Revising the Role of Cortical Cytoskeleton During Secretion: Actin and Myosin XI Function in Vesicle Tethering

Abstract

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase of transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.

Point mutations in the catalytic domain disrupt cellulose synthase (CESA6) vesicle trafficking and protein dynamics

“…In the 12 mutant lines with reduced CSC velocity, we observed a population of CSCs at the PM that were completely static (Figure 2A and Supplemental Figure S3; red arrows). The insertion of single CSC particles at the PM can be visualized with high spatiotemporal resolution live-cell imaging and the dynamic behavior of CSCs in a typical insertion process follows a specific pattern (Gutierrez et al, 2009; Zhang et al, 2019; Zhang and Staiger, 2022). After a CSC particle arrives at the PM insertion site, it initially pauses for ~ 80 s which is assumed to represent the tethering, docking and fusion steps.…”

“…We quantified the density of pause-forever CSCs in the AtCESA6 mutation lines, using a method modified from the quantification of CSC tethering frequency, as described previously (Zhang and Staiger, 2022). We extracted all of the kymographs from a region of interest (ROI) at the PM in a 5-min time lapse series and all vertical lines that lasted for at least 3 min were counted as pause-forever events.…”

“…All image analysis was performed using FIJI/ImageJ (Schindelin et al, 2012). Detailed methods for imaging quantification and analyses were as described previously (Huang et al, 2020; 2021; Zhang et al, 2019; 2021b; Zhang and Staiger, 2022). All imaging experiments were conducted with at least 3 biological repeats.…”