Visualizing Intercellular Tensile Forces by DNA-Based Membrane Molecular Probes.

Mechanical forces play critical roles in collective cell behaviors such as cell migration, proliferation, and differentiation. Extensive efforts have been made to measure forces between cells and extracellular matrices. However, force studies at cell-cell junctions remain a challenge. Herein, we reported a novel strategy to construct membrane DNA tension probes to visualize tensile forces at cell junctions. These lipid-modified probes can self-assemble onto cell membranes with high efficiency and stability. Upon experiencing tensile forces generated by neighboring cells, unfolding of the probes leads to a large increase in the fluorescence intensity. Compatible with readily accessible fluorescence microscopes, these easy-to-use membrane DNA tension probes can be broadly used to measure intercellular tensile forces.

Membrane association of a protein increases the rate, extent, and specificity of chemical cross-linking.

Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical cross-linking reactions with the trifunctional maleimide TMEA. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein-protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.

Genetically encoded RNA-based sensors for intracellular imaging of silver ions.

Silver has been widely used for disinfection. The cellular accumulation of silver ions (Ag+) is critical in these antibacterial effects. The direct cellular measurement of Ag+ is useful for the study of disinfection mechanisms. Herein, we reported a novel genetically encoded RNA-based sensor to image Ag+ in live bacterial cells. The sensor is designed by introducing a cytosine-Ag+-cytosine metallo base pair into a fluorogenic RNA aptamer, Broccoli. The binding of Ag+ induces the folding of Broccoli and activates a fluorescence signal. This sensor can be genetically encoded to measure the cellular flux and antibacterial effect of Ag+.