Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

Abstract

Robust spatiotemporal organization of cytoskeletal networks is crucial, enabling cellular processes such as cell migration and division. α-Actinin and fascin are two actin crosslinking proteins localized to distinct regions of eukaryotes to form actin bundles with optimized spacing for cell contractile machinery and sensory projections, respectively. In vitro reconstitution assays and coarse-grained simulations have shown that these actin bundling proteins segregate into distinct domains with a bundler size-dependent competition-based mechanism, driven by the minimization of F-actin bending energy. However, it is not known how physical confinement imposed by the cell membrane contributes to sorting of actin bundling proteins and the concomitant reorganization of actin networks in intracellular environment. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that the size of such a spherical boundary determines equilibrated structure of actin networks among three typical structures: single rings, astral structures, and star-like structures. We show that α-actinin bundling activity and its tendency for clustering actin is central to the formation of these structures. By analyzing physical features of crosslinked actin networks, we show that spontaneous sorting and domain formation of α-actinin and fascin are intimately linked to the resulting structures. We propose that the observed boundary-imposed effect on sorting and structure formation is a general mechanism by which cells can select between different structural dynamical steady states.

Confinement Geometry Tunes Fascin-Actin Bundle Structures and Consequently the Shape of a Lipid Bilayer Vesicle

“…To explore how the flexibility of actin bundles can influence their interaction at the midplane of GUVs and subsequent GUV shape changes, we sought to co-encapsulate fascin and α-actinin with actin in GUVs. The persistence length of encapsulated α-actinin-actin bundles is lower than the persistence length of fascin-actin bundles measured here (Takatsuki et al, 2014;Bashirzadeh et al, 2020). This causes α-actinin-actin bundles to bend around the membrane and merge into an actin ring in cell-sized GUVs in order to minimize their elastic energy (Miyazaki et al, 2015) ( Supplementary Figure 2A).…”

“…GUVs encapsulating fluorescent actin monomers with no sign of bundling activity were not counted). We calculated the persistence length of fascin-actin bundles using the skeletonized images of actin bundles by measuring their orientational correlation function as described previously (Bashirzadeh et al, 2020). Persistence length values are shown as average ± standard error of the mean of the data at each fascin concentration.…”

“…We modified continuous droplet interface crossing encapsulation (cDICE) technique for robust encapsulation in GUVs of various sizes (Abkarian et al, 2011;Bashirzadeh et al, 2020;Litschel et al, 2020). Briefly, a custom 3D-printed chamber is mounted on a benchtop stir plate and rotated at 1,200 rpm.…”

“…Images were processed using ImageJ/Fiji (Schindelin et al, 2012;Schneider et al, 2012;Litschel et al, 2020), SOAX (Xu et al, 2014(Xu et al, , 2015, and MATLAB routines (Bashirzadeh et al, 2020). 3D images were created using 3D Project command in ImageJ/Fiji.…”

“…Encapsulation of actin and accessory proteins inside membrane-enclosed systems such as GUVs allow for studying the orchestration of actin networks and their interaction with the lipid membrane in a cell-like compartment without the involvement of complex cell signaling pathways (Bashirzadeh and Liu, 2019). Encapsulated actin crosslinkers such as fascin, α-actinin, and filamin assemble actin into diverse structures (Honda et al, 1999;Abkarian et al, 2011;Bashirzadeh et al, 2020;Litschel et al, 2020). These structures have been shown to induce diverse GUV morphological changes (Honda et al, 1999;Limozin and Sackmann, 2002;Abkarian et al, 2011;Tsai and Koenderink, 2015;Litschel et al, 2020), and the shape changes were recently shown to be reversible by light-induced actin bundle disassembly (Litschel et al, 2020).…”