3D Single‐Molecule Imaging of Transmembrane Signaling by Targeting Nanodiamonds

Abstract

Fluorescent nanodiamonds (FNDs) have recently emerged as promising probes for imaging applications. A significant limitation of the applications is the use of FNDs as endogenous protein tags for long‐term 3D single molecule imaging to gain critical understanding of the underlying mechanism such as transmembrane signaling. Here, FNDs conjugated with transforming growth factor (TGF) are developed as an imaging probe for endogenous TGF‐beta (TGF‐β) receptor labeling and 3D single molecule imaging. FNDs display higher localization accuracy in 3D than organic dye making it an ideal candidate for nanoscopy applications. The real‐time dynamics of TGF‐β receptors after binding conjugated FNDs and in cells treated with therapeutic small molecule kinase inhibitors (SMI) are further monitored. The Bayesian treatment of hidden Markov models confirms and quantifies three different diffusive states and the transition rates between the three states. The kinetic reaction favors a faster diffusion population after therapeutic SMI treatment. The results show that immobilized TGF‐β is critical for active signaling. SMI treatment can release TGF‐β from the signaling complex. The results demonstrate the reported method that provides a powerful technique to study the mechanism of transmembrane signaling and valuable insights for developing better therapeutic for TGF‐β‐associated cancers.

Multifunctional nanodiamonds in regenerative medicine: Recent advances and future directions

“…By identifying ligand-receptor binding and activation of signaling pathways, FNDs can be utilized as a platform to screen receptor-targeting drugs. One strategy showed the efficacy of FNDs as a protein tag for 3D molecular imaging to test the effects of small molecule kinase inhibitors (SMIs) on transmembrane signaling of endogenous proteins [169]. Streptavidin-bound PEG was covalently linked to carboxylated FNDs by EDC-NHS coupling.…”

“…i) Single-particle trajectories of FND-labeled TGF-β in human adenocarcinoma cells, both unmodified (w/o SMI) and with the treatment of a small molecule kinase inhibitor (w/SMI). ii) Three distinct diffusion states were identified when the FND-labeled protein was tracked in adenocarcinoma cells [169]. …”

Biohybrid fluorescent nanodiamonds as dual‐contrast markers for light and electron microscopies

“…The neoglycoprotein‐FNDs showed high targeting ability for human liver cancer cells like HepG2 cells, as confirmed by flow cytometric analysis. With the aid of the BSA coating, Liu et al conjugated transforming growth factor‐beta (TGF‐β) receptors onto FNDs for three‐dimensional tracking in cells . They observed specific targeting of the FND‐bound TGF‐β to the plasma membrane of HCC827 cells.…”

Near‐Infrared Fluorescent Nanomaterials for Bioimaging and Sensing

“…Left: immobile particles (Diffusuion coefficient D = 0.01 µm 2 s −1 ); middle: anomalous diffusion (D = 0.2 µm 2 s −1 ); right: brownian diffusion (D = 3 µm 2 s −1 ). Reproduced with permission . Copyright 2015, Wiley‐VCH.…”

“…Simpson et al have tracked individual 130 nm sized nanodiamonds in drosophila melanogaster embryos and found both freely diffusing particles as well as actively transported particles inside the embryo . Recently, Lui and coworkers targeted 3D single particle tracking of nanodiamonds to monitor the real‐time dynamics of a trabsforming growth factor receptor (TGF‐β) . Nanodiamonds were also used to identify transplanted lung stem cells in vivo and track the cells' regenerative capabilities using confocal fluorescence microscopy and FLIM ( Figure )…”