Facile detection of mechanical forces across proteins in cells with STReTCh

Zhong, Brian L.; Vachharajani, Vipul T.; Dunn, Alexander R.

doi:10.1016/j.crmeth.2022.100278

Cited by 8 publications

(20 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Instead, it is an approach to assess whether mechanosensing is supported in a specific loadbearing protein and biological context. Compared to other imaging-based techniques for mechanosensing [11][12][13] , which rely on labeling unfolded protein domains with secondary binding probes, our approach based on FP mechanical switching has the advantage of being independent of secondary probe binding. Additionally, our approach is suited for dynamic measurements of mechanosensing in live cells.…”

Section: Discussionmentioning

confidence: 99%

“…The development of molecular tension sensors (MTSs) to visualize loads across specific proteins inside cells has advanced our understanding of mechanotransmission, specifically which proteins transmit loads and how these loads vary across biological contexts 10 . Progress in mechanosensing has been enabled by techniques to label unfolded protein domains via the binding of secondary probes, including antibodies that recognize the extended conformations of p130Cas or alpha-catenin 11,12 , and STReTCh, which operates by the force-induced exposure of SpyTag and subsequent covalent binding of SpyCatcher 13 . Likewise, advances in mechanotransduction have been made by monitoring the localization of endogenous transducer proteins in response to molecular tension across a load-bearing protein 14 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Detection of Fluorescent Protein Mechanical Switching in Cellulo

Shoyer,

Collins,

Ham

et al. 2024

Preprint

View full text Add to dashboard Cite

The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, the mechanisms by which forces affect protein function inside cells remain unclear. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated if force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Foerster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo in a synthetic actin-crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cellular force generation as well as force-sensitive bond dynamics of the biosensor. Together, this work describes a new framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Detection of Fluorescent Protein Mechanical Switching in Cellulo

Shoyer,

Collins,

Ham

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Magnetic tweezers were constructed and calibrated on a Nikon Ti-E microscope as previously described 48 . EGFP-YbbR-Lphn3 GAIN-HaloTag was biotinylated at the YbbR tag by reacting 4 μM protein with 10 mM MgCl 2 , 0.5 μM Sfp synthase, and 5 μM Biotin-PEG3-Coenzyme A (SiChem, SC-8618) at room temperature overnight.…”

Section: Methodsmentioning

confidence: 99%

Piconewton forces mediate GAIN domain dissociation of the latrophilin-3 adhesion GPCR

Zhong

Lee

Vachharajani

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-Autoproteolysis Inducing (GAIN) domain, but the two resulting fragments remain associated on the cell surface. It is thought that force-mediated dissociation of the fragments exposes a peptide that activates G-protein signaling of aGPCRs, but whether GAIN domain dissociation can occur on biologically relevant timescales and at physiological forces is unknown. Here, we show using magnetic tweezers that physiological forces dramatically accelerate the dissociation of the latrophilin-3 GAIN domain. Forces in the 1-10 pN range were sufficient to dissociate the GAIN domain on a seconds-to-minutes timescale, and the GAIN domain fragments reversibly reassociated after dissociation. Thus, mechanical force may be a key driver of latrophilin signaling during synapse formation, suggesting a physiological mechanism by which aGPCRs may mediate mechanically-induced signal transduction.

show abstract

“…SpyCatcher-ELP-functionalized #1.5 glass coverslips were prepared as previously described 71 , and stored in vacuum-sealed boxes at -20 °C. Briefly, 3-5 mm wide slits were cut with a razor blade into a piece of Parafilm M (Bemis) and sandwiched between a glass slide (1 mm thick, Fisher Scientific) and a SpyCatcher-ELP coverslip, to produce channels of ∼20 microliter volume.…”

Section: Methodsmentioning

confidence: 99%

PDZ Domains from the Junctional Proteins Afadin and ZO-1 Act as Mechanosensors

Vachharajani,

DeJong,

Dunn

2023

Preprint

Self Cite

View full text Add to dashboard Cite

Intercellular adhesion complexes must withstand mechanical forces to maintain tissue cohesion, while also retaining the capacity for dynamic remodeling during tissue morphogenesis and repair. Most cell-cell adhesion complexes contain at least one PSD95/Dlg/ZO-1 (PDZ) domain situated between the adhesion molecule and the actin cytoskeleton. However, PDZ-mediated interactions are characteristically nonspecific, weak, and transient, with several binding partners per PDZ domain, micromolar dissociation constants, and bond lifetimes of seconds or less. Here, we demonstrate that the bonds between the PDZ domain of the cytoskeletal adaptor protein afadin and the intracellular domains of the adhesion molecules nectin-1 and JAM-A form molecular catch bonds that reinforce in response to mechanical load. In contrast, the bond between the PDZ3-SH3-GUK (PSG) domain of the cytoskeletal adaptor ZO-1 and the JAM-A intracellular domain becomes dramatically weaker in response to ~2 pN of load, the amount generated by single molecules of the cytoskeletal motor protein myosin II. These results suggest that PDZ domains can serve as force-responsive mechanical anchors at cell-cell adhesion complexes, and that mechanical load can enhance the selectivity of PDZ-peptide interactions. PDZ mechanosensitivity may thus help to generate the intricate molecular organization of cell-cell junctions and allow junctional complexes to dynamically remodel in response to mechanical load.

show abstract

Facile detection of mechanical forces across proteins in cells with STReTCh

Cited by 8 publications

References 38 publications

Detection of Fluorescent Protein Mechanical Switching in Cellulo

Detection of Fluorescent Protein Mechanical Switching in Cellulo

Piconewton forces mediate GAIN domain dissociation of the latrophilin-3 adhesion GPCR

PDZ Domains from the Junctional Proteins Afadin and ZO-1 Act as Mechanosensors

Contact Info

Product

Resources

About