Biology subjects

Velusamy, A.

Publications and source records attributed to Velusamy, A..

4 recordsLinked to original sources

bioRxiv2024

Measuring integrin force loading rates using a two-step DNA tension sensor

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (pN) forces exerted by cells have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrins receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop a LR probe which is comprised of an engineered DNA structures that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN corresponding to hairpin unfolding and a high force threshold at 56 pN triggered through duplex shearing. These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live-cells. Automated analysis of tens of thousands of events from 8 cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a {tau} of 45.6 sec. A small subset of these events (<10%) mature in magnitude to >56pN with a median loading rate of 1.3 pNs-1 with these mechanical ramp events localizing at the periphery of the cell-substrate junction. Importantly, the LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of mechanotransduction. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/585042v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1df1342org.highwire.dtl.DTLVardef@696995org.highwire.dtl.DTLVardef@92bd9dorg.highwire.dtl.DTLVardef@1decc42_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

DNA Origami Tension Sensors (DOTS) to study T cell receptor mechanics at membrane junctions

The T cell receptor (TCR) is thought to be a mechanosensor, meaning that it transmits mechanical force to its antigen and leverages the force to amplify the specificity and magnitude of TCR signaling. The past decade has witnessed the development of molecular probes which have revealed many aspects of receptor mechanotransduction. However, most force probes are immobilized on hard substrates, thus failing to reveal mechanics in the physiological context of cell membranes. In this report, we developed DNA origami tension sensors (DOTS) which bear force sensors on a DNA origami breadboard and allow mapping of TCR mechanotransduction at dynamic intermembrane junctions. We demonstrate that TCR-antigen bonds experience 5-10 pN forces, and the mechanical events are dependent on cell state, antigen mobility, antigen potency, antigen height and F-actin activity. We tethered DOTS onto a microparticle to mechanically screen antigen in high throughput using flow cytometry. Finally, DOTS were anchored onto live B cell membranes thus producing the first quantification of TCR mechanics at authentic immune cell-cell junctions.

biophysics↗

bioRxiv2023

Molecular Mechanocytometry Using Tension-activated Cell Tagging (TaCT)

Flow cytometry is routinely used to measure single-cell gene expression by staining cells with fluorescent antibodies and nucleic acids. Here we present Tension-activated Cell Tagging (TaCT) to fluorescently label cells based on the magnitude of molecular force transmitted through cell adhesion receptors. As a proof-of-concept, we analyzed fibroblasts and mouse platelets after TaCT using conventional flow cytometry.

bioengineering↗

bioRxiv2022

Unbreakable DNA tension probes show that cell adhesion receptors detect the molecular force-extension curve of their ligands

Integrin receptors transduce the mechanical properties of the extracellular matrix. Past studies using DNA probes showed that integrins sense the magnitude of ligand forces with pN resolution. An open question is whether integrin receptors also sense the force-extension trajectory of their ligands. The challenge in addressing this question pertains to the lack of molecular probes that can control force-extension trajectories independently of force magnitude. To address this limitation, we synthesized two reversible DNA probes that fold with identical self-complementary domains but with different topologies. Thus, these probes unfold at the same steady-state force magnitude but following different kinetic pathways to reach the fully extended ssDNA state. Hairpin-like probes unzip with a low barrier of 14 pN while the pseudo-knot-like probes shear at 59 pN. Confirming that we had created probes with different barriers of unfolding, we quantified platelet integrin forces and measured 50-fold more tension signal with the unzipping probes over the shearing probes. In contrast, fibroblasts opened both probes to similar levels indicating more static forces. Surprisingly, fibroblast mechanotransduction markers, such as YAP levels, fibronectin production, actin organization, and integrin activation were significantly elevated on unzipping probes. This demonstrates that integrin receptors within focal adhesions sense the molecular force-extension profile of their ligands and not only the magnitude of equilibrium mechanical resistance.

biophysics↗