Optogenetic cross-linking of the actin cytoskeleton using DARPins

Precise tools to control actin filament organization and dynamics are essential for studying how the actin cytoskeleton regulates fundamental cell processes, such as morphological changes, migration and intracellular transport. Here, we present an optogenetic system for the reversible, spatiotemporal manipulation of actin cross-linking in living cells. Our approach employs actin-binding Designed Ankyrin Repeat Proteins (DARPins) fused to the improved Light-Induced Dimer (iLID) system, enabling actin cross-linking upon light-triggered dimerization. Following validation of cross-linking of bifunctional DARPins in reconstituted networks, the iLID system was integrated to create light-controlled associations inside cells. Optogenetic DARPin dimerization is rapid, reversible, and locally inducible. Activation of the DARPin-based actin cross-linkers revealed significant inhibition of cell traction forces when triggered throughout the cell. Our findings establish a versatile tool for investigating cytoskeletal dynamics with high spatial and temporal precision, paving the way for controlled manipulation of cellular architecture and mechanics. Graphical Abstract/Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/726659v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@c27701org.highwire.dtl.DTLVardef@112e82borg.highwire.dtl.DTLVardef@4892e4org.highwire.dtl.DTLVardef@a3d5a7_HPS_FORMAT_FIGEXP M_FIG C_FIG