Abstract
Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.
Highlights
Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro
Quantitative Image Analysis of Antigen-mediated Stabilization of a CTNNB1-specific CB—Previously, we have shown that an increase of endogenous CTNNB1 upon treatment with the GSK3- inhibitor CHIR99021 (CHIR) was accompanied by rising levels of a CTNNB1-specific CB stably integrated and constitutively expressed in HeLa cells using immunoblot analysis (see Figure 11 of [21])
We focused on an imaging-based readout and analyzed the level of both proteins in situ by immunofluorescence staining with a CTNNB1-specific antibody in combination with the detection of BC1-TagGFP2 fluorescence
Summary
Understanding cellular processes requires the determination of dynamic changes in the concentration of endogenous proteins. Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action. As exemplarily shown for CTNNB1-specific CBs, stable chromobody cell lines allow visualization of rapid and reversible changes in the concentration of endogenous proteins upon compound treatment by quantitative live-cell imaging
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