The Arp2/3 complex participates in the Exocytosis and endocytosis of cellulose synthase complex through regulating the dynamic homeostasis of cortical actin arrays in arabidopsis

The actin cytoskeleton acts as a dynamic platform to coordinate various cellular activities, such as exocytosis and endocytosis, that require cells to precisely control when and where specific actin arrays are generated. Actin filament nucleators, including 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, are key regulators of the dynamic homeostasis of cortical actin arrays. To elucidate the molecular mechanisms that underpin how these two nucleators cooperate, or compete, with each other to maintain the homeostatic network of cortical actin in plant cells, we combined the ability to image single filament dynamics in living plant epidermal cells with genetic and/or chemical approaches to stably or acutely disrupt nucleator activity. We found that both the Arp2/3 complex and formins contributed to side-branched filament nucleation in vivo but generated new daughter filaments with distinct dynamic properties. Surprisingly, simultaneous inhibition of both nucleators significantly enhanced the frequency of de novo nucleation events though a mechanism that remains unexplained. To understand how the Arp2/3 complex participates in exo- and endocytic trafficking, we employed the cellulose synthase (CESA) complex (CSC) as a cargo marker and discovered that the Arp2/3 complex contributed to the late stages of exocytosis of CSCs at the PM. To directly assess the internalization process of CSCs, we constructed a functional, fluorescense-tagged-CESA6 marker and developed single-particle live-cell imaging approaches to visualize and quantify the dynamic behavior of CSCs on the PM during internalization. We found that actin, myosin, as well as the Arp2/3 complex were involved in CSC internalization events. Collectively, these results reveal a previously undescribed role for the Arp2/3 complex in exocytosis and internalization in plant cells, possibly through regulating the dynamic homeostasis of the cortical actin cytoskeleton.