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

Abstract

SummaryMechanical 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 timedependent 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. IntroductionPhysiological processes generate and are modulated by mechanical stress (Kumar and Weaver, 2009;Shyu, 2009;Wallace and McNally, 2009). Of the three free energy sources available to cells, chemical, electrical and mechanical, the latter is mostly unmapped due to a lack of probes. To begin opening this field we developed the original FRET-based stress probe called stFRET. It used an a-helix (a molecular spring) to link two GFP mutants . We then developed sstFRET, which matched the mechanical compliance of common hosts by substituting a spectrin repeat for the linker (Meng and Sachs, 2011a). Grashoff and colleagues created another FRET sensor, TSMod, using a domain from spider silk as the linker (Grashoff et al., 2010). Iwai and Uyeda developed a strain sensor based on proximity imaging (PRIM) with GFP dimers (Iwai and Uyeda, 2008). These force sensors share a uniform mechanism for interpreting force: tension in the host induces strain in the linker, leading to increased distance between the donor and acceptor. The dynamic range of these sensors is limited by the nearly linear relationship between FRET efficiency and strain . These sensors are also relatively large, .70 kDa, and potentially perturb host function. This paper describes a new probe that uses angular orientation as the dominant variable and is physically smaller than previous probes. It expresses well in cells, making it a general tool for studying cell mechanics. We demonstrated probe efficacy by mapping stress gradients in spectrin in different cell types and demonstrated, for the first time, that stress in spectrin is modulated during cell migration.

Talin determines the nanoscale architecture of focal adhesions

“…Because the stress-fiber geometry should be relatively easy to estimate for cells on 2D substrate, these can simplify to one angle and one tension measurement. A number of strategies may be possible for the design of a single-molecule talin geometry reporter, such as a geometry-dependent FRET sensor (59) or by single-molecule localization of a pair of spectrally distinct FPs incorporated at welldefined sites within talin. Direct and simultaneous measurements of force and force-transmission structures in living cells will be instrumental in understanding how complex mechanosensitive responses originate in FAs.…”

Actin stress in cell reprogramming

“…The literature shows many macroscopic effects of mechanical stress on cellular processes including cell motility, embryogenesis, stem cell replication and differentiation (3,4), bone and muscle homeostasis, gene expression, protein folding (5), and membrane potential (6). However, these studies lacked the ability to measure stress in specific structural proteins, and the analyses have tended to treat cytoskeletal stresses as uniform, which we now know is not true (2,7). We, and other groups (8), have shown that the distribution of forces is nonuniform in both time and space and protein specificity (9).…”

“…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.…”

“…Mechanical loading twists the dipole orientation from near parallel to diagonal, decreasing FRET efficiency. The angular sensor has a much wider dynamic range and higher sensitivity than the sensors with linear linkers that use the distance dependence of FRET (7). The sensor shows ∼80% energy transfer at rest and reductions as low as 20% with physiologically relevant forces (7).…”

Mechanobiology of Ciliogenesis

“…There is, therefore, a persistent need for methods to apply and modulate mechanical forces inside living tissues. Methods to measure forces by fluorescence resonance energy transfer-based mechanosensors in living cells are becoming available (Meng and Sachs 2012), and these will also be extremely important for learning more about the relation of mechanics to ciliogenesis.…”