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
T
ransmembrane
Re
ceptor
D
ynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named
T
ransmembrane
Re
ceptor
D
ynamics (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.
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 k
on (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.
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