Hijacking Actin-Pre-Liquid–Liquid Phase Separation Suppresses Malignant Tumor Invasion and Growth

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.