title: Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking.
abstract: Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.
authors.affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas.
authors.forename: Yen-Liang
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas.
authors.forename: Evan P
authors.initials: EP
authors.lastname: Perillo
authors.affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas.
authors.forename: Cong
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas.
authors.forename: Peter
authors.initials: P
authors.lastname: Yu
authors.affiliation: Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas; Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan.
authors.forename: Chao-Kai
authors.initials: CK
authors.lastname: Chou
authors.affiliation: Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas; Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung, Taiwan.
authors.forename: Mien-Chie
authors.initials: MC
authors.lastname: Hung
authors.affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Dunn
authors.affiliation: Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas. Electronic address: tim.yeh@austin.utexas.edu.
authors.forename: Hsin-Chih
authors.initials: HC
authors.lastname: Yeh
mesh_terms: D000465:Algorithms; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D021621:Imaging, Three-Dimensional; D008853:Microscopy; D021381:Protein Transport
chemical_list: D066246:ErbB Receptors