Counting growth factors in single cells with infrared quantum dots to measure discrete stimulation distributions

Le, Phuong; Lim, Sung Jun; Baculis, Brian C.; Chung, Hee Jung; Kilian, Kristopher A.; Smith, Andrew

doi:10.1038/s41467-019-08754-5

Cited by 20 publications

(47 citation statements)

References 69 publications

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“…Z-stacks were recorded at 500 ms time resolution per single plane and with a step size of 0.2 μm (up to 250 planes in a single z-stack). The cell membrane shape in collected 3D z-stacks of QD-labeled HEK cells was mapped using the Matlab alphaShape function by importing ([ x ], [ y ], [ z ]) centroid coordinates of QD605 spots detected with the ImageJ 3D Object Counter plug-in. , QDs spots were considered to be internalized if their Euclidian distance from the boundary of the alpha shape was less than 10 pixels.…”

Section: Methodsmentioning

confidence: 99%

Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics

et al. 2020

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D2 dopamine receptors (DRD2s) belong to a family of G protein-coupled receptors that modulate synaptic dopaminergic tone via regulation of dopamine synthesis, storage, and synaptic release. DRD2s are the primary target for traditional antipsychotic medications; dysfunctional DRD2 signaling has been linked to major depressive disorder, attention-deficit hyperactivity disorder, addiction, Parkinson’s, and schizophrenia. DRD2 lateral diffusion appears to be an important post-translational regulatory mechanism; however, the dynamic response of DRD2s to ligand-induced activation is poorly understood. Dynamic imaging of the long isoform of DRD2 (D2L) fused to an N-terminal antihemagglutinin (HA) epitope and transiently expressed in HEK-293 cells was achieved through a combination of a high-affinity biotinylated anti-HA antigen-binding fragment (Fab) and streptavidin-conjugated quantum dots (QD). Significant reduction (∼40%) in the rate of lateral diffusion of QD-tagged D2L proteins was observed under agonist (quinpirole; QN)-stimulated conditions compared to basal conditions. QN-induced diffusional slowing was accompanied by an increase in frequency, lifetime, and confinement of temporary arrest of lateral diffusion (TALL), an intrinsic property of single receptor lateral motion. The role of the actin cytoskeleton in QN-induced diffusional slowing of D2L was also explored. The observed dynamic changes appear to be a sensitive indicator of the receptor activity status and might also spatially and temporally shape the receptor-mediated downstream signaling. This dynamic information could potentially be useful in informing drug discovery efforts based on single-molecule pharmacology.

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Section: Methodsmentioning

confidence: 99%

Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics

et al. 2020

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“…[1][2][3][4] Tools enabling acute control of such complex formation or its inhibition with effects on the signaling pathway are of primary interest in fundamental cell biology or nanomedicine. [5] While functionalized nano-probes such as nanoparticles, [6][7][8] quantum dots [9][10] or nanodiscs [11] already demonstrated to elucidate cell signaling behavior, the number and spatial localization of ligands on these probes are difficult to control. DNA origami, on the other hand, is a powerful tool to build versatile DNA-based platforms [12][13][14][15] which satisfy multiple structural and biofunctional constraints including the possibility of complex molecular conjugation with biomolecules such as peptides or proteins with nanometric precision.…”

Section: Introductionmentioning

confidence: 99%

Nanoscale Organization of FasL on DNA Origami as a Versatile Platform to Tune Apoptosis Signaling in Cells

Berger

Weck

Kempe

et al. 2020

Preprint

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AbstractNanoscale probes with fine-tunable properties are of key interest in cell biology and nanomedicine to elucidate and eventually control signaling processes in cells. A critical, still challenging issue is to conjugate these probes with molecules in a number- and spatially-controlled manner. Here, DNA origami-based nanoagents as nanometer precise scaffolds presenting Fas ligand (FasL) in well-defined arrangements to cells are reported. These nanoagents activate receptor molecules in the plasma membrane initiating apoptosis signaling in cells. Signaling for apoptosis depends sensitively on FasL geometry: fastest time-to-death kinetics are obtained for FasL nanoagents representing predicted structure models of hexagonal receptor ordering with 10 nm inter-molecular spacing. Slower kinetics are observed for one to two FasL on DNA origami or FasL coupled with higher flexibility. Nanoagents with FasL arranged in hexagons with small (5 nm) and large (30 nm) spacing impede signal transduction. Moreover, for predicted hexagonal FasL nanoagents, signaling efficiency is faster and 100× higher compared to naturally occurring soluble FasL. Incubation of the FasL-origami nanoagent in solution exhibited an EC50 value of only 90 pM. These studies present DNA origami as versatile signaling platforms to probe the significance of molecular number and nanoscale ordering for signal initiation in cells.

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“…MDA‐MB‐468 cells, a cell line well‐established for overexpression of EGFR, were incubated in Dulbecco's modified Eagle medium (DMEM)‐supplemented with 10% fetal bovine serum (FBS) in a glass‐bottom dish (either glass or substrate‐660) for 24 h, and was then treated in serum‐free DMEM for another 4 h before imaging. Competitive binding of EGF‐Cy3 (1.0 × 10 −9 m ) and dye‐free EGF (100 × 10 −9 m ) confirmed the specific binding (Figure S12, Supporting Information) of EGF on the cells.…”

mentioning

confidence: 99%

A Self‐Assembled Plasmonic Substrate for Enhanced Fluorescence Resonance Energy Transfer

Hou

Chen

et al. 2020

Advanced Materials

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Fluorescence resonance energy transfer (FRET) has found widespread uses in biosensing, molecular imaging, and light harvesting. Plasmonic metal nanostructures offer the possibility of engineering photonic environment of specific fluorophores to enhance the FRET efficiency. However, the potential of plasmonic nanostructures to enable tailored FRET enhancement on planar substrates remains largely unrealized, which are of considerable interest for high‐performance on‐surface bioassays and photovoltaics. The main challenge lies in the necessitated concurrent control over the spectral properties of plasmonic substrates to match that of fluorophores and the fluorophore–substrate spacing. Here, a self‐assembled plasmonic substrate based on polydopamine (PDA)‐coated plasmonic nanocrystals is developed to effectively address this challenge. The PDA coating not only drives interfacial self‐assembly of the nanocrystals to form closely packed arrays with customized optical properties, but also can serve as a tailored nanoscale spacer between the fluorophores and plasmonic nanocrystals, which collectively lead to optimized fluorescence enhancement. The biocompatible plasmonic substrate that allows convenient bioconjugation imparted by PDA has afforded improved FRET efficiency in DNA microarray assay and FRET imaging of live cells. It is envisioned that the self‐assembled plasmonic substrates can be readily integrated into fluorescence‐based platforms for diverse biomedical and photoconversion applications.

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Counting growth factors in single cells with infrared quantum dots to measure discrete stimulation distributions

Cited by 20 publications

References 69 publications

Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics

Single Quantum Dot Tracking Unravels Agonist Effects on the Dopamine Receptor Dynamics

Nanoscale Organization of FasL on DNA Origami as a Versatile Platform to Tune Apoptosis Signaling in Cells

A Self‐Assembled Plasmonic Substrate for Enhanced Fluorescence Resonance Energy Transfer

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