Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells.

Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of kon (linear to bent) from 3.2 to 10.7 s-1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.

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

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

Assessing metastatic potential of breast cancer cells based on EGFR dynamics.

Derailed transmembrane receptor trafficking could be a hallmark of tumorigenesis and increased tumor invasiveness, but receptor dynamics have not been used to differentiate metastatic cancer cells from less invasive ones. Using single-particle tracking techniques, we  developed a phenotyping asssay named Transmembrane Receptor Dynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named Transmembrane Receptor Dynamics (TReD). Here we show a clear evidence that increased EGFR diffusivity and enlarged EGFR confinement size in the plasma membrane (PM) are correlated with the enhanced metastatic potential in these cell lines. By comparing the TReD results with the gene expression profiles, we found a clear negative correlation between the EGFR diffusivities and the breast cancer luminal differentiation scores (r = -0.75). Upon the induction of epithelial-mesenchymal transition (EMT), EGFR diffusivity significantly increased for the non-tumorigenic MCF10A (99%) and the non-invasive MCF7 (56%) cells, but not for the highly metastatic MDA-MB-231 cell. We believe that the reorganization of actin filaments during EMT modified the PM structures, causing the receptor dynamics to change. TReD can thus serve as a new biophysical marker to probe the metastatic potential of cancer cells and even to monitor the transition of metastasis.