Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor

Abstract

SummaryMechanical forces are ubiquitous modulators of cell activity but little is known about the mechanical stresses in the cell. Genetically encoded FRET-based force sensors now allow the measurement of local stress in specific host proteins in vivo in real time. For a minimally invasive probe, we designed one with a mechanical compliance matching that of many common cytoskeleton proteins. sstFRET is a cassette composed of Venus and Cerulean linked by a spectrin repeat. The stress sensitivity of the probe was measured in solution using DNA springs to push the donor and acceptor apart with 5-7 pN and this produced large changes in FRET. To measure cytoskeletal stress in vivo we inserted sstFRET into -actinin and expressed it in HEK and BAEC cells. Time-lapse imaging showed the presence of stress gradients in time and space, often uncorrelated with obvious changes in cell shape. The gradients could be rapidly relaxed by thrombin-induced cell contraction associated with inhibition of myosin II. The tension in actinin fluctuated rapidly (scale of seconds) illustrating a cytoskeleton in dynamic equilibrium. Stress in the cytoskeleton can be driven by macroscopic stresses applied to the cell. Using sstFRET as a tool to measure internal stress, we tested the prediction that osmotic pressure increases cytoskeletal stress. As predicted, hypotonic swelling increased the tension in actinin, confirming the model derived from AFM. Anisotonic stress also produced a novel transient (~2 minutes) decrease in stress upon exposure to a hypotonic challenge, matched by a transient increase with hypertonic stress. This suggests that, at rest, the stress axis of actinin is not parallel to the stress axis of actin and that swelling can reorient actinin to lie more parallel where it can absorb a larger fraction of the total stress. Protein stress sensors are opening new perspectives in cell biology.

Actin stress in cell reprogramming

“…This changed with the development of genetically coded FRET-based force probes (2, 10). To calibrate the intrinsic force sensitivity of these probes, we used DNA springs to stretch the probes in solution and showed that the probes responded clearly to forces in the range of 10 pN (7,10,11). We have calibrated the strain sensitivity of the earlier linear linked probes and showed FRET to be linear as predicted (2).…”

“…The angular probe uses the dipole orientation dependence of FRET, allowing FRET efficiencies, in principle, from 0% to 100% (2,8,11). The dimeric structure of cpstFRET mimics the dimer subunit structure of many multimeric cytoskeleton proteins such as actin and tubulin ( Figs.…”

“…For human alpha-actinin constructs, we purchased pEGFP-N1 alpha-actinin plasmid from Addgene (Plasmid 11908). We created pEG-actinin-sstFRET with sstFRET inserted between two spectrin repeats in actinin and pEG-Actinin-C-sstFRET with sstFRET tagged to the C-terminal of actinin (11). We replaced sstFRET with cpstFRET and created pEG-Actinin-cpstFRET and pEG-Actinin-C-cpstFRET.…”

“…We have used a variety of different linkers and some that match the compliance of the host protein (7,11,12). All of the published work on force probes has required a host protein that is linear, so the probes can be coded into the DNA of the host protein.…”

A Threshold Shear Force for Calcium Influx in an Astrocyte Model of Traumatic Brain Injury

“…Cells withstand mechanical loading through support from cytoskeleton, a heterogeneous, anisotropic collection of dynamically cross-linked structural proteins. [41][42][43][44][45] The deformation of a cell depends on the intrinsic elastic deformation of fibrous cytoskeletal proteins and plastic deformation involving dynamics of reversible cross-linking and cytoskeleton reorganization. 46,47 Slow shear loading engages the plastic processes that significantly modify the local force around the Ca 2 + transducers acting like dashpots in series with the transducer.…”