Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

• Published in: Molecular biology of the cell 2013-02, Vol.24 (4), p.510-520
• Main Authors: Fendrych, Matyás, Synek, Lukás, Pecenková, Tamara, Drdová, Edita Janková, Sekeres, Juraj, de Rycke, Riet, Nowack, Moritz K, Zársky, Viktor
• Format: Article
• Language: English
• Subjects: Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis - ultrastructure; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cytoplasm - metabolism; Cytoplasm - ultrastructure; Cytoskeleton - metabolism; Cytoskeleton - ultrastructure; Exocytosis; Gene Expression; Microscopy, Fluorescence; Plant Epidermis - genetics; Plant Epidermis - metabolism; Plant Epidermis - ultrastructure; Plant Roots - genetics; Plant Roots - metabolism; Plant Roots - ultrastructure; Protein Binding; Protein Transport; rab GTP-Binding Proteins - genetics; rab GTP-Binding Proteins - metabolism; Secretory Vesicles - metabolism; Secretory Vesicles - ultrastructure; SNARE Proteins - genetics; SNARE Proteins - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.e12-06-0492

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.