Abstract
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.
Highlights
The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants
TPC is proposed to serve as an early adaptor complex (Gadeyne et al, 2014); temporal resolution remains the biggest challenge to monitor the dynamic recruitment of different endocytic protein players (Fujimoto et al, 2010; Fan et al, 2013; Gadeyne et al, 2014; Bashline et al, 2015)
As lowering the temperature generally slows down dynamics of cellular processes (Das et al, 1966), we hypothesized that by lowering the temperature we could slow down dynamics of endocytic events and enhance the temporal visualization of the differential recruitment of the endocytic players at the plasma membrane (PM)
Summary
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/Pan and AtEH2/Pan1) which, cytoplasmic proteins, are not associated with the hexameric subcomplex in the cytoplasm. 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/Pan 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. To investigate whether there is a differential order of recruitment between both AtEH/Pan proteins and the remaining hexameric subcomplex, we performed dual-color timelapse microscopy of CME in etiolated Arabidopsis epidermal hypocotyl cells. Our data strongly suggest that TPC is recruited as the octameric complex to the PM, where it functions as the early adaptor complex for plant CME
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