Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits

Abstract

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.

Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis

“…This is in contrast to the previously imputed role for clathrin in the internalization or 'endocytosis' of smaller molecular cargo in the range of 30-150 nm (Ehrlich et al, 2004). Our findings that reveal both lipid raft-and clathrin-dependent entry of T. forsythia are intriguing.…”

Virus trafficking – learning from single-virus tracking

“…Other viruses have also been observed to co-localize with CCPs in live cells, including reovirus and adenovirus 30,31 . In common with Influenza, reovirus enters by the de novo formation of CCPs 30 .…”

“…In common with Influenza, reovirus enters by the de novo formation of CCPs 30 . Experiments tracking Reoviruses, transferrin, LDL and labelled CCPs revealed a random initiation behaviour of coated pits, which are only stabilized after cargo binding 30 .…”

“…In common with Influenza, reovirus enters by the de novo formation of CCPs 30 . Experiments tracking Reoviruses, transferrin, LDL and labelled CCPs revealed a random initiation behaviour of coated pits, which are only stabilized after cargo binding 30 . For HIV, although direct fusion with the plasma membrane is the main pathway to deliver the capsid into the cytoplasm, a smaller fraction of viruses also infect cells by clathrin-mediated endocytosis 32 .…”

Initiation de l’endocytose par vésicules de clathrine

“…L'initiation de la formation de vésicu-les de clathrine est une étape difficile à étudier : les puits recouverts de clathrine se forment et deviennent des vésicules relativement rapidement (20 à 90 secondes), sont trop petits (environ 100 nm) pour être observés directement par microscopie optique et se forment aléatoirement sur la surface des cellules [19,20]. L'adaptateur AP2 a longtemps été considéré comme responsable de la nucléation des puits de clathrine.…”