 25th anniversary of the discovery by Preincubation with either drug alone produces, at best, Taylor and colleagues (1971) of the phenomenon of anti- only a partial inhibition. The fact that both drugs are gen capping—the remarkable process whereby surface required suggests, in the simplest interpretation, that antigens on a migrating eukaryotic cell, when extensively cap formation can be brought about by some action crosslinked by antibodies to form patches and made visi- involving either the actin or tubulin cytoskeletons inde- ble by a fluorescent tag, move to the trailing end of the cell. pendently of the other. It is these crucial facts—the need Although sometimes regarded as an artificially induced for crosslinking and for the cytoskeleton—that provide process and therefore of little consequence, this is, I think, a basis for thinking about how capping occurs. And it a mistake: cap formation is restricted to motile cells and is these same facts that have led to very different views can tell us much about how cells migrate. Furthermore, of the process of cap formation and hence of how cells cell polarity is of increasing importance in understanding locomote. developmental processes. Whether the polarity displayed in cap formation is related to the polarities exhibited in developmental processes (such as in some oocytes) re- Rakes Versus Flow mains to be discovered, but any easily observed polar For simplicity, I focus on the most plausible cytoskeletal process in animal cells is currently of interest. The aim of mechanism—the rake—and the alternative membrane this review is to compare two different views of the molec- flow scheme, which I favor (see Figure 1). ular mechanism of cap formation, how the experimental The rake scheme outlined here is based on the idea evidence lines up on each side of the argument and how that certain transmembrane proteins on the cell surface these views relate to cell locomotion. link the outside world with that part of the actin cytoskel- Although the process of cap formation was discovered eton adjacent to the plasma membrane (de Petris, 1977), by crosslinking antigens on the surfaces of lymphocytes and that this part of the cytoskeleton is continuously in suspension, the rearward migration of patches is more moving rearward—in the reference frame of the cell. easily identified on substrate attached cells. Because only This rearward movement causes the attached trans- those cells that are in a locomotory mode cap surface membrane proteins—the teeth—to comb the surface antigens—stationary cells do not do so—it has been evi- of the cell. Capping is envisaged as occurring when dent since its discovery that cap formation is closely re- crosslinked aggregates—or particles—become suffi- lated to cell locomotion. How they may be connected is ciently large that they are caught in the teeth of the rake not difficult to see: if the crosslinking molecules happen and hence are moved rearwards towards the back of to be attached to the substrate, the cell itself will move the cell. By contrast, uncrosslinked molecules would forward as crosslinked patches move rearward. In this remain unaffected by its passage. In this model the case, the crosslinked surface antigens have transiently rearward movement of actin filaments is balanced by become the cell’s feet. (Further advance could obviously filament assembly at the front of the cell and depolymeri- not occur unless the surface antigens were uncrosslinked zation towards its rear. and recycled somehow to the front of the cell). A different Experimentally, there is no doubt that in fibroblasts way of observing what is presumably the same process and several other cell types, actin filaments do form at is particle migration, which was discovered a few years the leading edge (Wang, 1985; Cao et al., 1993; Forscher before cap formation (Marcus, 1962; Ingram, 1969; Aber- and Smith, 1988), that actin filaments do continuously crombie et al., 1970; Bray, 1970): particles attached to the migrate rearwards and then disassemble towards the dorsal surface of a migrating cell move away from the rear of the cell near the nucleus (Heath and Holifield, front of the cell, towards its rear. In this case, the attached 1991). The observed actin flow generates a polarized particles—which are usually about 100 nm in size— actin cycle in which the initial rate of rearward filament become attached to many surface molecules and thus migration is about the same as that of particles attached act as massive crosslinking agents. Particle and patch to the cell’s dorsal surface. An essential requirement of migration are thus seen to be aspects of the same underly- this scheme is that, once the teeth of the rake have ing mechanism. But what is this? reached the sites of actin depolymerization, they find The movement of patches or particles is a directed their ways back to the front of the cell for reuse. This process: it always occurs away from the advancingedge return could be provided by forward diffusion in the of the cell, and requires metabolic energy. Any surface plasma membrane or by a polarized endocytic cycle. molecules that are extensively crosslinked cap—neigh- Other cytoskeletal models have been advanced: these boring untethered molecules do not do so. The rate of usually invoke specific recognition between the cyto- particle, or patch, migration away from the leading edge plasmic domains of just those molecules which have Cell 602

Figure 1. Two Schemes for Particle Migra- tion on Motile Fibroblasts In both schemes, the cells are moving to the left. (A) Polarized actin cycle in which actin fila- ments polymerize at the leading edge, push- ing the cell’s front forward to extend it. Actin depolymerization ahead of the cell’s nucleus generates actin subunits (g-actin) which dif- fuse to the cell’s front for reuse. Filamentous actin, moving rearward, pulls tines (small arrows) and associated particles on the cell surface towards the cell’s rear. Particles or patches often migrate with actin, although this is not always the case. (B) Polarized endocytic cycle in which exo- cytosis at the leading edge extends the cell surface, bringing a fresh source of substra- tum attachments with it. Endocytosis by coated pits occurs randomly on the cell sur- face, generating a lipid flow in the plasma membrane which decreases with increasing distance from the leading edge: this is indi- cated by the decreasing size of the arrows along the plasmalemma. This flow pushes on substratum-attached feet, providing a fluid drive to advance the cell. Particles or patches on the cell surface are swept towards the back of the cell, where rearward movement due to flow is balanced by random movement due to Brownian motion. been crosslinked and the cytoskeleton. These models occur at the leading edge of migrating fibroblasts do not explain how crosslinked lipid molecules or cross- (Bretscher and Thomson, 1983; Ekblom et al., 1983; Hop- linked glycophosphatidyl-inositol containing proteins kins et al., 1994) and coated pits are spread randomly on (such as Thy-1), which have no cytoplasmic domains, the surface of these cells. This polarized endocytic cycle can interact with the cytoskeleton and hence be capped. therefore leads to a rearward lipid flow in the cell’s plasma Nor do most of them include a polarized actin cycle and, membrane, whose rate decreases from the front of the as such, seem less viable. cell towards its rear. Such a decreasing flow rate would The membrane flow scheme suggests that capping be expected to result in a decreasing rate of rearward surface antigens is effected by a polarized endocytic particle migration as the object moved away from the cycle (Abercrombie et al., 1970; Bretscher, 1984), in front of the cell, as is observed (Harris and Dunn, 1972). which endocytosis occurs randomly on the cell surface Furthermore, it is possible to estimate how fast rearward and exocytosis at the front of the cell. As the sites of migration should be for a particle near the leading edge endocytosis and exocytosis are spatially separated, a of a fibroblast, given the known rate of endocytosis by flow of membrane necessarily occurs within the plasma coated pits. Calculation and observation agree within a membrane from the sites of exocytosis (the front of the factor of about two. (Coated pits internalize about 2% of cell) towards the sites of endocytosis (towards the rear the cell surface per minute; if added uniformly to the front of the cell). Since coated pits, which are assumed to be of a 100 ␮ long triangular flat cell, this would produce a the principle endocytic agents, internalize domains of frontal extension of 1 ␮/minute. Particles or Con A the plasma membrane which are greatly enriched in patches move rearwards on migrating fibroblasts at some proteins (such as the LDL- and transferrin-recep- about 2 ␮/minute [Abercrombie et al., 1970,1972]). tors) compared to other surfaceproteins (such as Thy-1), In many respects, the predicted behaviors of patches the actual situation is more complex. The flow of mem- as they cap, whether viewed by a rake or by a flow brane is most easily conceptualized as a rearward lipid mechanism, are quite similar. Both leave uncrosslinked flow (Bretscher, 1976), with some proteins (e.g. LDL- molecules more or less randomly placed; as the aggre- receptors) cycling rapidly and others very slowly. A gates become larger, so the effects of the teeth of the plasma membrane protein which does not actively cy- rake, or the flow, predominate. Although these schemes cle, such as Thy-1, can diffuse about on the cell surface place quite different emphases on the role of the cy- sufficiently quickly that any consequent drift in its posi- toskeleton, in neither of them is it clear how those drugs tion caused by the lipid flow is small. However, should which disrupt the actin or tubulin cytoskeletons actually such an antigen become extensively crosslinked by ex- have their effects. Before considering this, the relation- ternal agents, the diffusion coefficient of the resultant ship of each scheme to how cells locomote is presented. patch would decrease and calculation indicates that the flow would now sweep it to the rear of the cell(Bretscher, Cell Locomotion 1976). Capping, then, is seen as a direct consequence These two different models of how capping occurs have of reducing the diffusion coefficient of an object lying given rise to two views for how cells migrate. One class in the flowing bilayer. of models proposes that extension of the cell’s leading Experimentally, exocytosis of cycling membrane does edge is effected by the cytoskeleton, although several Review 603

different schemes—invoking different cytoskeletal ele- make it extremely adherent. Nevertheless, the “neurites” ments—exist (see, for example, Condeelis, 1993; Mitchi- can extend hundreds of microns. The authors suggest son and Cramer, 1996). In some, actin polymerization that the actin network in normal growth cones enhances itself provides the force to push the front of the cell the attachments they make with the substratum, and forward in much the same way that listeria (Tilney and assists filopodia and lamellipodia to form. This experi- Portnoy, 1989) and vaccinia virus (Cudmore et al., 1995) ment shows that an actin-based system is not an abso- are propelled in the cell’s cytoplasm. In others, the force lute requirement for neuronal growth. The observations is provided by actin filaments in conjunction with single- that the neuronal ending is packed with vesicles and that headed myosins. Traction is presumably required by a the existing microtubules and neurofilaments in these migrating cell in vivo as it pushes its way through a “neurites” only extend into the bases of these blunt- matrix or between other cells. The force for this needs ended growth cones (as they do in normal growth to be applied to those cell surface receptors bound to cones), suggest that extension in this case occurs by the extracellular framework—the feet of the cell, which the deposition of membrane material at the distal tip of are usually integrins. This could be provided by the rear- the “neurite.” wardly moving cytoskeleton: if the cell’s feet, while The suggestion (Marsh and Letourneau, 1984) that a bound by the substratum, attach to the actin cytoskele- role for the actin network at the leading edge is to en- ton (either directly or indirectly), a force would be trans- hance normal attachments is intriguing, especially as it mitted through them to push the cell forward. In this is consistent with observations of blebbing on migrating case, the feet could be the same molecules which con- cells. When blebs of membrane appear on the surface stitute the tines of the rake which effect capping. Like of a migrating cell, they usually do so at the leading the tines, a mechanism would be required to bring them edge. However, these balloons of apparently struc- from the rear of the cell up to the front for reuse and tureless membrane do not generally lead to new cellular this transport could occur either by diffusion or by the attachments which advance the cell; instead, they move endocytic cycle. rearwards (like a particle) on the cell’s dorsal surface The alternative viewpoint sees locomotion from a and disappear as they get resorbed into the cell (Harris, membrane perspective: endocytosed membrane is rein- 1973; Trinkaus, 1984). Blebs which appear at the cell’s serted at the leading edge of the cell, extending the front leading edge thus seem to be membrane which has of the cell forward. The flow of lipid generated—which exocytosed there but, for unknown reasons, lacks both causes particles to migrate rearward—pushes on those the normal lamellar structure provided (presumably) by feet attached to the substratum. These feet are, in effect, the actin network and its adhesive qualities: this results patches of integrins crosslinked by the substratum: the in a cell extension which has lost its grip. force provided by the lipid flow, which would normally Drugs which disrupt microtubules also permit some cap them, pushes against them to move the cell for- form of neurite growth (Lamoureux et al., 1990), although ward—a novel form of fluid drive. In addition, feet re- the extent of this seems to be rather modest. These leased from the substratum away from the front of the findings and those of Marsh and Letourneau (1984) have cell could be endocytosed and included in that mem- been extended to analyze the roles of each filamentous brane transported through the cytoplasm and added network separately in axonal transport, using mitochon- to the front of the cell (Bretscher, 1984; Lawson and dria as markers (Morris and Hollenbeck, 1995). The re- Maxfield, 1995). This would allow the extension of the sults dramatically show that microfilaments and micro- leading edge to make new attachments to the substra- tubules can independently advance mitochondria in tum for the cell to advance. both directions—towards the cell body or the distal tip. How can these two cycles—a polarized endocytic cy- In the absence of both networks (but in the presence cle and a polarized actin cycle—be distinguished? An of neurofilaments), no mitochrondrial movement occurs. examination of the effects of drugs or of specific muta- The fact that motors exist which can transport an organ- tions might help. elle—the mitochondrion in this case—along microfila- ments and microtubules suggests that the transcellular movement of vesicles in general may also be assisted Neuronal Processes by either set of filaments, although the efficiency with The advance of a neuronal growth cone at the tip of which they do so may vary with the kind of vesicle and an extending axon is similar to that of a fibroblast. In the cell type. However, if endocytosed membrane also particular, particles attached to their dorsal surfaces makes use of both filamentous systems for transport to migrate rearwards, suggesting that they use the same the leading edge in migrating cells, it could provide the basic mechanisms for advancing (Bray, 1970). The ef- basis for an explanation—in a flow scheme—for why fects of drugs which disturb the cytoskeleton have been cap formation is eliminated in cells preincubated with most carefully examined in neurons. In the prolonged drugs which disrupt both filamentous systems, but is presence of concentrations of cytochalasin B which ap- comparatively insensitive in cells treated with a drug pear to eliminate microfilaments, nerve cells can still which disrupts only one of them (de Petris, 1978). extend long processes (Marsh and Letourneau, 1984). A detailed analysis of the effects of cytochalasin B on They are, however, abnormal: the growth cone has lost Aplysia growth cones shows that, within seconds of its shape, being blunt-ended and packed with small drug addition, actin filament assembly at the leading vesicles, the processes themselves do not extend in a edge ceases (Forscher and Smith, 1988; Lin et al., 1996). linear fashion as do normal neurites but tend to grow Surprisingly, the flow of existing actin filaments away in curves, and they only form on a substratum treated to from the leading edge continues. This demonstrates that Cell 604

actin polymerization at the leading edge does not itself Genetic Evidence drive the rearward flow of filamentous actin and adjacent Genetics is usually the salvation of biologists trying to particles on the surface of the growth cone (Forscher solve complex problems: cell migration is something of and Smith, 1988). The results do, however, indicate that an exception. Two rather unsatisfactory “results” exist. filopodial growth depends on microfilament assembly. First, deletion of conventional myosin from Dictyostel- Recent studies in which (single-headed) myosins are ium might be expected either to eliminate locomotion inhibited (Lin et al., 1996) suggest that the rearward (if sliding filaments are directly involved in lamellar ex- flow of microfilaments and associated particle migration tension), or to have no effect: in fact, the result is in depend, at least in part, on myosin. Although these ex- between (Wessels et al., 1988). Furthermore, although periments do not indicate how the myosin acts, the rearward particle movement is unaffected in this mutant authors suggest that myosin in the growth cone could (Jay and Elson, 1992), cap formation—always regarded be fixed to a frame (fixed with respect to the substratum) as closely related to particle movement—is reported to and which could directly effect the rearward flow of be inhibited (Pasternak et al., 1989), an unhappy result. microfilaments and thereby movement of actin-linked Many other mutations affecting components of the particles. However, other possibilities are not ruled out. Dictyostelium cytoskeleton—and combinations of them— For example, endocytic vesicles associated with more do not seriously impair locomotion. Second, any pro- stable actin filaments could be cotransported by myosin cess that eliminates endocytosis should, in flow models, to the leading edge and thereby provide the putative halt locomotion. The shibire-ts1 mutation, a dynamin rearward flow of surface membrane needed, in a flow mutant originally discovered in Drosophila, reversibly scheme, to move the particle. blocks endocytosis by coated pits and thereby para- Recently, Dai and Sheetz (1995) have reexamined the lyzes flies above 30ЊC. This mutation seemed ideal for behavior of particles attached to the axons of growing investigating the role of endocytosis in cell locomotion. neurites: previousevidence indicated that such particles It has been transplanted into mammalian tissue culture remain stationary (Bray, 1970). By contrast, Dai and cells, where receptor-mediated endocytosis becomes Sheetz find that particles (attached through appropriate reduced above the restrictive temperature (Damke et antibodies to the axonal surface) move quite rapidly al., 1995), but its effect on locomotion has not been along the axon, from the growth cone towards the cell reported. However, any effect it has may only be small body. They propose that this movement is driven by an because, when these cells are shifted to the restrictive underlying endocytic cycle, in which endocytosis in the temperature, fluid phase endocytosis is initially only par- cell body is approximately balanced by exocytosis in the tially inhibited and fully recovers within an hour. Other growth cone. The authors report differences in surface kinds of endocytosis seem to exist, and the value of tension between different positions along an axon shibire in this context is thus greatly diminished unless which, they suggest, could support this flow. The parti- motile cells can be found whose only endocytic route cles, then, move up the axon with the surface mem- is via clathrin-coated pits. It seems evident that many brane—or lipid—flow in the same way proposed for par- basic cell biological processes are too vital to be left in ticles attached to the dorsal surfaces of fibroblasts the hands of a single gene, making a genetic approach (Bretscher, 1984). Although the actual site of exocytosis less straightforward. within the growth cone is not known, parallels with fibro- blasts would imply that it occurs at the leading edge. Conclusions In summary, actin polymerization occurs right at the I have tried here to provide a balanced view of cap leading edge of a growth cone, where it may help ad- formation and its extension to cell locomotion, as seen vance the leading edge (as described for fibroblasts). from two different perspectives. Its presence there, however, is not an absolute require- The cytoskeletal model described is based on a polar- ment for neurite extension; long-term treatment of neu- ized actin cycle (Bray and White, 1988). Filament assem- rons with cytochalasin B allows some neurite growth, bly occurs at the leading edge where it seems to and the results suggest that filamentous actin in a nor- strengthen substrate attachments. Actin may also lead mal growth cone may enhance its attachment to the to filopodial extension and help push the leading edge substratum. The rearward flow of actin and of surface- forward either directly by polymerization or in conjunc- attached particles appear not to be driven by actin poly- tion with other microfilament-associated proteins. Fila- merization at the leading edge, but the involvement of ments adjacent to the plasma membrane flow from the myosin in both processes is likely. Particles attached to leading edge towards the rear of the cell or growth cone, the surface of some axons migrate towards the cell where they disassemble. This cycle is completed by the body; this movement seems to be driven by a polarized transfer of actin from the back to the front for reuse, endocytic cycle. Axonal transport of mitochondria—and presumably by actin subunits diffusing forward through hence possibly other membraneous organelles—can be the cytoplasm. The cell’s feet, or the rake’s tines, poke effected by both microtubules and microfilaments. This through the plasma membrane and, being attached to raises the possibility —pure speculation at this stage— the actin network, also migrate rearwards. This could that the reason cap formation in lymphocytes is elimi- provide the motive force for capping and cell locomo- nated after the cells have been incubated with drugs tion. How the actin network is moved backward in this which inactivate both cytoskeletal systems is because scheme is unclear: myosin, the most likely motor, would each system contributes, in parallel, to the intracellular have to be linked to a structure to provide a buffer transport of endocytosed membrane. against which to push. Other models for effecting the Review 605

actin flow can be envisaged which incorporate different such as FM1–43, which rapidly partitions into only the microfilament-associated proteins, many of which are external leaflet of the lipid bilayer and can therefore be well-characterized molecules (Bretscher, 1991). This used to measure surface uptake (see, for example, Smith general scheme, as presented, has no obvious role for and Betz, 1996); it is unclear whether this method could microtubules in cap formation. be made sufficiently sensitive to measure very short I favor a flow mechanism, driven by a polarized endo- periods of endocytosis during locomotion. cytic cycle in which both microtubules and microfila- It seems probable that a polarized endocytic cycle ments effect the intracellular transport of endocytosed exists in migrating cells whose function is, at least in membrane, because it appears to explain cap formation part, to transport the cell’s feet from the rear to its front. and particle migration naturally. In addition, the experi- Whether this membrane cycle is sufficient to extend a mental evidence for the sites of endocytosis and exo- cell forward and to propel patches of crosslinked anti- cytosis and the speed at which the endocytic cycle gens rearward, or whether the polarized actin cycle is operates seem able to explain the observed phenom- the principle driving force for these movements, is the ena, at least in fibroblasts. I therefore see related pro- nub of how a cell puts its best foot forward. cesses from that viewpoint, and offer three examples of this. References First, the rearward movement of filamentous actin in migrating fibroblasts may be caused by its association Abercrombie, M., Heaysman, J.E. M., and Pegrum, S.M. (1970). The with, or proximity to, a rearwardly flowing membrane. locomotion of fibroblasts in culture. III. Movement of particles on This could fit with the observation (Forscher and Smith, the dorsal surfaces of the leading lamella. Exp. 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