Abstract 2021: Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions

Abstract

Abstract Determining the levels of protein-protein interactions (PPI) is essential for the understanding of signal transduction, regulation of gene expression and mutation effects, infection mechanisms, identification of diagnostic markers, etc. Taking the already challenging task of PPI identification a step further, the next aim is to determine the levels of non-interacting proteins in parallel. The ability to concurrently monitor free and interacting proteins opens the door to studying their functional states and interplay, thus gaining deeper insight from a single staining experiment. Additionally, to accomplish this in situ, retaining the structural integrity of the cell, helps understand spatiotemporal communication between proteins in their native environment. To this end, we designed Naveni TriFlex Cell – a highly sensitive and specific proximity-based technology, relying on two user-determined primary antibodies against the targets of interest, and on proprietary TriFlex Navenibodies, which are antibody-based proximity reagents. TriFlex Cell detects total protein A (i.e., both free and in complex with B), total protein B, and the AB interaction. The detected A, B and AB signals are amplified and generate fluorescent readout in three channels corresponding to each protein pool. As proof of principle, we stained MCF7 cells for E-cadherin and β-catenin, which are known to interact in the adherens junctions. We observed highly abundant interactions at the cell membrane, some complexes and free proteins in the cytoplasm, and very low background in controls where either or both primary antibodies were omitted. These results attested to the high sensitivity and specificity of our method. Next, we explored the expression and interaction of Histone H3 and Lamin B, a nuclear envelope protein. Despite the compact structure of the nucleus, we were able to distinguish individual signals and abundant interactions, with free Lamin B particularly enriched in the nuclear membrane. In dividing cells, we observed diffuse Lamin signals in line with nuclear envelope breakdown, and more densely packed Histone H3 associated with chromatin. In contrast to non-mitotic cells, interactions were few, but increasing with the advance from metaphase to telophase. Finally, we stained against GM130, a Golgi complex protein, and COX1, a mitochondrial marker, as a biological non-interaction control experiment. TriFlex Cell successfully detected the individual proteins in their respective subcellular locations, and no complexes. In summary, our data demonstrate that Naveni TriFlex Cell is a sensitive and specific method that visualizes low and high abundant targets in various cell compartments and adds value by uncovering protein interplay, such as complex formation/dissolution under dynamic biological conditions. Citation Format: Axel Klaesson, Doroteya Raykova, Agata Zieba Wicher. Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2021.