Louise P. Cramer

The Randall Institute, Kings College London, 26-29 Drury Lane, London WC2B 5RL, UK.

Received 5/21/97; Accepted 5/26/97

4. TYPES OF ACTIN-DEPENDENT MOTILE FORCE TO DRIVE RETROGRADE PARTICLE FLOW RELATIVE TO THE SUBSTRATUM IN LAMELLIPODIA

4.1. Actin flow-coupled mechanism

The classic photobleaching work of Wang (20) together with more recent work has lead to the view that actin filaments are formed at the front of lamellipodia (21-23), and then the filaments continuously flow retrograde relative to the substratum (19, 24-26), before disassembling further back in the lamellipodium. This, in conjunction with results showing that in some cell types cell surface-attached particles flow retrograde at the same rate as internal filamentous structures (8, 27), has led to the prevalent idea that particles flow retrograde in lamellipodia because they are coupled to the retrograde flow of actin filaments. In Aplysia bag cell neuronal growth cones, this is supported by a direct test; actin filaments marked by photobleaching of phalloidin move at the same rate as surface-attached foreign beads (26). The natural question then is 'how does the actin flow?' While it was initially thought that actin assembly itself might drive retrograde actin flow (20), flow occurs in the absence of actin polymerization (28). This result switched investigators attention to alternative candidates for driving retrograde actin flow in lamellipodia. In Aplysia growth cones, one candidate is that the motor activity of a myosin drives actin flow (e.g. Fig 5A). In these cells actin flow is inhibited (29) by a low affinity inhibitor of myosin ATPase (BDM, (30, 31) and microinjection of cells with NEM inactived-myosin heads. It is not known which myosin drives retrograde actin flow or where the myosin is spatially located.

Fig 5. Types of actin-dependent motile force to drive retrograde particle flow relative to the substratum in lamellipodia. (A and B, actin flow-coupled mechanism) (A) Myosin (grey ball and stick), theoretically associated with an adhesion site (black bar) moves (short arrow to right) toward the barbed end of actin filaments (chevrons) and drives the filament retrograde (lower short arrow to left). Particles (lollipop) are coupled to this retograde actin flow. (B) In the same lamellipodium, some particles (lollipop) may be coupled to a population of actin filaments (chevrons) flowing at a different rate (lower long arrow to left) to the filaments in A. (C and D, alternative mechanisms to drive retrograde particle flow) (C) Particles attached to the cell surface (lollipop) or located inside the lamellipodium (not shown), are actively driven retrograde by a putative motor (black ball and stick) directed toward (lower long arrow to left) the pointed end of actin filaments (chevrons). In keratocyte lamellipodia, actin filaments are stationary relative to the substratum and are likely attached to adhesion sites (vertical black bars). In tissue culture fibroblasts actin filaments are not stationary, but flow retrograde slower than particles. If a pointed end-directed motor moves particles retrograde on these actin filaments in these cells, a mechanism must exist to prevent the filaments from undergoing net forward movement (which has not been reported to occur in lamellipodia). (D) Surface tension is higher at the back than the front of the lamellipodium and drives surface lipids (lower thick arrow to left) and surface-attached particles (lollipop) retrograde.

An alternative candidate for driving retrograde actin flow has come from a mathematical model (32). In this model,flow is driven by loss of actin filaments from a crosslinked actin network at the back of the lamellipodium. This is predicted to induce greater stress in the remaining network at the back of the lamellipodium, creating a tension gradient, sufficient to drive retrograde flow of the actin network. Also, since in this model the crosslinks in the actin network allow the stress to develop, a gradient in actin crosslinks, higher at the back of the lamellipodium, is also predicted to generate a tension gradient. For some motile cell types, this is a very attractive model. For example in Ascaris sperm cells, where actin filaments are replaced by major sperm protein filaments (that flow retrograde relative to the substratum (33)), no cytoskeletal motors have been identified. Also this model may not be at odds with a role for a myosin in driving retrograde flow of actin filaments, as above in Aplysia growth cones. Instead of using the motor activity of a myosin, as drawn in Fig 5A, a myosin may instead act to crosslink the filament network. Certainly, myosin II crosslinks actin filaments into a non-sarcomeric, 'zig-zag' array at the back of lamellipodia of certain tissue culture fibroblasts (34, 35).

4.2. Alternative mechanisms to drive retrograde particle flow in lamellipodia

Outside of the Aplysia system it is unclear if particles couple to the retrograde flow of actin filaments. In lamellipodia of keratocytes, and MC7 and IMR 90 tissue culture fibroblasts, surface-attached particles, and phase-dense inhomogeneities flow retrograde relative to the substratum faster than actin filaments (25), compare (36) and (37). Retrograde particle flow is dependent on an intact actin cytoskeleton. Particles flowing at different rates may simply be driven by different populations of actin which are flowing at different rates in the same lamellipodium, but are not equally detected by methods used in different motile cell types (Fig 5B). Consistent with this possibility, particles have been observed to flow retrograde relative to the substratum at different rates over dorsal and ventral surfaces respectively in the same fibroblast lamellipodium (38). Alternatively, these data also fit a model in which particles, either on the surface, or inside the lamellipodium, are actively driven retrograde by the action of an actin-based motor. Genetic studies in amoeba do not support a role for myosin II (3), nor for myosin IA/1B, IB/IC or IB/ID (39) in driving retrograde particle flow in lamellipodia. Chromophore assisted laser inactivation studies in chick dorsal root ganglia do not report such a role for myosin IB or V (40). BDM does not inhibit retrograde particle flow in lamellipodia in either newt lung cells (Waterman, Storer and Salmon, submitted) or heart or MC7 fibroblasts (Cramer and Mitchison, unpublished). While it is too early to exclude a role for myosin in driving retrograde particle flow in lamellipodia, one possibility is that the motor is a yet to be identified pointed end-directed actin motor protein (Fig 5C). In mitotic cells, the theoretical existence of such a motor to drive a type of retrograde particle flow is the simplest explanation of experimental data (41). It is consistent with the polarity of most actin filaments detected in lamellipodia (15-19). Supporters of this idea need to find alternative roles for the myosins enriched in lamellipodia. One obvious role, but so far not reported in the literature outside of the Aplysia system, is to drive retrograde flow of actin filaments. Alternatively, video tracking has shown that certain cell surface proteins can move rapidly forward in lamellipodia (42-44). This movement requires actin filaments and may be driven by a myosin, allowing receptors to rapidly promote substrate sensing and adhesion. This is consistent with the localization of a myosin I isoform to forward moving particles in lamellipodia of coelomocytes (45). Other work implicates roles for unconventional myosins in protrusion and retraction of leading edge structures (40), also see (46), vesicle transport/secretion (reviewed in (47), and stabilization of actin containing structures (48).

A distinct alternative mechanism for driving retrograde flow of surface-attached particles is tension-driven surface lipid flow (Fig 5D). Recent studies show that surface lipid in chick dorsal root ganglia neurites flows retrograde relative to the substratum at 4-7 mm/min along a shallow surface tension gradient (49). The observed rate of lipid flow is certainly sufficient to drive observed retrograde flow relative to the substratum of particles attached to the surface of lamellipodia in keratocytes (5 mm/min, calculated from (36)) and fibroblasts (1-2 mm/min, (25)). The Dai and Sheetz data differ significantly from previous views of lipid flow; where, in locomoting cells, surface lipid has instead been invoked to flow retrograde relative to the cell, but remain essentially stationary relative to the substratum (50, 51). Also the data are in contrast with previous convincing reports which do not reveal retrograde surface lipid flow relative to the substratum over the cell body, lamella, or portions of lamellipodia (9, 36, 52). In these studies, however, measurements were not reported from 0 to 1-4 mm from the front of lamellipodia. Since this is typically where retrograde particle flow is fastest, it remains a formal possibility that there is local retrograde surface lipid flow relative to the substratum in lamellipodia. Supporters of this idea need to find a source of lipid to move retrograde from the front of the lamellipodium, and for removing excess lipid that would otherwise pile up at the back of the lamellipodium. In the neurite study one source of lipid is likely to come from the secretory pathway, and in motile cells polarized insertion of lipid vesicles has been observed at the front of lamellipodia (reviewed in (51)). Polarized removal has been observed at the back of protrusive structures in several motile cells types (see (53)). Also the exact source of tension in the cell surface needs to be found. In the neurons studied above, tension is at least partly generated by activity of the actin cytoskeleton (54). In lamellipodia, could the known organization of actin filaments (Fig 4) generate a tension gradient? The bulk, uniform polarity actin filament network may generate a tension gradient as predicted by mathematical modeling (32) (as described above). Presumably this tension gradient could be transmitted to the cell surface through integral membrane proteins. Alternatively, alternating polarity actin bundles may generate contractile force (discussed in (19)). These bundles are in a prime position to contract under the dorsal surface at the front of lamella/back of lamellipodium.