Abstract
We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.
Highlights
Directional flow of membrane material between sub-cellular compartments by different vesicular structures is essential for fundamental cellular functions such as cell division, polarization, differentiation, motility, and response to environmental stress
After seedling treatment with 50 μM ES2, we observed in a routine time-laps experiment without photoconversion the development of discrete ES2 agglomerates (ES2As) in the cytoplasm (Fig 1A) with about 1 μm size
Analysis of PIN2-Dendra2 dynamics in the plasma membrane (PM) by time-lapse experiment highlighted decreasing signal intensity compared to the controls exhibiting the constant fluorescence (Fig 1F–1I)
Summary
Directional flow of membrane material between sub-cellular compartments by different vesicular structures is essential for fundamental cellular functions such as cell division, polarization, differentiation, motility, and response to environmental stress. A pharmacological approach using chemical compounds combined with the monitoring of membrane proteins labeled by fluorescence proteins or visualized by immunohistology techniques has become a favorite method to study dynamics in plant cell membrane structures. Many new bioactive small molecules with an impact on the vesicle trafficking network have been discovered by high-throughput chemical genetic screens [1,2,3,4,5]. Mostly precise targets for these bioactive compounds have not been identified.
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