Chromobodies to Quantify Endogenous Proteins in Living Cells

Abstract

As key cellular processes are dynamic in nature and underlie sophisticated spatial and temporal orchestration within living cells and whole organisms “seeing is believing” has turned into a main principle in cell biology. By employing live-cell fluorescence imaging, these important mechanisms can be deciphered. Importantly, an in-depth understanding of cellular processes requires the determination of dynamic changes in the concentration of endogenous proteins. However, today this task is mainly fulfilled by in vitro end point assays, which contradicts the requirement of assessing dynamic protein changes under native conditions within living cells. During the last years fluorescently labeled nanobodies (chromobodies, CBs), implemented as intracellular molecular probes, have emerged as valuable tools to visualize the spatial and temporal localization of endogenous proteins in living cells. In this dissertation, a novel aspect of CBs was observed and illuminated: The intracellular CB amount directly depends on the concentration of its cognate antigen, which was termed “Antigen-Mediated CB Stabilization” (AMCBS). Employing this phenomenon allows an expansion of the CB technology from the visualization of the spatiotemporal distribution of the antigen towards a relative quantification of time-resolved changes in endogenous protein concentration by simply determining the CB fluorescence. This idea was confirmed by analyzing four previously generated CBs, targeting β-catenin, proliferating cell nuclear antigen (PCNA), vimentin and HIV capsid protein p24, in their performance to monitor dynamic up- and downregulation of their respective antigens. To improve the accuracy and dynamic range of AMCBS, turnover-accelerated versions of these CBs were generated by utilizing the concept of the N-end rule and comparative analysis revealed that fast and reversible changes can be assessed more precisely by quantitative live-cell imaging applying the novel generated turnover-accelerated CBs. To illustrate a possible application, the turnover-accelerated β-catenin-specific CB was employed to monitor drug action and kinetics in living cells through AMCBS. Lastly, a protocol to generate stable cell lines expressing turnover-accelerated CBs was established by site-directed genetic integration into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of human cell lines using CRISPR/Cas9. In summary, AMCBS-based protein quantification in combination with highly specific and functional CBs allow an imaging-based quantification of endogenous proteins in living cells, thereby enabling unprecedented insights into protein dynamics. For the future, this method is broadly applicable in biomedical research as it is straight-forward to implement and the only technical requirement is a fluorescence microscope.