Analysis of Ion Channel Dynamics by Single Molecule Tracking in Live Cells

Protein dynamics play an important role in signal transduction in association with their activation mechanisms, functions, and so on. Single molecule tracking technique have been widely used for investigating diffusion behavior of protein in live cells. Especially membrane proteins have been studied since they are important to understand cell responding to the surroundings. However, not many ion channel proteins such as AMPAR, NMDAR, etc., have been monitored due to their complex system. Here, we observed ionotropic glutamate receptor in neuroblastoma SH-SY5Y cell using single-molecule imaging. The diffusion coefficient of the receptor was significantly low because the receptor has four subunits (tetramers) and each subunit possesses a four transmembrane domain. Furthermore, we also analyzed interaction between subunits using single molecule tracking, further investigation of the protein dynamics of the membrane proteins such as ionotropic receptors, their structure and protein activation mechanism will be possible.1

Monitoring Rotation Dynamics of Membrane Protein in Live Cells

Dynamic behavior of membrane protein provides critical information in molecular and cellular mechanisms. To have access to the mobility of a membrane protein, single-particle tracking has been advanced for the microscopic mechanism understandings. Among various molecular motions, however, only the lateral motion of the protein has been monitored due to the lack of in situ imaging tool enabling observation for rotation and vibration. Here, we developed plasmonic nanoparticles which can monitor rotational diffusion dynamics as well as lateral motion. This nanoparticle probe allows direct evidence and quantitative analysis of rotation dynamics, and furthermore, observation of conformation changes of proteins and the protein-protein interactions in live cells. This study provides an insight into the molecular mechanism regarding the intracellular signaling process.1

Single-Molecule Rotation for EGFR Conformational Dynamics in Live Cells

Monitoring the dynamics of proteins in live cells on appropriate spatiotemporal scales may provide key information regarding long-standing questions in molecular and cellular regulatory mechanisms. However, tools capable of imaging the conformational changes over time have been elusive. Here, we present a single-molecule stroboscopic imaging probes by developing gyroscopic plasmonic nanoparticles, allowing for replication of protein-protein interactions and the conformational dynamics based on rotational and lateral velocities. This study fundamentally monitors the rotational motion of a membrane protein, epidermal growth factor receptor (EGFR), to decipher undiscovered structural dynamics in live cells without any molecular perturbations. This method offers a strategy to visualize assemblies and conformational changes, and provides unique insights into the mechanism underlying the molecular dynamics for receptors. © 2018 American Chemical Society.1