Perturbation and imaging of exocytosis in plant cells

Abstract

The exocytosis process delivers proteins, lipids, and carbohydrates to the plasma membrane or the extracellular space to sustain plant cell growth, development, and response to environmental stimuli. Plant exocytosis is highly dynamic and requires the coordinated functions of multiple cellular components such as tethering complexes, GTPase signaling, and vesicle fusion machinery. Accurate spatio-temporal control of plant exocytosis is critical for the proper functions of plant cells. Live-cell imaging of fluorescence-tagged cargo proteins allows for quantitative analysis of exocytosis dynamics in plant cells. Small molecule inhibitors that target important components in the exocytosis machinery allow for transient manipulation of the exocytosis process. In this chapter, we describe procedures that use Endosidin2 (ES2) and Brefeldin A (BFA) as small molecule inhibitors to disrupt plant exocytic processes and use fluorescent protein-tagged PIN-formed 2 (PIN2) and Cellulose Synthase (CESA) as cargo proteins to quantify exocytosis dynamics in plant cells.

Keywords: Brefeldin A; Endosidin2; Exocytosis; Fluorescence recovery after photobleaching.