Actomyosin and the Arp2/3 Complex Are Involved in the Internalization of Cellulose Synthase Complexes

The coupling of exo- and endocytic trafficking of Cellulose Synthase Complexes (CSCs) has been proposed to be important for maintaining the population of active CSCs at the plasma membrane (PM) and thus appropriate levels of cell wall assembly. Although actin and myosin are known to participate in the late stages of exocytosis of CSCs, their exact role during CSC internalization events remains controversial. We constructed a functional, photoconvertible fluorescent mEOS2-CESA6 reporter and developed single-particle live-cell imaging approaches to visualize and quantify the dynamic behavior of CSCs at the PM during internalization. Using the small molecule inhibitor of clathrin, Endosidin 9-17 or ES9-17, we confirmed that clathrin-mediated endocytosis is a major pathway for CSC internalization. We also found that the actin cytoskeleton is involved in CSC internalization. Genetic or chemical inhibition of actin, myosin, or the Arp2/3 complex significantly reduced the frequency of CSC internalization events and prolonged the CSC pause time prior to internalization. Additionally, we found that the Arp2/3 complex contributes to the late stage of exocytosis of CSCs into the PM. These results reveal a role for actomyosin and the Arp2/3 complex in both CSC secretion as well as internalization that was previously undescribed in plant cells. One sentence summaryDirect visualization of individual CSC internalization events reveals that actomyosin participates in CSC internalization and the Arp2/3 complex contributes to both exocytosis and internalization of CSCs through regulating the dynamic homeostasis of the cortical actin cytoskeleton.