title: Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.
abstract: Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.
authors.affiliation: The Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Fletcher
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Leicestershire, United Kingdom.
authors.forename: Mudassar
authors.initials: M
authors.lastname: Iqbal
authors.affiliation: School of Mathematics and Institute for Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Sara
authors.initials: S
authors.lastname: Jabbari
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Leicestershire, United Kingdom.
authors.forename: Dov
authors.initials: D
authors.lastname: Stekel
authors.affiliation: Northwestern University, Feinberg School of Medicine, 303 E. Chicago Avenue, Chicago, Illinois, United States of America.
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Rappoport
mesh_terms: D000818:Animals; D004285:Dogs; D004705:Endocytosis; D061985:Madin Darby Canine Kidney Cells; D062793:Occludin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D059748:Proteolysis; D055571:Secretory Pathway; D019108:Tight Junctions / Q000378:metabolism
chemical_list: D062793:Occludin