Simultaneous inhibition of the Arp2/3 complex and formins enhancesde novoactin filament nucleation in Arabidopsis

Abstract

AbstractPrecise 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 the homeostatic cortical actin array of living Arabidopsis (Arabidopsis thaliana) 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. Surprisingly, simultaneous inhibition of both nucleators increased overall actin filament abundance and enhanced the frequency ofde novonucleation events. Collectively, these observations suggest that plant cells have a unique actin filament nucleation mechanism that maintains cortical actin organization and dynamics when compared to yeast or animal cells.One sentence summaryUsing advanced live-cell imaging and genetic mutation or small molecule inhibitor approaches, the roles for two classes of actin filament nucleator in maintaining the homeotic cortical cytoskeleton array are defined.