Deciphering the molecular mechanisms of actin cytoskeleton regulation in cell migration using cryo-EM

The actin cytoskeleton plays a key role in cell migration and cellular morphodynamics in most eukaryotes. The ability of the actin cytoskeleton to assemble and disassemble in a spatiotemporally controlled manner allows it to form higher-order structures, which can generate forces required for a cell to explore and navigate through its environment. It is regulated not only via a complex synergistic and competitive interplay between actin-binding proteins (ABP), but also by filament biochemistry and filament geometry. The lack of structural insights into how geometry and ABPs regulate the actin cytoskeleton limits our understanding of the molecular mechanisms that define actin cytoskeleton remodeling and, in turn, impact emerging cell migration characteristics. With the advent of cryo-electron microscopy (cryo-EM) and advanced computational methods, it is now possible to define these molecular mechanisms involving actin and its interactors at both atomic and ultra-structural levels in vitro and in cellulo. In this review, we will provide an overview of the available cryo-EM methods, applicable to further our understanding of the actin cytoskeleton, specifically in the context of cell migration. We will discuss how these methods have been employed to elucidate ABP- and geometry-defined regulatory mechanisms in initiating, maintaining, and disassembling cellular actin networks in migratory protrusions

ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning

Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.</jats:p