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Originally published In Press as doi:10.1074/jbc.M312717200 on January 12, 2004
J. Biol. Chem., Vol. 279, Issue 13, 13190-13204, March 26, 2004
Endocytic Adaptor Molecules Reveal an Endosomal Population of Clathrin by Total Internal Reflection Fluorescence Microscopy*
Peter A. Keyel ,
Simon C. Watkins, and
Linton M. Traub
From the
Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Most eukaryotes utilize a single pool of clathrin to assemble clathrin-coated transport vesicles at different intracellular locations. Coat assembly is a cyclical process. Soluble clathrin triskelia are recruited to the membrane surface by compartment-specific adaptor and/or accessory proteins. Adjacent triskelia then pack together to assemble a polyhedral lattice that progressively invaginates, budding off the membrane surface encasing a nascent transport vesicle that is quickly uncoated. Using total internal reflection fluorescence microscopy to follow clathrin dynamics close to the cell surface, we find that the majority of labeled clathrin structures are relatively static, moving vertically in and out of the evanescent field but with little lateral motion. A small minority shows rapid lateral and directed movement over micrometer distances. Adaptor proteins, including the  subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2  2 subunit, incorporated into both AP-2 and AP-1 adaptor complexes, exhibits both types of behavior. This suggests that the highly motile clathrin puncta may be distinct from plasma membrane-associated clathrin structures. When endocytosed cargo molecules, such as transferrin or low density lipoprotein, are followed into cells, they exhibit even more lateral motion than clathrin, and gradually concentrate in the perinuclear region, consistent with classical endosomal trafficking. Importantly, clathrin partially colocalizes with internalized transferrin, but diverges as the structures move longitudinally. Thus, highly motile clathrin structures are apparently distinct from the plasma membrane, accompany transferrin, and contain AP-1, revealing an endosomal population of clathrin structures.
Received for publication, November 20, 2003
, and in revised form, January 8, 2004.
* This work was supported in part by National Institutes of Health Grant R01 DK53249 and the Senior Vice Chancellor for the Health Sciences, University of Pittsburgh in support of the Competitive Medical Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
 The on-line version of this article (available at http://www.jbc.org) contains movies of live cell TIR-FM corresponding to the experiments shown in Figs. 1 (GFP-LCa), 3 (AP-2 C subunit-YFP), 6 (AP-2 2 subunit-YFP and DsRed-LCa), and 7 (Tfn488).
 Supported by the Renal and Epithelial Biology Training Grant 5T32 DK061296-02.
 To whom correspondence should be addressed: S325 BSTWR, Dept. of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261. E-mail: traub{at}pitt.edu.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
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