- Steps in Filopodia Formation and Function
- Actin nucleation initates filopodium formation
- Cross-linking and extension of actin filaments
- The rate of filopodia extension
- Lateral movement of filopodia
- Filopodia stasis
- Filopodia adherence to the cell substrate
- Filopodia Pulling
- Filopodia Retraction and Collapse
- Growth Cone Collapse
Current research into these models suggests that both remain plausible mechanisms for actin filament nucleation and filopodia initiation in vivo, however only the "convergent elongation model" is supported with direct experimental evidence. Much of this evidence is based on the effect of modulating activators or regulators of the Arp2/3 complex, although some findings are directly attributed to the expression and function of the Arp2/3 complex itself.
Actin filaments in filopodia are unbranched , indicating filaments elongate primarily through the activity of formins. Following the rapid polymeriztaion facilitated by formins, a steady state mechanism that is common to many actin based structures will maintain filament length. This is known as actin treadmilling  and is described in greater detail in the functional module; actin filament polymerization.
As actin filaments extend, force is exerted on the cellular membrane, leading filopodia protrusion. For efficient protrusion, membrane curvature rigidity must be overcome. This is aided by actin filament cross-linking, which gives the structure the strength required to push against the compressive force of the plasma membrane [17, 19]. In nerve growth cones, more than 15 parallel filaments may be bundled together by crosslinking . Bundle stiffness increases with the number of bundled filaments and so contributes to the overall length of the filopodium . Filament bundling results from crosslinking proteins, many of which co-localize at the base of filopodia and work in concert to produce crosslinked filaments . Examples include α-actinin and fascin. The filamin family of proteins are also crucial actin crosslinking and scaffolding proteins and bind to both actin and a number of signaling molecules, including Rho GTPases. Crosslinking also increases the ATPase activity of myosins and increases the tension on filaments .
As well as mediating cargo transport along actin filament bundles, recent studies have implicated myosin-X as integral to the initiation of filopodia and the elongation of long filopodia. This has been attributed to a mechanism of myosin-X that promotes actin filament bundling, similar to cross-linking.
Studying the localization and motility of single myosin-X molecules using TIRF microscopy, Watanabe et al, hypothesized that binding of cargo, and the preceding dimerization of myosin-X monomers at the cell periphery is important for filopodia initiation as it promotes actin filament bundling . This was proposed to occur in a similar manner to regular crosslinking. Previous reports had indicated that even without the cargo-binding FERM domain, lateral movement of myosin-X along the leading edge of lamellipodia promoted actin reorganization and through its motor activity, filopodia initiation. In this case the length of the head and neck domain of myosin-X was proposed to be important for initiation. In the study by Watanbe et al a rapid increase in the rate of recruitment and assembly of myosin-X at filopodia initiation sites moments before protrusion commenced was observed and again this was shown to be independent of the presence of the FERM domain.
After removing the FERM domain from myosin-X (using a FERM domain-truncated construct called M10-ΔFERM) the protein was still able to walk along actin filament bundles and continued to localize at both the leading edge and filopodia tip. Significant changes to the length and stability of the growing filopodia were however noted. Not only were filopodia significantly shorter and more unstable, as previously reported [24, 25], but the phased-extension mechanism of elongation observed when complete myosin-X was present, did not occur.
Although it was noted that without the FERM domain the transport of essential cargo to the tips of the filopodia would be insufficient to permit continued elongation, it was also proposed that through FERM-β-integrin interactions at the tips of filopodia (when filopodia are attached to substrates via focal adhesion sites), myosin-X may also possess adhesive qualities. In this hypothesis, as filopodia enter the retraction phase and actin filaments move back towards the cell body by retrograde flow, myosin-X remains at the tips of filopodia together with the adhesion complex. Upon shrinkage of the tips, the protein will rebind to actin filament bundles and allow a new phase of extension to begin from adhesion sites. This mechanism of phased-extension is supported by observations that without the FERM domain myosin-X diffuses back into the cell body during the retraction phase whilst intact myosin-X remains at the tips .
One such study indicates that although the spatial distribution of static Myosin-X is universally consistent, and not altered by organelle length, the concentration of walking motors may vary. "Traffic jams" of myosin-X may occur for example at the base of the filopodia. Although logically this will impede progression of the proteins and their cargo down the filament, it was calculated that following a build up of G-actin at the blockage site, a concentration gradient is generated that enables its diffusion down the filopodia, (so long as the G-actin is not sequestered by the blocked motors) which subsequently sustains filopodia extension . Similarly, the constant association and dissociation of capping protein at the barbed ends of actin filaments has been shown, also using stochastic simulations, to influence the dynamics of filopodia extension. In this case amplification of these regulatory proteins from initially low concentrations may trigger the fast retrograde flow of actin and induce repeated extension-retraction cycles that occur on a micro-meter scale. Compared to actin-only models, these dynamic cycles enable the growth of substantially longer filopodia .
|Mouse cortical neurons||500 nm/s||Sheetz et al., 1992 |
|NG108||30 nm/s||Mallavarapu & Mitchison, 1999|
|Dictyostelium cells||~30 nm/s||Schirenbeck et al., 2005 |
|Helisomatrivolvis||~40 nm/s||Tornieri et al., 2006 |
|Dictyostelium cells||1000 nm/s||Medalia et al., 2007 |
|Chick DRG neurons||300 nm/s||Constantino et al., 2008 |
|Primary mesenchyme cells||~150 nm/s||Miller et al., 1995 
Lateral movement and merging of filopodia may also stimulate further elongation of filopodia, suggesting that this may be an alternative method for promoting filopodial maturation and growth cone advancement on less adherent substrates .
1) Ligand binding to filopodial receptors and subsequent adhesion of the ligand/receptor complex to actin bundles:
After a ligand binds to a receptor on the filopodium, lateral connections between the receptor and the actin bundle(s) prevent retrograde actin movement relative to the ligand. This inhibition converts the previous retrograde movement of the retracting filaments into tension on the bundles. This tension might influence polymerization and stability of actin filaments and/or myosin activity.
2) Ligand binding to filopodial receptors followed by uncapping of filament barbed ends:
Retracting filopodia become static when receptor binding leads to filament uncapping and rapid polymerization at the barbed end. The retraction force is converted to retrograde flux and the filament length remains constant.
3) Capping of unstable actin filaments :
If retraction occurs by barbed end disassembly, capping of unstable filaments by an activated receptor will block retraction and induce stasis.
Three distinct types of adhesions can be identified within filopodia. Adhesion components may be localized to the tips of filopodia or be actively transported down the bundled actin filaments by myosin proteins. Each adhesion may function independently or work in concert to produce the overall guidance response:
Increasing the force on an adhesion, either by an external source or by increasing cellular contractility, strengthens and enlarges the adhesion . Similarly, in basal adhesions of filopodia in neuronal growth cones, the size and stability of nascent adhesion increases in a maturation process that is reminiscent of the focal adhesions (FAs) found in non-neuronal cells [43, 44]. Though smaller than FAs, these adhesions share a number of signal components, pathways, and proteins leading to adhesion site formation and maturation [47, 45, 48].The structure of the growth cone adhesion site varies with the substrate  and adhesion directs growth cone navigation and movement (reviewed in [49, 50]).
In each case the mechanism behind pulling remains the same. While the powerstroke of Myosin II essentially drives the movement of filaments, the pulling process occurs over three distinct stages, each defined by varying rates of retraction. These can be described using the process by which pathogens are pulled towards macrophage cell bodies as an example. Here, immediately following filopodial adhesion to a pathogen, a slow motion retraction of the filopodia commences. This is followed by a phase of rapid movement where the pathogen is pulled towards the cell. A final phase of slow retraction occurs, resulting in the pathogen being fixed to the cell surface. This final phase of retraction is also characterized by a thickening of the filopodial base which is necessary to build-up large retraction forces for internalization [52, 55]. Filopodial attachment to a surface (in addition to the ligand) produces counteracting adhesion forces which influence the retraction speed. In general, the rearward movement or ‘step size’ of a retracting filopodia decreases as the filopodia encounters a greater counteracting force . Importantly, the mechanism may also be hijacked by a pathogen to ensure an efficient infection. This has been described for the Murine Leukemia Virus, amongst others . In this case the virus binds to surface receptors at the tips of the filopodia and essentially rides the retracting actin filaments to entry points in the cell where it is internalized.
Table: Filopodial Retraction Rates
|Mouse cortical neurons; primary mesenchyme cells||400 nm/s||[29, 34]|
|Mouse macrophage||600 nm/s|||
|Chick DRG neurons||200 nm/s|| 
A number of factors regulate collapse and retraction. For example, capping proteins promote filopodial retraction by shielding the barbed end of filaments from further assembly and elongation . Inhibition of F-actin polymerization and protrusion during collapse are mediated by RhoA kinase activity . Collapse may result from the exposure of a “naive” growth cone to a high concentration of a repellent followed by an overactive response . The repulsive component appears to shut down the growth program and is therefore dominant over the growth-stimulating effects of adhesion molecules. In addition, the repellent also interferes with mechanisms that would normally result in filopodial retraction .