dna-tension-probes-to-map-transient-piconewton-receptor-forces-immune

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Georgia Institute of Technology and Emory University

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

DNA probes that store mechanical information reveal transient piconewton forces applied by T cells.

Cytokine signaling is challenging to study and therapeutically exploit as the effects of these proteins are often pleiotropic. A subset of cytokines can, however, achieve signal specificity via association with latency-inducing proteins, which cage the cytokine until disrupted by discreet biological stimuli. Inspired by this precision, here, we describe a strategy for synthetic induction of cytokine latency via modification with photolabile polymers that mimic latency while attached then restore protein activity in response to light, thus controlling the magnitude, duration, and location of cytokine signals. We characterize the high dynamic range of cytokine activity modulation and find that polymer-induced latency, alone, can prolong in vivo circulation and bias receptor subunit binding. We further show that protein derepression can be achieved with a near single-cell resolution and demonstrate the feasibility of transcutaneous photoactivation. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and more precise immunotherapies.

Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

Article	Year
DNA probes that store mechanical information reveal transient piconewton forces applied by T cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31391300	2019
Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency. Biomacromolecules\| PubMed ID: 32374589	2020
Live-cell super-resolved PAINT imaging of piconewton cellular traction forces. Nature methods\| PubMed ID: 32929270	2020

Anna V. Kellner

Biography

JoVE Journal Publications

Other Publications