Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates Academic Article uri icon

Overview

MeSH Major

  • Biosynthetic Pathways
  • Cell Polarity
  • Epithelial Cells
  • Rhodopsin
  • rab GTP-Binding Proteins

abstract

  • Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.

publication date

  • March 18, 2014

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3964106

Digital Object Identifier (DOI)

  • 10.1073/pnas.1304168111

PubMed ID

  • 24591614

Additional Document Info

start page

  • 4127

end page

  • 32

volume

  • 111

number

  • 11