Research Highlights
Makito Miyazaki
Precise control of actin cytoskeleton assembly with blue light
OptoVCA, a new system developed by IMS investigators, uses light to control actin network assembly and density on lipid bilayers or in cells in a precise, tunable, and reversible manner. This technique allows direct testing of how network density affects the behavior and function of actin-binding proteins.
Controlling actin network assembly by blue light using a novel optogenetic tool, OptoVCA.
The iLID–SspB light-inducible dimerization system was used to control the localization and surface density of the VCA domain of nucleation-promoting factors (NPFs) on lipid membranes. This technique was applied to investigate how actin network density regulates the penetration and activities of two representative actin cytoskeleton proteins, myosin and ADF/cofilin.
Overview
The actin cytoskeleton, a dynamic network of actin filaments and their associated proteins, plays an essential role in fundamental cellular processes including cellular morphology, motility, and cell division. To initiate actin network assembly, actin polymerization is triggered by nucleation-promoting factors (NPFs), such as the Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous (WAVE) proteins. The VCA domain of WAVE binds actin monomers and activates the Arp2/3 complex to initiate actin branching, a specialized form of actin polymerization where new branches grow off the side of existing filaments at ~70° to create a dense, tree-like network. Actin branching is important for cellular processes that involve movement, e.g., cell migration, where it pushes the plasma membrane forward at the leading edge, endocytosis and vesicle trafficking, where it provides pushing forces to deform membranes and pinch off vesicles, etc. Much is known about the biochemistry of these processes, yet how network density governs protein penetration and dynamics has been unclear until the studies reported here.
A key component of OptoVCA is the use of the iLID-SspB light-induced dimerization pair. iLID (improved Light-Induced Dimer) contains a short SsrA peptide that is sequestered in the dark, but is exposed when exposed to blue light. SspB is a bacterial protein that binds tightly to SsrA. Blue light causes an SspB-VCA fusion protein to bind to membrane-anchored iLID, which increases local VCA concentration to initiate the actin branching described above. Adjusting: 1. Light intensity controls VCA density, which controls actin density. 2. Illumination duration controls network thickness. 3. The illumination pattern controls the network shape. OptoVCA has significant advantages over previous methods, since actin polymerization stops immediately when the light is turned off, it allows reversible assembly and disassembly, and it can vary VCA density anywhere on the same membrane, permitting a direct comparison of different types of networks.
The IMS investigators used OptoVCA to examine how network density affects two actin-binding proteins, myosin and ADF/cofilin. Even modest increases in density inhibited myosin filament penetration by steric hindrance. By contrast, the smaller ADF/cofilin could access the networks regardless of density, but network disassembly, a function of ADF/cofilin, was reduced by increased density. These studies have revealed that network density differentially regulates actin-binding protein penetration and activity. The unique capabilities of OptoVCA may be used in future studies to understand not only cellular mechanics but also cellular compartmentalization mechanisms.
Original paper
Yamazaki K, Miyazaki M. Optogenetic actin network assembly on lipid bilayer uncovers the network density-dependent functions of actin-binding proteins. Nat Commun 16, 7583 (2025). doi: 10.1038/s41467-025-62653-6