Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 11, Pages 4127-4132Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1304168111
Keywords
-
Categories
Funding
- National Institutes of Health (NIH) [EY08538, GM34107]
- Margaret Dyson Foundation
- NIH [EY11307, EY016805]
- Research To Prevent Blindness
- Excellence Initiative of the German Research Foundation [EXC 294]
- Ministry of Science, Research and the Arts of Baden-Wurttemberg [Az: 33-7532.20]
- European Research Council [ERC-2011-StG 282105]
- European Molecular Biology Organization fellowship
- Austrian Science Fund (PhD Program Molecular Bioanalytics)
- Government of Upper Austria
- European Fund for Regional Development (Hochsensitive Analysemethoden fur die Untersuchung von biologischen Proben)
- Marshall Plan Scholarship
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available