Our attempts to obtain a rough force calibration of the probe using DNA springs were successful in that we could readily resolve the forces due to a change in persistence length. What is that force? The experiments and analysis by Zocchia's group suggest that they are in the range of 5–7 pN.36
When the bending of the DNA is relatively small, it can be well approximated by the mechanics of bending an elastic rod. However, stress from high curvature at length scales below the accepted persistence length for ds-DNA, can cause a kink in the DNA to form and thus limits the force.36,50
There is no force/distance data available for the linker that we used, but single molecule force spectroscopy of helices from spectrin37
show a constant compliance up to ~40 pN. More precise calibrations of stFRET will require varying the length of the ss and complementary DNA and a better theory of DNA bending at these short length scales. Zocchi's group has shown that stresses seem to be distributed over the host protein,48
but we should point out that the DNA is not attached at the C and N termini where the host protein is attached, but at the cysteines of GFPs. These different geometries could produce somewhat different values of R0
due to the geometric factor of κ2
However, the primary utility of these probes is to measure gradients of stress at the resolution of the light microscope, so that this means averaging the effects over a voxel volume. This will include many molecules that could be at different regions of the force distance curve,37
fibers containing bilabeled peptides, monolabeled peptides or unlabeled peptides that are in parallel with the labeled ones since we did not suppress the endogenous gene expression. The data could also include crosslinkers that dissociated from actin and are averaged over the exposure time and over the voxel volume. Molecular erythroid spectrin tetramers can dissociate under shear stress at their dimer-dimer interaction ends.1
Filamin binding to actin can be ruptured at forces similar to the stress required to unfold its Ig-like subdomains.11
The adaptation effects we observed with repetitive shear stress with BAECs were possibly due to reforming of the cytoskeleton with new links, stabilization of some existing links, or both. But to visualize stress gradients at optical resolution, we do not need to know the absolute stress on a given molecule. In the long run, making a more homogenous population by suppressing expression of the wild type gene with iRNAs could lead to an improved signal to noise ratio.
The proteins we have chosen to label are hopefully representative of the fibrous proteins in and around cells. Actinin and filamin are actin bundling agents that are recruited to focal adhesion complexes and stress fibers. While actinin primarily acts to crosslink fibers that run in parallel longitudinally in stress fibers, filamin appears to crosslink actin fibers that are crossing.11,14
Spectrin is a crosslinking protein that primarily resides in the cortical cytoskeleton and acts as a scaffolding protein for membrane complexes, e.g., it links cell–cell junctions to perijunctional actin band in endothelial cells.3
Collagen is the most common protein in animals22
and our successful expression of an extracellular protein with the stFRET probe suggests that it, and related probes, will be extremely useful in understanding the mechanical function of the extracellular matrix and the glycocalyx.
We found that stFRET was least disruptive to normal protein distributions of all four host proteins when positioned centrally in the host. Collagen-19 was especially sensitive possibly due to the two cysteine clusters located at the C and N-terminals that form disulfide bridges between collagen monomers in the extracellular matrix.30
All four protein constructs had constitutive FRET values within the dynamic range determined from the free stFRET probe in solution (pulled tightly together by the linker) and the cleaved dimers (effectively infinitely apart). The distribution of FRET values within a single cell or organism differed by as much as 50% showing the presence of constitutive spatial and temporal stress gradients, features that are not currently visible through cell morphology. Since most filamentous proteins cannot be compressed due to buckling, prestressing these elements reflects not only mechanical reinforcement but provides for high speed (m/s) signal processing since viscosity effects are minimized.31
The detergent solubilization of worm cuticles showed that there is constitutive stress even in these nonliving demembranated preparations (supporting Fig. S6
Much of the internal stresses in cells are reaction forces applied to adhesion plaques. These constitutive stresses could be mostly relieved by detachment of the cell from the substrate as evidenced by the rather uniform FRET in rounded cells. Stress applied to the plaques is generated mostly by actin and is sensitive to actin reagents,47
however our data shows that the crosslinking proteins showed a modest but rapid increase
in stress with the actin inhibitors. The stress increase could have several origins. The actin reagents are effective only on cycling actin21
and cortical actin has a slow turnover23
so that release of stress for deeper actin and other linked cytoskeletal proteins would be transferred to the cell cortex that is still intact. Furthermore, actinin and filamin will produce tension in an actin network simply from crosslinking10,40
which again could be redistributed upon treatment with actin inhibitors. Also, some actin assembly is known to occur in the presence of latrunculin due to resistant actin oligomers.34
In this regard, we noted that that while treated cells showed an overall decrease in contact area with the glass, there were still incidences of filipodial extension (see actinin video in Fig. S2C
). Finally, if the regions of crosslinker binding are not broken down, the Poisson effect (stretched things getting thinner) will tend to stretch cross linkers as tension is removed in the primary filaments.
Mechanically induced strains from fluid shear on BAECs showed that the stFRET can dynamically report gradients of stress distributed differently over complex cell shapes. Quantitative analysis of these stresses remains a difficult analysis problem, but in our first order analysis of the initial shear pulse we saw a >50% increase in tension at the upstream edge and ~20% increase at the downstream side. The FRET response was viscoelastic (time dependent) and showed slow adaptation as expected for cytoskeletal networks26,44
that correlated with changes in cell stiffness.
The experiments on C. elegans showed that stFRET can be used to examine micromechanics in situ. We have begun extending these transgenic studies to mice where insertion of the labeled actinin has no effect on behavior or development or reproduction. These preparations will allow us to study the stress in different organs and tissues such as the heart and bone in a variety of normal and pathological states. As we have shown, we can detect internal stress in specific proteins from the application of external stresses to the animal with a time resolution <10 ms.
stFRET is providing data on processes that were heretofore invisible. How can the probe be improved? An ideal probe would have the compliance of the host protein so that the physical behavior of the host is not compromised. We have done this recently by replacing the α-helix linker in stFRET with the spectrin repeat. Ideally we would like a higher contrast probe. However, for linkers with a linear compliance, the effective contrast with stress is limited by thermal energy that will cause the probe to change conformation in the same manner as an applied force. If the probe is operating in a nonlinear portion of the force-distance relationship as occurs with single molecule unfolding, we should be able to increase contrast at the expense of sacrificing linearity. And finally, as with nearly all fluorescent probes, increased photostability is a persistent and unmet goal.