Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca microscopy.

Objective: Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca indicator GmNL(Ca), and its application in a customized microscope for high-throughput drug screening.

ABCA1 dimer-monomer interconversion during HDL generation revealed by single-molecule imaging.

The generation of high-density lipoprotein (HDL), one of the most critical events for preventing atherosclerosis, is mediated by ATP-binding cassette protein A1 (ABCA1). ABCA1 is known to transfer cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating discoidal HDL (dHDL) particles, composed of 100-200 lipid molecules surrounded by two apoA-I molecules; however, the regulatory mechanisms are still poorly understood. Here we observed ABCA1-GFP and apoA-I at the level of single molecules on the plasma membrane via a total internal reflection fluorescence microscope.

Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging.

Receptor dimerization is important for many signaling pathways. However, the monomer-dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein-coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method.

Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules.

Recent advancements in single-molecule tracking methods with nanometer-level precision now allow researchers to observe the movement, recruitment, and activation of single molecules in the plasma membrane in living cells. In particular, on the basis of the observations by high-speed single-particle tracking at a frame rate of 40,000 frames s(1), the partitioning of the fluid plasma membrane into submicron compartments throughout the cell membrane and the hop diffusion of virtually all the molecules have been proposed. This could explain why the diffusion coefficients in the plasma membrane are considerably smaller than those in artificial membranes, and why the diffusion coefficient is reduced upon molecular complex formation (oligomerization-induced trapping).

Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules.

Tracking single molecules in the plasma membrane in live cells is becoming a useful technique for studying the spatial-temporal control of membrane molecular processes, such as signal transduction and the formation of large molecular complexes. In this review, three topics largely based on recent single-molecule observations are described, with a special emphasis on the results that are considered to be difficult to obtain using conventional methods monitoring the ensemble-averaged behavior of molecules. First, we describe the high-speed single-molecule tracking data, mostly obtained by our group that necessitated the paradigm shift of the plasma membrane structure, from the two-dimensional continuum fluid model to the compartmentalized fluid model.

Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains.