title: Local clustering of transferrin receptors promotes clathrin-coated pit initiation.
abstract: 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.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Allen P
authors.initials: AP
authors.lastname: Liu
authors.forename: François
authors.initials: F
authors.lastname: Aguet
authors.forename: Gaudenz
authors.initials: G
authors.lastname: Danuser
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Schmid
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism; D034002:Adaptor Protein Complex sigma Subunits / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation / Q000235:genetics / Q000379:methods; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D033941:Clathrin Light Chains / Q000235:genetics / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000502:physiology* / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D007700:Kinetics; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D011940:Receptor Aggregation / Q000502:physiology*; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D033962:Adaptor Protein Complex 2; D034002:Adaptor Protein Complex sigma Subunits; D033941:Clathrin Light Chains; D029968:Escherichia coli Proteins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001710:Biotin; D019809:Streptavidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli