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 singleparticle 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.

Brain-Penetrating Peptide Shuttles Across the Blood-Brain Barrier and Extracellular-like Space

“…The velocities for active transport are within the observed values of active intracellular transport via motor proteins (0.5 -2 µm/s) in EGFR trafficking 62 and in other studies [63][64][65] . The velocities for passive diffusion were within the range measured for confined diffusion, which is observed during events associated with ligand-receptor binding 62,66 . The intracellular trajectories of a representative M13 clone and segmentation of their motion into active transport and passive diffusion are shown in Figure S4.…”

“…The SPT software was a gift from Prof. Keith Lidke at the University of New Mexico. The trajectories were analyzed using a mean-squared displacement (MSD)-based trajectory classification algorithm 62 to extract the diffusion coefficient (D) and identify the subtrajectories exhibiting active transport. [27][28][29][30] were seeded in a 12-well plate (Corning #3513) at a seeding density of 4.0*10 4 /cm 2 and cultured for 5 days.…”

Spatial EGFR Dynamics and Metastatic Phenotypes Modulated by Upregulated EphB2 and Src Pathways in Advanced Prostate Cancer

“…To elucidate the connection between EGFR dynamics and malignancy, we firstly performed the TReD assay on EGFRs in five prostate epithelial cell lines ( Figure 1B). Trajectories of 455-2961 single EGFR complexes (EGFRs recognized by anti-EGFR IgG antibody-conjugated fluorescent nanoparticles) from each cell line were analyzed using a modified mean-squared displacement (MSD) algorithm [41,42] to calculate EGFR diffusivity (D) and size of the linear confinement (L) [12] ( Figure 1B and Figure S1, Method S1 and S2). From our tracking results, PC3 cells had the highest D and the largest L. In particular, the EGFR diffusivity of PC3 cells (0.0177 ± 0.0011 µm 2 /s, n = 1298, the statistic estimators represent sample mean ± standard error of the mean) was 144% (p < 0.00001), 124% (p < 0.00001), 97% (p < 0.00001), higher than those of BPH1 (0.0073 ± 0.0006 µm 2 /s, n = 928), LNCaP (0.0080 ± 0.0008 µm 2 /s, n = 455), and DU145 (0.0090 ± 0.0004 µm 2 /s, n = 2961), respectively ( Figure 1B).…”

“…Additionally, since clathrin-and caveolin-mediated endocytosis controls the degradation and recycling of EGFRs [8], the deregulated EGFR endocytosis results in prolonged activation, which can also lead to the development of cancer [9,10]. Despite accumulating evidence suggesting that many types of cancers are caused by the dysregulation of spatial distribution and trafficking of EGFRs [6,11], few studies have reported real-time intracellular trafficking and dynamic signaling processes of EGFRs in live cells using single-particle/molecule tracking (SPT/SMT) techniques [12,13].…”

Brain-Penetrating Peptide Shuttles Across the Blood-Brain Barrier and Extracellular-like Space

“…The SPT software was a gift from Prof. Keith Lidke at the University of New Mexico. The trajectories were analyzed using a mean-squared displacement (MSD)-based trajectory classification algorithm 62 to extract the diffusion coefficient (D) and identify the subtrajectories exhibiting active transport.…”