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Vol. 14, Issue 1, 142-155, January 2003

Visualization of TGN to Endosome Trafficking through Fluorescently Labeled MPR and AP-1 in Living Cells

Satoshi Waguri,^* Frédérique Dewitte,^* Roland Le Borgne,^* Yves Rouillé,^* Yasuo Uchiyama, Jean-François Dubremetz,^* and Bernard Hoflack^*§

 ^*Institut de Biologie, EP CNRS 525, Institut Pasteur de Lille, 59021 Lille Cedex, France; and  Department of Cell Biology and Neuroscience (A1), Osaka University Graduate School of Medicine, Osaka, Japan

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 -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.

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Online version of this article contains video material. Online version is available at www.molbiolcell.org.

^§ Corresponding author and present address. E-mail address: hoflack{at}mpi-cbg.de.

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Molecular Biology of the Cell
Vol. 14, 142-155, January 2003
Copyright © 2003 by The American Society for Cell Biology

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