Orientation-based FRET sensor for real-time imaging of cellular forces

J Cell Sci. 2012 Feb 1;125(Pt 3):743-50. doi: 10.1242/jcs.093104.

Abstract

Mechanical stress is an unmapped source of free energy in cells. Mapping the stress fields in a heterogeneous time-dependent environment like that found in cells requires probes that are specific for different proteins and respond to biologically relevant forces with minimal disturbance to the host system. To meet these goals, we have designed a genetically encoded stress sensor with minimal volume and high sensitivity and dynamic range. The new FRET-based sensor, called cpstFRET, is designed to be modulated by the angles between the donor and acceptor rather than the distance between them. Relative to other probes, it is physically smaller and exhibits a greater dynamic range and sensitivity and expresses well. For in vivo testing, we measured stress gradients in time and space in non-erythroid spectrin in several different cell types and found that spectrin is under constitutive stress in some cells but not in others. Stresses appear to be generated by both F-actin and tubulin. The probe revealed, for the first time, that spectrin undergoes time-dependent force modulation during cell migration. cpstFRET can be employed in vitro, in vivo and in situ, and when incorporated into biologically expressed extracellular polymers such as collagen, it can report multidimensional stress fields.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Base Sequence
  • Biomechanical Phenomena
  • Biophysical Phenomena
  • Cattle
  • Cell Line
  • Cell Movement / physiology*
  • Computer Systems
  • Cytoskeleton / metabolism
  • DNA Primers / genetics
  • Dogs
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Models, Molecular
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Spectrin / chemistry
  • Spectrin / metabolism*
  • Stress, Mechanical
  • Tubulin / metabolism

Substances

  • Actins
  • Bacterial Proteins
  • DNA Primers
  • Fluorescent Dyes
  • Luminescent Proteins
  • Recombinant Proteins
  • Tubulin
  • yellow fluorescent protein, Bacteria
  • Spectrin