A biosensor generated via high-throughput screening quantifies cell edge Src dynamics

Abstract

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge.

Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation

“…As described previously, biosensor concentrations were adjusted to report Cdc42 activity while minimizing perturbation of normal Cdc42 signaling. 5,27–29 Consistent with earlier studies, Cdc42 was locally activated in protrusions. A control biosensor with point mutations known to reduce the CRIB domain’s affinity for active Cdc42 (mutant CRIB199 , H246D and H249D) 4,30 reduced the level of Cdc42 activation seen in the cells (Figures 3 and S5).…”

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

“…In combination with recent progress in cell-type-specific DNA delivery strategies (41), intrabodies perturbing specifically PTMs may become a valuable tool in signaling research or human and animal health. Furthermore, the uniformity and stability of the DARPin scaffold may greatly simplify the construction and optimization of fluorescent biosensors for living cells based on resonance energy transfer or attachment of environmentally sensitive dyes (42).…”

Use of micro-emulsion technology for the directed evolution of antibodies

“…Thus, there has been a great deal of recent interest in tapping the potential of recombinant affinity reagents for characterizing the human proteome [1][2][3][4][5], and the early comparisons to traditional antibodies are promising [6,7]. Furthermore, it takes less time to identify a recombinant affinity reagent to a target than to generate a rabbit polyclonal or mouse monoclonal antibody [8], and one can do some experiments that are impossible with traditional antibodies, such as evolve higher affinities [9,10], express inside cells and perturb targets [11,12], mislocalize targets in embryos [13], create biosensors [14], and incorporate unnatural amino acids for chemical derivatization [15], which allows new labeling, capturing and immobilization approaches.…”