Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions

Pui‐Yan, Victor; Liu, Yang; Blanchfield, Lori; Su, Hanquan; Evavold, Brian D.; Salaita, Khalid

doi:10.1021/acs.nanolett.6b01817

Cited by 71 publications

(80 citation statements)

References 60 publications

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“…Major breakthroughs in SLB technologies include the ability to precisely pattern fluid and anchored ligands, to incorporate properly oriented and fluid transmembrane proteins, to generate multiple stacked bilayers, and to measure mechanical forces at the cell-SLB interface using ratiometric tension probes [25, 109, 110, 123, 136, 137]. Current studies of integrin and cadherin mediated adhesion using SLBs offer new insight into NA and AJ formation and demonstrate the power of spatiomechanical mutation using SLBs.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, this method has been applied to map T cell receptor, epidermal growth factor receptor, and integrin forces with high spatiotemporal resolution [11, 15, 131–136]. In MTFM, an immobilized probe molecule comprised of a flexible linker and flanked by a fluorephore-quencher pair presents a ligand to a receptor of interest.…”

Section: Methods To Measure Receptor Forces At Fluid Interfacesmentioning

confidence: 99%

“…A second fluorophore is non-specifically attached to the streptavidin, and this “density reporter” fluorophore directly reports probe surface density. An additional benefit to this probe is the high signal-to-noise ratio; donor fluorescence is dual quenched by the molecular quencher and by nanoparticle surface-energy transfer (NSET) with the AuNP [136] (Figures 4B,C). Nowosad, et.…”

Section: Methods To Measure Receptor Forces At Fluid Interfacesmentioning

confidence: 99%

“…Scale bar 5 μm. (C,D reprinted from [136] with permission from publisher). (E) Ratiometric DNA tension probes containing a density reporter fluorophore on the hairpin strand.…”

Section: Figurementioning

confidence: 99%

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Supported lipid bilayer platforms to probe cell mechanobiology

Glazier¹,

Salaita²

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Mammalian and bacterial cells sense and exert mechanical forces through the process of mechanotransduction, which interconverts biochemical and physical signals. This is especially important in contact-dependent signaling, where ligand-receptor binding occurs at cell-cell or cell-ECM junctions. By virtue of occurring within these specialized junctions, receptors engaged in contact-dependent signaling undergo oligomerization and coupling with the cytoskeleton as part of their signaling mechanisms. While our ability to measure and map biochemical signaling within cell junctions has advanced over the past decades, physical cues remain difficult to map in space and time. Recently, supported lipid bilayer (SLB) technologies have emerged as a flexible platform to measure and manipulate membrane receptor mechanotransduction, allowing one to mimic cell-cell junctions. Changing the lipid composition and underlying substrate tunes bilayer fluidity, and lipid and ligand micro- and nano-patterning spatially control positioning and clustering of receptors. Patterning metal gridlines within SLBs introduces corrals that confine lipid mobility and introduce mechanical resistance. Here we review fundamental SLB mechanics and how SLBs can be engineered as tunable cell substrates for mechanotransduction studies. Finally, we highlight the impact of this work in understanding the biophysical mechanisms of cell adhesion.

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Section: Discussionmentioning

confidence: 99%

Section: Methods To Measure Receptor Forces At Fluid Interfacesmentioning

confidence: 99%

Section: Methods To Measure Receptor Forces At Fluid Interfacesmentioning

confidence: 99%

“…Scale bar 5 μm. (C,D reprinted from [136] with permission from publisher). (E) Ratiometric DNA tension probes containing a density reporter fluorophore on the hairpin strand.…”

Section: Figurementioning

confidence: 99%

See 2 more Smart Citations

Supported lipid bilayer platforms to probe cell mechanobiology

Glazier¹,

Salaita²

2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…To better mimic this biophysical observation and study the dynamics of TCR forces during the formation of the immunological synapse, we tethered DNA hairpin tension probes on SLBs that allow concurrent measurement of TCR forces and lateral clustering of TCR‐pMHC complexes ( Figure A) . These force probes include two fluorescence reporters, one that reports on clustering and was insensitive to DNA hairpin unfolding, and the second fluorophore that reports on mechanical forces and probe clustering.…”

Section: Dna‐based Force Probes To Map Piconewton Forces Within Cellsmentioning

confidence: 99%

DNA Nanotechnology as an Emerging Tool to Study Mechanotransduction in Living Systems

Pui‐Yan

Salaita

2019

Small

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The ease of tailoring DNA nanostructures with sub‐nanometer precision has enabled new and exciting in vivo applications in the areas of chemical sensing, imaging, and gene regulation. A new emerging paradigm in the field is that DNA nanostructures can be engineered to study molecular mechanics. This new development has transformed the repertoire of capabilities enabled by DNA to include detection of molecular forces in living cells and elucidating the fundamental mechanisms of mechanotransduction. This Review first describes fundamental aspects of force‐induced melting of DNA hairpins and duplexes. This is then followed by a survey of the currently available force sensing DNA probes and different fluorescence‐based force readout modes. Throughout the Review, applications of these probes in studying immune receptor signaling, including the T cell receptor and B cell receptor, as well as Notch and integrin signaling, are discussed.

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T‐Cell Antigen Recognition – Lessons from the Past and Projections into the Future

Platzer

Huppa

2020

Encyclopedia of Life Sciences

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T‐cells are central to adaptive immunity and arguably to this date the most intensely studied cells in life sciences. Paying tribute to their developmental plasticity and the complexities associated with many of their physiological functions, numerous aspects of their physiology are still far from being understood to an extent that would be sufficient to rationally design therapies effectively targeting allergies, autoimmunity and cancer. T‐cell antigen recognition is no exception: this field took up speed with the rise of monoclonal antibodies and the first successful cloning of the T‐cell antigen receptor (TCR) genes roughly 40 years ago. In the meantime, hundreds of TCRs have been crystallised in complex with their nominal peptide/MHC (pMHC) binding partners and many TCR‐pMHC interaction kinetics have been measured. Furthermore most, if not all signalling molecules acting downstream of the TCR have been identified. Despite these accomplishments, we are still searching for convincing explanations as to how T‐cells mange to reliably detect the presence of even a single antigen on the surface of antigen‐presenting cells (APCs). Elaborating underlying mechanisms will invariably require a more advanced understanding of the molecular, subcellular and cellular context in which T‐cell antigen recognition operates. What renders this endeavour both challenging and exciting is the rather weak strength and promiscuous nature of TCR‐pMHC binding and the fact that antigenic pMHCs are typically vastly outnumbered on APC surfaces by structurally similar, yet nonstimulatory pMHCs. While research of the last 20 years has provided some clarity, it has also caused at times controversies, which need to be resolved to unleash the full potential that T‐cells offer for clinical progress. Key Concepts T‐cells are indispensable for orchestrating and executing cellular and humoral adaptive immune responses; in recurrent communication with other cells of the immune system, T‐cells continuously patrol our body in search for antigenic peptide fragments derived from pathogens or cancer‐derived neoantigens. T‐cells are exquisitely sensitive for their nominal antigen as they can detect the presence of even a single stimulatory pMHC amongst thousands of structurally similar yet nonstimulatory pMHCs on the very crowded surface of an APC. Despite considerable progress in the field over the last 40 years, the molecular, biophysical and (sub‐) cellular principles underlying the detection efficiency associated with T‐cell antigen recognition and are far from being resolved. Given the complexities inherent to processes associated with T‐cell antigen recognition, which involve (1) the short‐lived nature of key protein–protein and protein–lipid interactions and (2) mechanical forces acting within the narrow confines of the immunological synapse, we consider integrative approaches combining classical biochemistry, structural biology and genetics with biophysics, advanced live‐cell imaging and systems biology most likely to provide much‐needed answers to most fundamental questions. Easy access to both experimental/analysis modalities and primary data of published work as well as improved literacy in the areas of biophysics and systems biology will help accelerate progress in the field of T‐cell antigen recognition with immediate and far‐reaching clinical implications benefiting allergy, autoimmune and cancer patients.

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Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions

Abstract: TOC GRAPHICS

Cited by 71 publications

References 60 publications

Supported lipid bilayer platforms to probe cell mechanobiology

Supported lipid bilayer platforms to probe cell mechanobiology

DNA Nanotechnology as an Emerging Tool to Study Mechanotransduction in Living Systems

T‐Cell Antigen Recognition – Lessons from the Past and Projections into the Future

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