Check out Laura McCormick’s first author work now on BioRxiv, in collaboration with Nick Brown and Dave Kovar. We identified 2 ubiquitination sites on VASP (K240 and K286) and created purified, ubiquitinated VASP mimics. Using several in vitro biochemical assays, we showed multi-monoubiquitination (mmUb) reduces the ability of VASP to bind, bundle, and elongate actin filaments. We also electroporated our purified protein into MEFs. Compared to unmodified protein, the presence of multi-monoubiquitinated VASP altered cell spreading morphology and showed diminished localization to lamellipodia/filopodia. Collectively, our results show how multi-monoubiquitination negatively regulates VASP activity and influences actin dynamics in cells.