title: DNA-based probes for flow cytometry analysis of endocytosis and recycling.
abstract: The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Parkville, Victoria, Australia.
authors.forename: Claire
authors.initials: C
authors.lastname: Dumont
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Ewa
authors.initials: E
authors.lastname: Czuba
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Moore
authors.initials: M
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Parkville, Victoria, Australia.|Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Villadangos
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.|ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, Australia.
authors.forename: Angus P R
authors.initials: AP
authors.lastname: Johnston
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Parkville, Victoria, Australia.
authors.forename: Justine D
authors.initials: JD
authors.lastname: Mintern
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D011956:Receptors, Cell Surface / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014168:Transferrin / Q000378:metabolism
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; D004247:DNA
keywords: endocytosis; probes; recycling; transferrin