Abstract

The cytoskeleton, an indispensable component during cell migration and proliferation, relies on the regionalized programming of intracellular proteins─liquid–liquid phase separation (LLPS) that enables cells to shift between phenotypes to determine their fate of migration and reproduction. However, the precise manipulation of the G/F-actin is extremely challenging, and effective direct regulatory measures are currently lacking to intervene in the physiological and pathological processes of cells. Herein, through LLPS visualization experiments, we reveal a storage and regulatory mechanism of cytoplasmic G-actin in cells via LLPS. Based on the conversion mechanism between stored G-actin and active G-actin within the free actin pool in the cytoplasm, G-actin’s LLPS forms droplets to participate in cellular activities. Furthermore, based on orthogonal disulfide chemistry reactions, we developed a cross-linker capable of altering the formation of actin bioaggregates. By screening targeted modification groups and backbone libraries, we designed the penicillamine-linked 4Arm-PEG (4Arm-Pen) capable of cross-linking with actin. Visualization of LLPS reversal intra- and extracellularly, 4Arm-Pen target proteomics, and mathematical modeling of F-actin polymerization kinetics collectively confirm that 4Arm-Pen not only cross-links G-actin, impeding droplet formation, but also influences the polymerization kinetics of F-actin, causing the cells to lose their invasive capacity. Furthermore, we demonstrated that G-actin depletion induced by cross-linking renders cancer cells immotile and triggers alterations in endogenous cellular mechanical parameters. Collectively, these findings provide deeper insights into the analysis and modulation of G-actin LLPS in cells, paving a path toward promising therapeutic strategies for cancer treatment.