Hartman Institute for Therapeutic Organ Regeneration

Accurate measurement of fast endocytic recycling kinetics in real time.

TitleAccurate measurement of fast endocytic recycling kinetics in real time.
Publication TypeJournal Article
Year of Publication2020
AuthorsJonker CTH, Deo C, Zager PJ, Tkachuk AN, Weinstein AM, Rodriguez-Boulan E, Lavis LD, Schreiner R
JournalJ Cell Sci
Volume133
Issue2
Date Published2020 Jan 22
ISSN1477-9137
KeywordsEndocytosis, Humans, Kinetics, Protein Transport
Abstract

<p>The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid, with some molecules returning to the plasma membrane with a half time <5 min. Existing methods to study these trafficking pathways utilize chemical, radioactive or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay based on a newly designed cell-impermeable fluorogenic ligand for HaloTag, Janelia Fluor 635i (JFi, where i indicates impermeant), which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found that this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.</p>

DOI10.1242/jcs.231225
Alternate JournalJ Cell Sci
PubMed ID31843759
PubMed Central IDPMC6983720
Grant ListR01 DK029857 / DK / NIDDK NIH HHS / United States
R01 EY008538 / EY / NEI NIH HHS / United States
R01 GM025662 / GM / NIGMS NIH HHS / United States
R01 GM034107 / GM / NIGMS NIH HHS / United States

Weill Cornell Medicine
Hartman Institute for Therapeutic Organ Regeneration
1300 York Ave, Box 136 New York, NY 10065