Evaluation of Molecular Tension Sensors using Single-Molecule Force Spectroscopy and Live Cell FRET imaging

Mehlich, Alexander; Austen, Katharina; Ringer, Pia; Rief, Matthias; Grashoff, Carsten

doi:10.1038/protex.2015.095

Cited by 3 publications

(1 citation statement)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Bead-DNA-protein-bead-DNA dumbbells were generated as described previously (Mehlich et al, 2015). In short, maleimidemodified oligonucleotides were attached to the cysteines of the purified protein via a disulfide bond to form a proteinoligonucleotide construct.…”

Section: Ross: Mack Mllyvliisn Dkklieeark Maekanlel R Tvktedelkk Yleementioning

confidence: 99%

Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein

et al. 2020

Self Cite

View full text Add to dashboard Cite

De-novo designed proteins have received wide interest as potential platforms for nano-engineering and biomedicine. While much work is being done in the design of thermodynamically stable proteins, the folding process of artificially designed proteins is not well-studied. Here we used single-molecule force spectroscopy by optical tweezers to study the folding of ROSS, a de-novo designed 2x2 Rossmann fold. We measured a barrier crossing time in the millisecond range, much slower than what has been reported for other systems. While long transition times can be explained by barrier roughness or slow diffusion, we show that isotropic roughness cannot explain the measured transition path time distribution. Instead, this study shows that the slow barrier crossing of ROSS is caused by the population of three short-lived high-energy intermediates. In addition, we identify incomplete and off-pathway folding events with different barrier crossing dynamics. Our results hint at the presence of a complex transition barrier that may be a common feature of many artificially designed proteins.

show abstract

Section: Ross: Mack Mllyvliisn Dkklieeark Maekanlel R Tvktedelkk Yleementioning

confidence: 99%

Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

et al. 2015

Self Cite

View full text Add to dashboard Cite

The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule–calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages upon cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton (pN) forces and bear, on average, 7–10 pN during cell adhesion depending on their association with f-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform-specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing.

show abstract

Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1

Ringer¹,

Weißl²,

Cost³

et al. 2017

Nat Methods

135

148

View full text Add to dashboard Cite

Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3-5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment.

show abstract

Evaluation of Molecular Tension Sensors using Single-Molecule Force Spectroscopy and Live Cell FRET imaging

Cited by 3 publications

References 0 publications

Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein

Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1

Contact Info

Product

Resources

About