Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function

Nat Chem Biol. 2018 May;14(5):497-506. doi: 10.1038/s41589-018-0032-5. Epub 2018 Apr 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Fluorescent Dyes
  • Integrin beta1
  • Integrin beta3
  • Integrins
  • Oxygen