title: Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates.
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.
authors.affiliation: Margaret M. Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY 10065.
authors.forename: Roland
authors.initials: R
authors.lastname: Thuenauer
authors.forename: Ya-Chu
authors.initials: YC
authors.lastname: Hsu
authors.forename: Jose Maria
authors.initials: JM
authors.lastname: Carvajal-Gonzalez
authors.forename: Sylvie
authors.initials: S
authors.lastname: Deborde
authors.forename: Jen-Zen
authors.initials: JZ
authors.lastname: Chuang
authors.forename: Winfried
authors.initials: W
authors.lastname: Römer
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
authors.forename: Enrique
authors.initials: E
authors.lastname: Rodriguez-Boulan
authors.forename: Ching-Hwa
authors.initials: CH
authors.lastname: Sung
mesh_terms: D000818:Animals; D053898:Biosynthetic Pathways / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology*; D006056:Golgi Apparatus / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D012243:Rhodopsin / Q000096:biosynthesis / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D017931:DNA Primers; D012243:Rhodopsin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins