Dimerization of β-adrenergic receptor is responsible for the constitutive activity subjected to inverse agonism.

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.

Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging.

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.

Improved resolution in single-molecule localization microscopy using QD-PAINT.

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.

Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay.

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).