The goal of these studies was a biosensor based on an engineered scaffold designed for high throughput screening. This proof of principle study can pave the way for generating other biosensors via screening, i.e. for targets where no suitable naturally occurring binders are known, and with greatly simplified biosensor engineering. To study Src family signaling at the cell’s leading edge, we based this prototype biosensor on a known fibronectin monobody that binds specifically to the SH3 domains of Src family kinases 6
. The fibronectin monobody was a good choice for a generally applicable biosensor scaffold because it contains no native cysteines (facilitating site-specific dye attachment) and folds well in living cells. This contrasts with scFv and other antibody fragments 37
. The FN3 monobody has flexible loops that accommodate insertion and randomization of amino acid residues, and has proven capacity to generate binders against diverse protein targets 5,6,38
. We used a solvent-sensitive merocyanine dye to report target binding, which proved to provide enhanced sensitivity, with brightness > 2 times higher than direct excitation of cerulean fluorescent protein, and therefore substantially higher than fluorescent proteins indirectly excited for FRET. This proved valuable at the thin edge of cells where signal/noise is an important limitation. This biosensor design could report activation of endogenous target protein, thereby reducing cell perturbation.
We showed that the monobody had the desired specificity for active SFKs, and then examined where dyes could be attached around the binding site to report target binding without greatly diminishing affinity. Three different sites were suitable, indicating that dyes will likely be suitable as readouts for monobodies binding to different proteins. The four fluorophores tested were designed for use in living cells --bright (ε > 100,000, QY = 0.17–0.61 in hydrophobic environments), with excitation at ≥ 550 nm to avoid auto-fluorescence and minimize cell damage, and with solvent-sensitive fluorescence suitable for reporting protein binding in vivo
. Remarkably, for the two dyes based on the coupled indolenine and benzothiophen-3-one-1,1-dioxide rings (mero87 and mero53), shifting the position of attachment determined whether the dye showed an increase or decrease of fluorescence upon target binding. The attachment site producing a decrease in fluorescence (position 52) is positioned at the interface between the beta sheet and the flexible DE loop. Dye attachment may have caused partial unfolding of the monobody, leading the dye to find a hydrophobic pocket in the monobody before interaction with target protein. Binding to the target could force the dye out of the pocket, thereby decreasing fluorescence. This would be partially driven by restoration of binding interactions that stabilize the monobody. For positions and dyes showing an increase in emission intensity upon binding (C53-m87 and C24-m53), the dye likely experienced a more hydrophobic environment on target binding. There were marked differences in the response and brightness exhibited by two dyes that differed only in placement of a sulphonate moiety(mero53 and 87). Repositioning of the sulphonate could have altered the dyes’ photophysics4
or interaction with the monobody interface (see below). The environment of the dye on the monobody also influenced its brightness. Although dye mero87 at position 53 gave the largest response (), its low overall brightness on the monobody led us to select dye mero53 at position 24 for use in living cells. This dye showed a 50% fluorescence increase upon target binding and was 2.4x as bright as Cerulean fluorescent protein. The labeled monobody had 500–600 nM affinity for Src SH3, a range proven valuable for biosensor reversibility and specificity in previous studies 1,21,39
We generated computer models of 1F11-mero53 conjugates and docked these models to Src SH3, thereby examining the merobody-target interface (, supplementary Figs. 16, 17
). In the biosensor used for live cell studies, our modeling suggests that the dye does not directly interact with the SH3 domain. Rather, it experiences a change in local environment due to differing interactions with the FN3 monobody itself (). Modeling suggests that the water-exposed surface area of the dye decreases as the merobody binds the target SH3 (), consistent with the observed increase in dye emission upon binding. Merocyanine dyes, including mero53, generally show enhancement in emission intensity when shifting from polar to a polar environments. The decrease in solvent-accessible surface area (see ) is in fact more pronounced for the specific moiety on the merocyanine (the sulphone) that is believed to confer sensitivity to solvent polarity2
. The models do not show changes in solvation for mero53 attached at positions that produced poor fluorescence response (Supplementary Fig. 16, 17
Modeling of the 1F11-dye/SH3 interface
Finally, we attached a fluorescent protein via an optimized linker to eliminate the formation of fluorescent puncta, potentially due to autophagy, that would have severely hindered live cell imaging. We are hopeful that these changes, and the identification of optimum dye attachment sites, have generated a scaffold that can now be targeted to other intracellular proteins, providing a generalizable tool to study endogenous protein conformation. This work has demonstrated the feasibility of generating practical biosensors from engineered scaffolds. It is important to note that essentially all biosensors perturb cell physiology, as they must interact with the molecules whose behaviors they report. Different designs either inhibit or mimic normal protein action. The SFK monobody biosensor described here may compete with endogenous ligands that normally bind to the SH3 domain of Src family proteins. This could generate either ‘false negative’ data, in which native ligands outcompete biosensor, or the biosensor could inhibit normal interactions. The enhanced sensitivity of the SFK merobody will enable us to use less biosensor, more closely approaching the equilibria in unperturbed cells.
The SFK monobody revealed localized and transient activation of SFK at the cell edge and in PDGF-induced dorsal ruffles. Immunostaining has shown that phosphorylated, active Src localizes to dorsal ruffles and at the cell edge28–31
, where it phosphorylates cortactin or N-WASP, leading to Arp2/3 activation and consequent actin polymerization.27,40–42
Src is known to be necessary for the formation of dorsal ruffles, and SFKs are known to regulate signaling molecules involved in actin assembly and organization within these ruffles27
(Abl tyrosine kinase43
, Rac GTPase 44
). The merobody biosensor provided direct evidence, consistent with these previous studies, that endogenous SFK are in the active conformation within ruffles specifically during actin-based protrusion.
The biosensor was used to study SFK activation in the lamellipodia of migrating cells. Through development of a quantitative line scanning approach, statistically valid correlations of protrusion velocity and Src activity distribution could be based on thousands of line scans. Though it was elevated in both protrusion and retraction, SFK activity was significantly higher during cell protrusion. Most strikingly, during protrusion the activity was proportional to the rate of lamellipodial extension. SFK activity may regulate protrusion speed by controlling the rate and extent of actin polymerization, potentially through phosphorylation of actin-regulatory proteins (potentially the WAVE complex40,41
, gelsolin, pCAS46,47
, Abl tyrosine kinase29,43
, or regulators of Rho family GTPases12,34,44,48–50
). The biosensor showed that activation occurred with a defined profile, peaking within 2 microns of the edge in the lamellipodium of the PTK1 cells. Src kinases play a critical role in regulating both actin dynamics and the assembly and disassembly of adhesions 33,34,47
, so SFK at this position may regulate actin, focal adhesions, or their coordination. Further work will be required to define the interactions of SFK with specific molecules at the leading edge and their positions relative to actin and adhesion dynamics. SFK activation in retraction is much less pronounced and may be part of constitutive signaling responsible for edge retraction 48
In conclusion, we have demonstrated the feasibility of producing a biosensor based on modification of the fibronectin monobody, a scaffold suitable for high throughput screening and for use in living cells. This exemplifies a generalizable approach capable of producing biosensors when no suitable affinity reagents are known, to increase the throughput of biosensor production, and to greatly simplify biosensor design. These biosensors report the activation of endogenous, unmodified proteins, thereby reducing perturbation of cell physiology. Dyes here provided exceptional sensitivity, but made it more difficult to introduce the biosensor into living cells. Ultimately it may be possible to use fluorescent proteins for genetically encoded readouts of endogenous target binding. Automated image analysis revealed that SFK are more strongly activated during protrusion than retraction, and that the level of activity is proportional to the velocity of the extending edge. Automated analysis of multiple points along the cell edge revealed an activity profile with a single peak of maximal activation at the edge of constitutively migrating PTK cells.