Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells.
We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose gamma-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.
Waguri
Satoshi
S
Institut de Biologie, EP CNRS 525, Institut Pasteur de Lille, 59021 Lille Cedex, France.
Dewitte
Frédérique
F
Le Borgne
Roland
R
Rouillé
Yves
Y
Uchiyama
Yasuo
Y
Dubremetz
Jean-François
JF
Hoflack
Bernard
B
D033961:Adaptor Protein Complex 1 / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
D033961:Adaptor Protein Complex 1; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins