Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution ...depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.
Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in ...unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.
Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate ...that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control.
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•NUDT2 is identified as a novel regulator of Rag GTPase-mTORC1 signaling.•NUDT2 was associated with recruitment of mTORC1 to the lysosomal membrane.•The physical interaction between NUDT2 and Rag GTPase heterodimers is required for the lysosomal localization of mTORC1.•NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells.
Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion ...phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion.
Blue-conversion, a photooxidative conversion leading to the hypsochromic shift of absorption and emission spectra, occurs in popular organic dyes under conventional laser illumination and produces severe artifacts in multicolor fluorescence imaging.
Dimerization of beta 2-adrenergic receptor (β
-AR) has been observed across various physiologies. However, the function of dimeric β
-AR is still elusive. Here, we revealed that dimerization of β
-AR ...is responsible for the constitutive activity of β
-AR generating inverse agonism. Using a co-immunoimmobilization assay, we found that transient β
-AR dimers exist in a resting state, and the dimer was disrupted by the inverse agonists. A Gα
preferentially interacts with dimeric β
-AR, but not monomeric β
-AR, in a resting state, resulting in the production of a resting cAMP level. The formation of β
-AR dimers requires cholesterol on the plasma membrane. The cholesterol did not interfere with the agonist-induced activation of monomeric β
-AR, unlike the inverse agonists, implying that the cholesterol is a specific factor regulating the dimerization of β
-AR. Our model not only shows the function of dimeric β
-AR but also provides a molecular insight into the mechanism of the inverse agonism of β
-AR.
Surface-enhanced Raman scattering (SERS) has been extensively applied to detect complex analytes due to its ability to enhance the fingerprint signals of molecules around nanostructured metallic ...surfaces. Thus, it is essential to design SERS-active nanostructures with abundant electromagnetic hotspots in a probed volume according to the dimensions of the analytes, as the analytes must be located in their hotspots for maximum signal enhancement. Herein, we demonstrate a simple method for detecting robust SERS signals from multi-scaled bioanalytes, regardless of their dimensions in the liquid state, through a photothermally driven co-assembly with colloidal plasmonic nanoparticles as signal enhancers. Under resonant light illumination, plasmonic nanoparticles and analytes in the solution quickly assemble at the focused surface area by convective movements induced by the photothermal heating of the plasmonic nanoparticles without any surface modification. Such collective assemblies of plasmonic nanoparticles and analytes were optimized by varying the optical density and surface charge of the nanoparticles, the viscosity of the solvent, and the light illumination time to maximize the SERS signals. Using these light-induced co-assemblies, the intrinsic SERS signals of small biomolecules can be detected down to nanomolar concentrations based on their fingerprint spectra. Furthermore, large-sized biomarkers, such as viruses and exosomes, were successfully detected without labels, and the complexity of the collected spectra was statistically analyzed using t-distributed stochastic neighbor embedding combined with support vector machine (t-SNE + SVM). The proposed method is expected to provide a robust and convenient method to sensitively detect biologically and environmentally relevant analytes at multiple scales in liquid samples.
A method utilizing photothermally driven assembly of colloidal plasmonic nanoparticles and multi-scaled bioanalytes is presented for enhancing SERS signals. This approach enables label-free detection of various biomolecules, including small biomolecules and larger biomarkers like viruses and exosomes, offering a robust and sensitive means to detect analytes in liquid samples. Display omitted
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