title: Live-cell single-molecule labeling and analysis of myosin motors with quantum dots.
abstract: Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level.
authors.affiliation: Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai 980-8579, Japan hatake@tohoku.ac.jp.|Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Hatakeyama
authors.affiliation: Department of Information and Intelligent Systems, Tohoku University, Sendai 980-8579, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Nakahata
authors.affiliation: Department of Information and Intelligent Systems, Tohoku University, Sendai 980-8579, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yarimizu
authors.affiliation: Graduate School of Biomedical Engineering, Tohoku University, Sendai 980-8579, Japan.|Department of Information and Intelligent Systems, Tohoku University, Sendai 980-8579, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kanzaki
mesh_terms: D041721:3T3-L1 Cells; D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018274:Electroporation; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000378:metabolism; D021381:Protein Transport; D045663:Quantum Dots; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D000199:Actins; D051275:Glucose Transporter Type 4; D009218:Myosins