How does cross-linking of actin filaments aid in their extension?2018-02-06T10:37:13+08:30

How does cross-linking of actin filaments aid in filopodia extension?

Once nucleation has taken place, actin filaments begin to extend. This process is primarily facilitated by members of the formin family of proteins, however numerous other proteins also play important roles. In the ‘convergent elongation model’ filament barbed ends are locally associated with each other and must be protected from capping in order for extension to occur [1]. This protection is provided by the activity of proteins such as the Ena/VASP family of proteins which is delivered to the filopodia tip by myosin X [2]. Here, Ena/VASP enhances filament polymerization, and promotes F-actin bundling, thereby stimulating filopodial protrusion [1][3][4][5][6].

Steps in filopodium formation. Actin filament assembly can be initiated by uncapping pre-formed actin filaments or by de novo formation (which includes both formin- and Arp2/3-mediated [not shown] nucleation). The force produced by actin assembly at the barbed end of actin filaments drives membrane protrusion. Numerous proteins (including IRSp53, Ena/VASP proteins, WASp/Scar proteins) cooperate to promote actin-assembly and enhance bundling of actin filaments by fascin. When the barbed end of the filament is capped, this stops filament assembly and protrusion; continued retrograde movement of the filaments results in retraction of the filopodium. (Figure adapted from Faix J and Rottner K, 2006)

Actin filaments in filopodia are unbranched [7], 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 [8] and is described in greater detail in the functional module; actin filament polymerization.

Cross linking strengthens the actin cytoskeleton

As actin filaments extend, force is exerted on the cellular membrane, leading to 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 [6][9]. In nerve growth cones, more than 15 parallel filaments may be bundled together by crosslinking [10]. Bundle stiffness increases with the number of bundled filaments and so contributes to the overall length of the filopodium [6]. Filament bundling results from crosslinking proteins, many of which co-localize at the base of filopodia and work in concert to produce crosslinked filaments [11]. 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 [12].

Myosin-X in filament bundling

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 [13]. 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 [14][15], 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 [13].

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