Simultaneous Inhibition of the Arp2/3 Complex and Formins Enhances de novo Actin Filament Nucleation in Arabidopsis thaliana

Precise control over how and where actin filaments are created in eukaryotic cells leads to the construction of unique cytoskeletal arrays with specific functions within a common cytoplasm. Actin filament nucleators are key players in this activity and include the conserved Actin-Related Protein 2/3 (Arp2/3) complex, that creates dendritic networks of branched filaments, as well as a large family of formins that typically generate long, unbranched filaments and bundles. In some eukaryotic cells, these nucleators compete for a common pool of actin monomers and loss of one favors the activity of the other. To test whether this is a common mechanism across kingdoms, we combined the ability to image single filament dynamics in living plant epidermal cells with genetic and/or small molecule inhibitor approaches to stably or acutely disrupt nucleator activity. We found that Arp2/3 mutants or acute CK-666 treatment markedly reduced the frequency of side-branched nucleation events as well as overall actin filament abundance. We also confirmed that plant formins contributed to side-branched filament nucleation in vivo. Surprisingly, simultaneous inhibition of both nucleators increased overall actin filament abundance and enhanced the frequency of de novo nucleation events. Collectively, these observations suggest that plant cells have a unique actin filament nucleation mechanism compared to yeast or animal cells.