Liquid-liquid phase separation of vasodilator-stimulated phosphoprotein and its role in stress fiber and focal adhesion dynamics

Focal adhesions (FAs) and stress fibers (SFs) are key components of the actin cytoskeleton. They work in concert to maintain the mechanical homeostasis of cells, from adapting cell morphology to mediating force generation and transmission. Recent studies suggest that biomolecular condensates formed through liquid–liquid phase separation (LLPS) could be a generic mechanism for nucleation of nascent FAs and regulation of mature FAs. As an actin elongator that is enriched at both FAs and SFs, vasodilator-stimulated phosphoprotein (VASP) has been revealed to undergo LLPS and polymerize actin in vitro. However, whether this process occurs under physiological conditions inside cells and how it regulates cytoskeletal mechanics are unknown. Using high resolution confocal microscopy, we observed that VASP appeared as speckles at both FAs and damaged sites on SFs. As an interacting partner of VASP, zyxin also appeared as speckles, which showed good colocalization with the VASP speckles. In the photoablation experiments where we induced SF strained sites using lasers, we also observed good temporal and spatial coincidence of VASP and zyxin. We thus hypothesize that VASP forms force-sensitive co-condensates with zyxin that detect and repair strained sites of the SFs. Next, we will explore the role of phosphorylation, especially the zyxin binding and intrinsically disordered regions of VASP, and mechanical tensions of the SFs on the VASP-zyxin speckle formation and dynamics. Our final goal is to relate LLPS of VASP to the FA and SF assembly and disassembly dynamics, potentially shining new insights on our understanding of the actin cytoskeleton regulation.