Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy.
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 alpha subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2 beta2 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.
Keyel
Peter A
PA
Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Watkins
Simon C
SC
Traub
Linton M
LM
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D002966:Clathrin; D008024:Ligands; D008164:Luminescent Proteins; D020033:Protein Isoforms; D014168:Transferrin; D049452:Green Fluorescent Proteins; D013395:Sucrose