The movement of cells along surfaces is a complex phenomenon
that consists of several interrelated processes, including
cell-substratum adhesion, and extension and retraction of the cell
edge, in which the actin cytoskeleton plays a crucial role. The past
decade has seen increasingly detailed molecular-based
investigations into cell motility, but it is still not known how
molecular events are integrated to give cell movement. Molecular
studies are now beginning to be linked to a more global concept of
how whole cells move, and this combined approach promises to
yield new insights into cell locomotion.
The authors are at the Department of
Cell Biology and Anatomy,
University of North Carolina at
Chapel Hill, Chapel Hill, NC
27599, USA.
The basic molecular mechanisms underlying the locomotion of cells along solid substrata are thought to be shared by different cell types, including those that appear morphologically distinct. Such loco- motion is usually regarded as a single complex phenomenon, but it is perhaps better thought of as the dynamic interplay of various processes such as cell-substratum adhesion, extension of the leading cell edge and retraction of the trailing cell edge (Fig. 1). To move along a substratum, cells must first adhere to it with sufficient strength to allow sub- sequent spreading of the cell margin. Second, one part of the cell margin must become specialized to extend outwards while the opposing side retracts inwards. Finally, for continued locomotion, ex- tension and retraction must be regulated both spatially and temporally. It is now clear that for most motile cells these processes depend on the dynamic behaviour of an actin-containing cytoskeleton I-s. This review focuses on current ideas about the role of the actin cTtoskeleton in the locomotion of eukary- eric cells over surfaces in vitro.
Are retrograde actin fluxes Involved in locomotion?
Observations of rearward-moving particles, surface ruffles 6,7 and membrane receptors a on the dorsal sur. face of motile cells have long been thought to reflect an underlying mechanism for locomotionL An attractive idea has been that if membrane receptors (which would otherwise be carried rearwards) are immobilized on the substratum, an opposing reac- tion force would push the cell forwards. There have been two very different views on how the retrograde motion of receptors or particles occurs.
One hypothesis proposed that the insertion of membrane lipid at the leading edge together with endocytosis over the remaining cell surface generates a retrograde lipid flow that sweeps dorsal membrane particles or receptors rearwards 1°. This idea became less plausible after the failure to detect a retrograde flow of lipid in locomoting cells n,lz. Instead it was found that plasma membrane lipid advances in con- cert with the extending edge. The other view empha- sized the role of a dynamic meshwork of actin fila- ments 1. The development of this idea began with the demonstration that actin filaments move rearwards with respect to the cell edge in motile cells 13,14. In addition the rearward motion of particles or recep- tors was found to accompany the retrograde actin flUXa, ls-17.
What causes a retrograde actin flux? One expla- nation is that it arises from the treadmilling of actin filaments, in which actin addition to filaments at the leading edge is counterbalanced by a net loss of actin monomers from filaments at the base of the lamel- lipodium. Evidence that actin polymerization occurs predominately at the extending edge 1s-z° is consist- ent with this explanation. Another suggestion is that myosin motors pull the actin meshwork rearwards together with membrane receptors and particles that are linked to this structure 14,17. Support for this idea comes from the finding that in Diclyostelium dis- coideum, myosin II is responsible for both capping of surface receptors and the accompanying increases in tension within the actin cortex zl,zz. Thus it appears that an actomyosin contractile mechanism drives the retrograde actin flux and associated rearward motion of particles and receptors.
However, it is not clear to what extent a retrograde actin flux is involved in cell locomotion. This is because in fibroblasts the rate of retrograde actin flux, as determined by photobleaching experiments is, is much slower than the rate of flux associated with rearward-moving dorsal str~ctures 16. Furthermore, the rate of retrograde actin flux does not correlate with the rate of leading e~o~e extension in a range of cell types 23-2a. This suggests that a retrograde flux of actin is not part of the mechanism involved with edge extension but instead may simply reflect the contractile properties of lamellar actin.
Origin of protrusive forces for edge extension The most actively extending region of a moving
cell is the lamell ipodium, an actin-rich region along the outer boundaw of the leading lamella. A number of hypotheses have been proposed for the produc- t ion of protrusive forces at the extending edge 4. One idea is that strong actomyosin contractions at the rear of a cell increase the hydrostatic pressure w i th in the cytoplasm such that it is forced towards the t ip of the lamellal,26,zL A localized weakening of the cortical c~oskeleton at the t ip of the cell may then allow extension to occur 1. A related idea suggests that the severing of actin filaments leads to osmotic swelling of the actin gel w i th in the lamella zs. This would result in protrusion of the lamella unt i l a new equil ibrium is reached between osmotic pressure and the strength of the actin meshwork (Fig. 2a).
Another possibility is that actin polymerization at the cell edge: directly provides the necessary force for protrusion (Fig. 2b). In many cells actin polymeriz- ation has been shown to occur predominately at the extending edge tg,z°,zg. In addition, the width of the actin meshwork corresponds to the rate of lamellar extension in motile mouse macrophages ~°. Although actin polymerization does indeed appear to be 'directly involved in lameUar extension, there is still some doubt as to whether it could generate the pro- trusive force necessary for locomotion. However, bio- physical considerations about actin polymerization support the idea that lamellar protrusion could result from a Brownian ratchet mechanism 3t.32. According to this idea, thermal fluctuations between an actin filament and the plasma membrane allow insertion of actin monomers that then ratchet the cell edge outwards (Fig. 2b).
Alternatively, protrusive forces may result from actin polymerization in conjunction with the action of myosin I motors at the leading edge (Fig. 2c). This is consistent with the localization of some myosin I isoforms at the extending edges of Dictyostelium amoebae and of fibroblasts 33,s4. Binding of myosin I isoforms to the membrane-associated cortex may facilitate protrusion by 'walking' towards the barbed ends (preferred growing ends) of actin filaments that are immobilized by linkages to the substratum 3s. Myosin I is also suggested to drive the spreading of post-mitotic cellsa6; in these cells, two populations of actin filaments appear to slide relative to each other. However, such sliding has not been detected in loco- rooting cells 24,2s. Furthermore, genetic ablation of the myosin I B gene in Dictyostelimn shows that it is not required for lamellar extension (Ref. 37; see also the review by Condeells in this issue).
Although actin polymerization is clearly of major importance in edge extension, this does not necess- arily preclude the operation of other mechanisms for generating protrusive forces.
Retraction of the cell margin Retraction of the rear margin of the cell must occur
in addition to extension of the leading edge for con- tinued locomotion (Fig. 1). In fibroblasts, retraction is preceded by the development of tension between the front and rear of the cell. When the relative con- tractile strength of the actin meshwork exceeds cell-substratum adhesion strength at the rear, attach- ments are broken and retraction occurs. Detachment is ini t ial ly followed by a recoil of the cell margin, which is then retracted further towards the cell body in an ATP-dependent phase 38,~9. Thus retraction consists of two components: the release of elastic potential energy stored during lamellar extension, and an active actomyosin contraction. In addition, retraction of the cell margin generate~ ~olds in the dorsal cell surface, and these are thoug.qt to provide surface area for subsequent lamellar ex~:ension.
Although retraction appears to be largely a matter of 'ripping' the trailing cell margin from the sub- stratum, there is evidence that the dissociation of integrin-ligand complexes in response to an increase in intraceliular Ca z+ concentration occurs on the yen-
(a) Attachment and spreading
III I l l III II!
• Involves extension of the cell edge in all directions
• Occurs when sufficient cell-substratum contacts are made
(b) Retraction
. . . . . l ~ f O " ~ . ~
. . . . . . . . . . . . . . . . ' " . ) • Involves elastic recoil of the cell edge, an
actomyosin contraction, and dissociation of cell-substratum contacts
• Occurs when contractility of actin meshwork is greater than cell-substratum adhesion strength
,l (o) Lamellar extension
I l l - " I l l ! i?
• Involves generation of protrusive forces at the leading edge
• Occurs when sufficient cell-substratum conta~s are made to resist contractility of actin meshwork
FIGURE 1
Diagram illustrating the coordination of motile phenomena essential for the locomotion of a whole cell. The cell outline represents a side view of a generalized e~karyotic cell. Attachment and spreading of the cell margin in all directions (arrows) precedes the development of a polarized cell shape, which involves the development of extending and retracting cell margins at the front and rear of the cell, respectively. The formation of new cell-substratum adhesions at the extending edge and release of adhesions at the rear are required for continued locomotion.
tral cell surface of neutrophils (Refs 40,41; see also the review by Maxfield in this issue).
Role of cell-substratum contacts Cell-substratum contacts not ordy maintain cell
attachment to a surface; they also allow traction forces generated by the cell to be exerted on the sub- stratum. Focal adhesions 42, the most extensively studied and well-characterized cell contacts, are the predominant type of cell-substratum contact found in slower-moving cell types such as fibroblasts. In rapidly moving cells, adequate cell contact is pro-
TRENDS IN CELL BIOLOGY VOL. 3 NOVEMBER 1993 367
u~'iuw5
(a) Actin gel swelling Severing of actin filaments by proteins such as gelsolin
Osmotic pressure drives expansion of actin gel to new equilibrium position
(b) Actin polymerization at leading edge Insertion of actin monomers
Actin polymerization between growing end of l / / ,=, ~ filament and cell edge
(c) Action of myosin I motors Actin filaments sliding forwards
~ ~ Sliding of actin filaments results in protrusive force
Actin filament Myosin I moving anchored to towards barbed substratum end of actin filament
FIGURE 2 Diagram illustrating the key molecular events underlying three possible mechanisms for
generating protrusive forces at the extending cell edge. Ventral cell surfaces are shown to be attached to the substratum via transmembrane linkages that link actin filaments (solid lines) to
adhesion molecules on the substratum; the molecular assembly (shaded) shown here represents an integrin-adheslon-molecule complex, and other components of focal adhesions. (a) Actln gel swelling. Actln filaments forming the actin gel within the lamella are severed (thick dashed lines)
by actin-binding proteins such as gelsolin (open ellipse), Osmotic pressure within the lamella causes it to extend outwards until a new mechanical equilibrium is reached between osmotic
pressure within the lamella and the elasticity of the actin gel. (b) Actin polymerization at the cell edge. Insertion of actin monomers (black ellipses) at the tips of elongating actin filaments
(elongation is indicated by chevrons) is shown occurring predominately at the cell edge. Here, pcotrusive forces are suggested to arise from a ratchet mechanism in which actin monomers
become inserted between the tips of actin filaments and the cell edge. Actin.nucleation proteins such as profilin, ponticulin or talin are also present at the cell edge (lightly shaded area).
(c) Actinn of myosin I motors. Myosin I molecules (darkly shaded) are shown moving towards the barbed end (chevron) of an actin filament that is anchored by transmembrane linkages to
the substratum. Actin filaments that are bound to myosin I but not fixed to the substratum are pushed forwards, thus generating a protrusive force.
368
vided by diffuse, highly labile regions of close con. tact, of unknown molecular composition 4a.
Lamellar extension can succeed only if the ventral surface is attached to the substratum 44 (Fig. 1). The continuous formation of cell-substratum contacts at the front of the cell serves to anchor newly pol- ymerized actin to the substratum and acts to resist lamellar contractility. When contacts cannot be
formed lamellar extension will not occur. If contacts are lost from any part of the cell margin, it will retract (Fig. 1).
In addition to their role in cell adhesion, cell-substratum contacts are involved in the transduction of chemical and mechanical signals across the cell membrane that are important in regulating actin fila- ment dynamics 4s,46. Signalling events are triggered by the clustering of in- tegrin molecules at sites of cell- substratum contacP 7,4s. For example, phosphorylation of molecular com- ponents within focal adhesions in response to integrin clustering is cru- cial for the maintenance of normal cell adhesion, morphology and cell motility. The induction of phospha- tidylinositol 4,5-bisphosphate (PIP2) synthesis is also thought to result from integrin clustering. This may in turn lead to an increased rate of actin polymerization via binding of PIP 2 to the actin-binding proteins profilin and gelsolin. Furthermore, integrins have been shown to be mechanically coupled to the actin cytoskeleton 49. Thus at sites of cell-substratum con- tact, mechanical stresses can directly influence the organization of the cytoskeleton, in addition to generat- ing chemical signals that regulate actin filament dynamics.
Regulation of locomotion The posit ioning and t iming of
lamellar extension and retraction will profoundly affect cell shape and mode oi locomotion. The spatial and temporal regulation of actin filament dynamics therefore forms the mob ecular basis for the control of cell locomotion. Many classes of mob ecules have been found to influence actin architecture and dynam- ics s,s°-s2. These include a wicle variety of actin-binding proteins, and second messengers such as intracellular Ca 2÷, inositol trisphosphate and diacyl. glycerol. Current research is attempt. ing to determine which factors in- fluence the interactions of regulatory molecules with the actin cyto- skeleton and how these interactions
are spatially and temporally organized. Although it is not clear how differences arise
between the front and rear of a cell, once formed these specializations tend to b,.. self-perpetuating. For example, a gradient of intracellular Ca 2+ in motile newt eosinophils is suggested to be involved in main- taining a polarized cell shape (Ref. 53; but see also the review by Maxfield in this issue). Relatively low
TRENDS IN CELL BIOLOGY VOL. 3 NOVEMBER 1993
levels of intracellular Ca 2÷ at the extend- ing edge are thought to promote lameilar extension, by increasing the activity of actin-filament-severing proteins, which may then provide more sites for actin pol- ymerization. Localization of the actin- binding proteins ponticulin and profilin at the extending edges of motile cells is also believed to increase actin pol- ymerization in these regions 4s. At the rear of the cell, increases in Ca 2÷ are thought to promote retraction of the cell margin by increasing the contractility of the actin filament meshwork.
Local molecular regulation of actin fil- ament dynamics is suggested to be or- ganized at the scale of the whole cell by tension within the cytoskeleton s4'ss. For
example, increased tension has been shown to decrease actin polymeriz- ation s4. Therefore, increased contractility of the actin meshwork at the rear of a cell will inhibit extension but promote retraction. Conversely, decreases in ten- sion of the actin meshwork, such as that occurring following retraction, can induce lamellar extension at the front of the cell 39. Consistent with this scheme are observations that actin stress fibres are abundant within retracting regions of the lamella where con- tractility of the actin meshwork is relatively high, but absent from rapidly extending cell edges. Tension within the actin meshwork may regulate stretch-sen- sitive Ca 2÷ channels that then in turn influence actin filament dynamics s6.
FIGURE 3
The movement of whole cells To understand how whole cells move it is necess-
ary to learn how various motile phenomena are inte- grated during locomotion. One general difficulty is that the majority of cell types studied display com- plicated morphologies and modes of locomotion. For example, the movement of fibroblasts occurs in two phases that are temporally and spatially distinct. Retraction at the rear of the cell is followed after a short lag by extension at the front (Fig. 3). This type of movement is further complicated by small 'pock- ets' of retraction at the extending edge, and regions of extension at the retracting edge. Such complexi- ties impede attempts to relate how events at the molecular level lead to locomotion of a whole cell. By contrast, fish epidermal keratocytes possess a uniquely simple shape and gliding mode of locomo- tion (Fig. 4a). This is because lamellar extension and retraction occur in a very coordinated manner, so that cell size and shape are maintained during loco- motion. A model has recently been proposed that relates the movement of keratocytes to the behaviour of the actin cytoskeleton sT. The basic idea is that a graded outgrowth of actin filaments perpendicular to the extending edge occurs simultaneously with a graded contraction of lamellar actin perpendicular to the rear edge (Fig. 4b). Thus the gliding mode of keratocyte locomotion appears to result from a highly synchronized regulation of actin filament dynamics ss.
b ¢
:.: ~.
TRENDS IN CELL BIOLOGY VOL. 3 NOVEMBER 1993
The shape and mode of locomotion displayed by a fibroblast. (a) Phase-contrast image of a fibmblast moving in the direction indicated consists of an irregularly shaped lamella (L) at the front of the cell and a retracting 'tail' (T) at the rear. Bar, 10 I~m. (b) Fibroblast locomotion is discontinuous, consisting of a discrete phase of retraction (arrow) at the rear of the cell, followed after a short lag by a phase of lamellar extension at the front (arrow). Thus extension and retraction of the cell margin are temporally and spatially separate events.
Although the processes of lamellar extension and retraction occur in all moving cells, a key difference underlying the distinct morphologies of different cell types appears to be in the way actin filament dynam- ics are regulated. This idea is consistent with the find- ing that cells such as fibroblasts may resemble ker- atocytes morphologically for a short time following plating out. Conversely, keratocytes can mimic fibrohlasts both in shape and mode of locomotion. These observations confirm the notion of similar basic mechanisms of locomotion in distinct cell types. Future comparative studies of the slower- moving, complex-shaped cells such as fibroblasts and the rapidly moving keratocyte will yield useful infor- mation about differences in the regulation of actin dynamics in these cells.
Whatever the focus of future research into cell locomotion, it is clear that molecular-based studies of locomotion will need to accompany the develop- ment of models that link this information to the movement of a whole cell. Only a combination of molecular and global approaches will yield further
b
FIGURE 4
Simple shape and mode of locomotion displayed by a fish epidermal keratocyte. (a) Phase-contrast image of a fish keratocyte moving in the direction indicated (arrow). It consists of one large ellipsoidal lamella with a smooth outer margin. Keratocytes display a continuous, gliding mode of locomotion. Bar, 10 lam. (b) Extension and retraction of the cell margin (indicated by size and direction of arrows) occurs simultaneously and in a graded manner, perpendicular to the cell
margin.
369
e'~'i~qv"
Acknowledgements
We thank Jennifer Upfert for help in
preparing this manuscript. Work
in this laboratory is supported by the
NIH.
insights into the complex phenomenon of cell loco- motion.
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