Facile detection of mechanical forces across proteins in cells with STReTCh.
Cell reports methods
2022; 2 (9): 100278
Numerous proteins experience and respond to mechanical forces as an integral part of their cellular functions, but measuring these forces remains a practical challenge. Here, we present a compact, 11-kDa molecular tension sensor termed STReTCh (sensing tension by reactive tag characterization). Unlike existing genetically encoded tension sensors, STReTCh does not rely on experimentally demanding measurements based on Forster resonance energy transfer and is compatible with typical fix-and-stain protocols. Using a magnetic tweezers assay, we calibrate the STReTCh module and show that it responds to physiologically relevant, piconewton forces. As proof of concept, we use an extracellular STReTCh-based sensor to visualize cell-generated forces at integrin-based adhesion complexes. In addition, we incorporate STReTCh into vinculin, a cytoskeletal adaptor protein, and show that STReTCh reports on forces transmitted between the cytoskeleton and cellular adhesion complexes. These data illustrate the utility of STReTCh as a tool for visualizing molecular-scale forces in biological systems.
View details for DOI 10.1016/j.crmeth.2022.100278
View details for PubMedID 36160040
Improved immunoassay sensitivity and specificity using single-molecule colocalization.
2022; 13 (1): 5359
Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-alpha, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.
View details for DOI 10.1038/s41467-022-32796-x
View details for PubMedID 36097164
Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2020; 142 (13): 5952–57
Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.
View details for DOI 10.1021/jacs.0c00904
View details for Web of Science ID 000526393700009
View details for PubMedID 32182063