3D single molecule tracking with multifocal plane microscopy reveals rapid intercellular transferrin transport at epithelial cell barriers.
The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single molecule tracking of rapid protein dynamics in a 10 micron thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf molecules between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf molecules at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.
Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Sripad
S
Ram
Dongyoung
D
Kim
Raimund J
RJ
Ober
E Sally
ES
Ward
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002555:Cerebrospinal Fluid / Q000166:cytology; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D042541:Intracellular Space / Q000378:metabolism*; D007700:Kinetics; D008853:Microscopy / Q000379:methods*; D021381:Protein Transport; D045663:Quantum Dots; D051381:Rats; D014168:Transferrin / Q000378:metabolism*
D014168:Transferrin