RRC ID |
52663
|
著者 |
Tsunoyama TA, Watanabe Y, Goto J, Naito K, Kasai RS, Suzuki KGN, Fujiwara TK, Kusumi A.
|
タイトル |
Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function.
|
ジャーナル |
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.
|
巻・号 |
14(5)
|
ページ |
497-506
|
公開日 |
2018-5-1
|
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 |
12.587
|
引用数 |
22
|
リソース情報 |
ヒト・動物細胞 |
HeLa(RCB0007) |