Arp2/3 Branched Actin Network Mediates Filopodia-Like Bundles Formation In Vitro

Abstract

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.

From Branched Networks of Actin Filaments to Bundles

“…Now the filaments can bend toward each other, enabling fascin to crosslink them into finger-like bundles, resulting in a structural transition from a dense aster to a large star-like structure. [28,29] The similarity between the aster-to-star and the lamellipodium-tofilopodia transitions underlies our in vitro experimental studies and their theoretical interpretations.…”

“…alyze theoretically a series of in vitro experiments, partly described here and in more detail elsewhere, [28,29] which appear to support the first mechanism. More explicitly, in the next section we show, both experimentally and theoretically, that in the presence of fascin, a dense and highly branched aster-like network of actin filaments exhibit a transition to a star-like structure, where bundles of filaments emanate and grow radially from the aster-like core.…”

“…[30,31] Previous work showed that upon mixing these components Arp2/3 plays a dominant role in the early stages of actin network formation, resulting in the appearance of densely branched, nearly spherical, aster-like structures. [28,29] Fascin comes into play at a later stage, when the filaments at the aster surface are long and sparsely branched. Now the filaments can bend toward each other, enabling fascin to crosslink them into finger-like bundles, resulting in a structural transition from a dense aster to a large star-like structure.…”

Filopodia initiation

“…This idea is consisted with results obtained in reconstitution studies that showed that more and longer actin bundles were formed in vitro, when the concentration of the Arp2/3 complex was decreased, while the concentrations of other components, such as WASP-VCA, actin and fascin, kept constant. 87 A naturally high rate of lamellipodia-tofilopodia transition is observed in neuronal growth cones, which do not form extensive lamellipodia, but rapidly reorganize their small lamellipodial regions into filopodia. 27 Another factor that may contribute to this phenomenon in Arp2/3 complex knockdown cells is that when actin monomers are less consumed by Arp2/3 complex, a higher concentration of them becomes available for actin filament elongation, thus shifting the actin dynamics at the leading edge toward filopodia.…”

Hierarchical self-assembly of actin in micro-confinements using microfluidics

“…3 Networks and clusters of actin bundles are observed at high concentrations of different cross-linkers such as espin, fascin, aactinin, and filamin. 4 Self-organized asters emerge in actin-myosin-fascin 5 and actin-Arp2/3 complex-fascin 6 systems. Microtubule-motor systems also self-organize into asters, vortices, and networks 7 and the self-organization is influenced by the size of the confinement.…”