Dataset Information

Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques.

ABSTRACT: Diffusion of a G-protein coupled receptor, mu-opioid receptor (muOR), in the plasma membrane was tracked by single-fluorescent molecule video imaging and high-speed single-particle tracking. At variance with a previous publication, where gold-tagged muOR was found to be totally confined within a domain, which in turn underwent very slow diffusion itself, we found that muOR undergoes rapid hop diffusion over membrane compartments (210-nm and 730-nm nested double compartments in the case of normal rat kidney cell line), which are likely delimited by the actin-based membrane-skeleton "fence or corrals" and its associated transmembrane protein "pickets", at a rate comparable to that for transferrin receptor (every 45 and 760 ms on average, respectively), suggesting that the fence and picket models may also be applicable to G-protein coupled receptors. Further, we found that strong confinement of gold-labeled muOR could be induced by the prolonged on-ice preincubation of the gold probe with the cells, showing that this procedure should be avoided in future single-particle tracking experiments. Based on the dense, long trajectories of muOR obtained by high-speed single-particle tracking, the membrane compartments apposed and adjoined to each other could be defined that are delimited by rather straight boundaries, consistent with the involvement of actin filaments in membrane compartmentalization.

SUBMITTER: Suzuki K

PROVIDER: S-EPMC1305513 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques.

Suzuki Kenichi K Ritchie Ken K Kajikawa Eriko E Fujiwara Takahiro T Kusumi Akihiro A

Biophysical journal 20050128 5

Diffusion of a G-protein coupled receptor, mu-opioid receptor (muOR), in the plasma membrane was tracked by single-fluorescent molecule video imaging and high-speed single-particle tracking. At variance with a previous publication, where gold-tagged muOR was found to be totally confined within a domain, which in turn underwent very slow diffusion itself, we found that muOR undergoes rapid hop diffusion over membrane compartments (210-nm and 730-nm nested double compartments in the case of normal ...[more]

PMID: 15681644

Similar Datasets

Project description:Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus, a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca(2+)-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves, identifies and quantitates the rapidly diffusing monomers and slowly diffusing heterodimers. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing ratio of monomers and heterodimers, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ~10(2)-fold greater in the membrane system than in solution, apparently due to both faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.

Dataset Information

Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques.

Publications

Rapid hop diffusion of a G-protein-coupled receptor in the plasma membrane as revealed by single-molecule techniques.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets