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 τ 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.

Time-resolved multirotational dynamics of single solution-phase tau proteins reveals details of conformational variation.

Intrinsically disordered proteins (IDPs) are crucial to many cellular processes and have been linked to neurodegenerative diseases. Single molecules of tau, an IDP associated with Alzheimer's disease, are trapped in solution using a microfluidic device, and a time-resolved fluorescence anisotropy decay is recorded for each molecule. Multiple rotational components are resolved and a novel k-means algorithm is used to sort the molecules into two families of conformations. Differences in rotational dynamics suggest a change in the rigidity and steric hindrance surrounding a sequence (306VQIVYK311) which is central to paired helical filament formation. This single-molecule approach can be applied to other IDPs to resolve heterogeneous populations and underlying differences in conformational dynamics.

Revealing Conformational Variants of Solution-Phase Intrinsically Disordered Tau Protein at the Single-Molecule Level.

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution-phase structural studies. We employed an anti-Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time-resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.