Nucleation geometry governs ordered actin networks structures

Abstract

Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators 1-3 in vivo [4][5][6][7] . Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices [8][9][10][11][12][13][14][15][16][17][18] . However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied 14,19 . Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures.In cells, actin nucleation occurs at various locations at the plasma membrane, and bundles of parallel actin filaments are initiated at focal adhesion sites 1 or result from the rearrangement of the dynamic branched actin network of the lamellipodium 6 . Here, we modulate the positioning of nucleation sites at scales corresponding to cellular dimensions. As a first step and to precisely regulate the position of actin nucleation sites in vitro, we used a recently developed ultraviolet-based micropatterning approach 20 to create a template for the localization of the nucleation promoting factor pWA (Fig. 1a). pWA comprises the C-terminal domains from the WASP/Scar proteins, a ubiquitous family of proteins that initiate actin polymerization on a pre-existing actin filament in the presence of the Arp2/3 complex and an actin monomer [21][22][23] . A small volume of solution made of a minimal set of purified proteins ensuring actin polymerization 8,10 was placed between the pWA-coated micropatterned slide and a glass support. Functionalized micropatterns specifically initiate actin filament nucleation on their surface and promote two-dimensional growth of actin filaments (Fig. 1b). Real-time visualization of actin-filament nucleation and growth highlighted the autocatalytic process of network formation (see Supplementary Fig. S1). These networks consist of filaments growing from the pWA-coated regions, with their fast-growing, barbed end oriented outwards ( Fig. 1c and Supplementary Fig. S2 and Videos S1-S3). In agreement with actin-filament growth on glass rods 15 , as the nucleation waves propagate, dense and interconnected

Actin Dynamics, Architecture, and Mechanics in Cell Motility

“…Two nonexclusive mechanisms are generally admitted, one that requires the Arp2/3 complex and another that involves barbed end elongation enhancement proteins like formins or Ena/VASP proteins that will be discussed in the next paragraph (3,4,39,115,200,256,264,276,305,323,324,345). The first situation occurs when capping protein is absent from Arp2/3 complex-generated networks (FIGURE 3A, ϪCP).…”

“…As uncapped filaments elongate freely at their barbed ends, they tend to bundle via electrostatic interactions, and then a transition from branched to parallel actin filaments is observed. Geometrical constraints and the angle by which filaments are contacting each other can alter this transition and generate either parallel or antiparallel actin structures (256). As the filaments start to organize in parallel structures, they can be captured by crosslinkers such as fascin that further stabilize the bundled conformation and stiffen the structure (FIGURE 3, A, mechanical representation, AND C).…”

“…Indeed, in the absence of capping protein, branches elongate with no limitation, resulting in most cases in an array of parallel bundles growing away from the NPF sites, and thus this condition is not optimal for force production (FIGURE 3A and Refs. 3,4,115,150,233,256). Instead, capping protein or proteins with similar activities work in synergy with the Arp2/3 complex to build a dense actin network of capped filaments that are sterically displaced by new branches formed at NPF sites (3,4,25,96,150,351).…”

“…studies concerning the mechanical properties and force generation of branched networks, no clear consensus has emerged relating the detailed actin structure to how good the actin network is at moving an object (3,4,20,87,195,257,338). New methods using NPF-micropatterning to orient actin network growth and control its geometry in combination with physical methods to measure force production, such as magnetic colloids in magnetic fields, atomic force microscopy, or optical tweezers, should make it possible in the future to correlate the biochemical composition of the actin network and its microscopic organization to the force generated by the network (34,59,96,217,252,256). …”

Elucidating the molecular mechanisms underlying cellular response to biophysical cues using synthetic biology approaches

“…84 Controlled 2D micropatterning of an actin-promoting factor led to the possibility of nucleating desired actin orientations. 85 The schematic in Figure 6C displays the architecture used to create regions of unordered, parallel and antiparallel actin filaments in the absence or presence of myosin. Remarkably, myosin VI was only effective at contracting and disassembling antiparallel filaments whereas parallel filaments tended to align and elongate, recruiting monomers from the disassembled antiparallel filaments.…”