RRC ID 52663
Author Tsunoyama TA, Watanabe Y, Goto J, Naito K, Kasai RS, Suzuki KGN, Fujiwara TK, Kusumi A.
Title Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function.
Journal Nat Chem Biol
Abstract Single-fluorescent-molecule imaging tracking (SMT) is becoming an important tool to study living cells. However, photobleaching and photoblinking (hereafter referred to as photobleaching/photoblinking) of the probe molecules strongly hamper SMT studies of living cells, making it difficult to observe in vivo molecular events and to evaluate their lifetimes (e.g., off rates). The methods used to suppress photobleaching/photoblinking in vitro are difficult to apply to living cells because of their toxicities. Here using 13 organic fluorophores we found that, by combining low concentrations of dissolved oxygen with a reducing-plus-oxidizing system, photobleaching/photoblinking could be strongly suppressed with only minor effects on cells, which enabled SMT for as long as 12,000 frames (~7 min at video rate, as compared to the general 10-s-order durations) with ~22-nm single-molecule localization precisions. SMT of integrins revealed that they underwent temporary (<80-s) immobilizations within the focal adhesion region, which were responsible for the mechanical linkage of the actin cytoskeleton to the extracellular matrix.
Volume 14(5)
Pages 497-506
Published 2018-5
DOI 10.1038/s41589-018-0032-5
PII 10.1038/s41589-018-0032-5
PMID 29610485
MeSH Actin Cytoskeleton / metabolism Animals CHO Cells Cell Adhesion Cricetulus Extracellular Matrix / metabolism Fluorescent Dyes / chemistry* HeLa Cells Humans Integrin beta1 / metabolism Integrin beta3 / metabolism Integrins / metabolism* Mice Microscopy, Fluorescence* NIH 3T3 Cells Oxidation-Reduction Oxygen / chemistry Photobleaching Video Recording
IF 12.154
Times Cited 22
Human and Animal Cells HeLa(RCB0007)