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

Area of Science:

• Cellular mechanics and biophysics
• Molecular imaging and biosensing
• Biochemistry and structural biology

Background:

• Mechanical stress is a significant, yet poorly understood, source of free energy within cells.
• Accurate mapping of cellular stress fields requires sensitive, specific probes with minimal biological disturbance.
• Existing probes often lack the sensitivity, dynamic range, or specificity needed for complex cellular environments.

Purpose of the Study:

• To design and validate a novel genetically encoded sensor for mapping mechanical stress in living cells.
• To characterize the sensitivity, dynamic range, and applicability of the new sensor in various biological contexts.
• To investigate the spatial and temporal distribution of mechanical stress in spectrin within different cell types.

Main Methods:

• Development of a genetically encoded Förster Resonance Energy Transfer (FRET)-based sensor (cpstFRET) sensitive to angular changes between donor and acceptor fluorophores.
• In vivo testing of cpstFRET to measure stress gradients in non-erythroid spectrin across diverse cell types.
• Analysis of stress generation mechanisms involving F-actin and tubulin, and observation of spectrin's force modulation during cell migration.

Main Results:

• The cpstFRET sensor is physically smaller, more sensitive, and possesses a greater dynamic range compared to existing probes.
• Spectrin was found to be under constitutive stress in certain cell types, while others showed no significant stress.
• The study revealed time-dependent force modulation of spectrin during cell migration, a previously unobserved phenomenon.
• Stresses within spectrin appear to be influenced by both F-actin and tubulin cytoskeletal components.

Conclusions:

• cpstFRET provides a powerful new tool for quantifying mechanical stress in biological systems with high spatiotemporal resolution.
• The findings demonstrate the dynamic nature of cellular mechanical stress and its regulation by cytoskeletal elements.
• This sensor technology has broad applications for in vitro, in vivo, and in situ studies, including mapping multidimensional stress fields in extracellular matrices.