High Temporal Resolution Reveals Simultaneous Plasma Membrane Recruitment of TPLATE Complex Subunits1[OPEN]

Lowering the temperature increases the temporal resolution of visualizing protein recruitment at the plasma membrane, demonstrated by dual-color live-cell imaging of endocytic complex subunits. The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC in Arabidopsis (Arabidopsis thaliana) contains six evolutionarily conserved subunits and two plant-specific subunits (AtEH1/Pan1 and AtEH2/Pan1) which, although cytoplasmic proteins, are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live-cell imaging at physiological and lowered temperatures. Lowering the temperature slowed down endocytosis, thereby enhancing the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE and the AtEH/Pan1 proteins exhibited simultaneous recruitment at the PM. These results, together with colocalization analysis of different TPC subunits, allow us to conclude that the TPC in plant cells is not recruited to the PM sequentially but as an octameric complex.