Fig. 2.

VAEM allows for reliable co-localization analysis on protein candidates at single particle level. a Clathrin light chain (CLC) -GFP and mCherry-FLOT1a both localized to PM and intracellular punctate structures under LSCM; The close-up inlets showed that CLC-GFP and mCherry-FLOT1a colocalized at PM and intracellular structures; b BFA treatment induced both CLC-GFP and mCherry-FLOT1a to accumulate into BFA compartments, indicating both clathrin and FLOT1a were involved into active endocytic events; c Transgenic Arabidopsis line co-expressing CLC-GFP and mCherry-FLOT1a showing characteristic localization to largely separated punctate structures; d VAEM observations on BFA-induced compartment formation. e Co-localization analysis using ImageJ intensity correlation analysis plugin and Protein Proximity Index (PPI) analysis on the LSCM images (a) indicated clathrin and FLOT1a were co-localized in a high percentage; f Co-localization analysis using ImageJ intensity correlation analysis plugin and PPI analysis on the VAEM images (c) indicated clathrin and FLOT1a were associated rather than co-localized. Bars = 20 μm (a-b), 3 μm (c-d); Interval between frames = 200 ms