Local clustering of transferrin receptors promotes clathrin-coated pit initiation

• Published in: The Journal of cell biology Vol. 191; no. 7; pp. 1381 - 1393
• Main Authors: Liu, Allen P, Aguet, François, Danuser, Gaudenz, Schmid, Sandra L
• Format: Journal Article
• Language: English
• Published: United States The Rockefeller University Press 27.12.2010; Rockefeller University Press
• Subjects: Adaptor Protein Complex 2 - genetics; Adaptor Protein Complex 2 - metabolism; Adaptor Protein Complex sigma Subunits - genetics; Adaptor Protein Complex sigma Subunits - metabolism; Animals; Biochemistry; Biotin - metabolism; Biotinylation - genetics; Biotinylation - methods; Carbon-Nitrogen Ligases - genetics; Cell cycle; Cell division; Cell Line; Cell Membrane - metabolism; Cercopithecus aethiops; Clathrin Light Chains - genetics; Clathrin Light Chains - metabolism; Coated Pits, Cell-Membrane - physiology; Coated Pits, Cell-Membrane - ultrastructure; Endocytosis; Endocytosis - physiology; Epithelial Cells - cytology; Epithelial Cells - physiology; Escherichia coli Proteins - genetics; Fluorescence; Freight; Humans; Imaging; Internalization; Kinetics; Ligands; Maturation; Molecules; Physiological regulation; Protein Binding - physiology; Proteins; Rats; Receptor Aggregation - physiology; Receptors; Receptors, Transferrin - genetics; Receptors, Transferrin - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Repressor Proteins - genetics; Streptavidin - genetics; Streptavidin - metabolism; Transduction, Genetic; Transferrin receptors
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.201008117

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.