Chromobody projects

For in cellulo studies we make use of intracellularly functional nanobodies (chromobodies) to monitor the spatiotemporal dynamics and the abundance of endogenous components in living cells.

In combination with high-throughput microscopy and automated image analysis we apply chromobodies as intracellular biosensors for phenotypic screening and high-content imaging (HCI) in real time.

To date chromobody-based cellular models have been established to monitor cell cycle, signal transduction or to trace epithelial mesenchymal transition upon compound treatment within living cells.

Current chromobody cell models

A549_VB6-eGFP (monitoring EMT)

A549_VB6-tagRFP (monitoring EMT)

HeLa_BC1-tagGFP2 (Wnt/beta-Catenin pathway)

U2OS_BC1-tagGFP2 (Wnt/beta-Catenin pathway)

A549_VB6_Act-CB (dual CB cell line, EMT)

Chromobodies and epithelial-mesenchymal transition (EMT)

Epithelial-mesenchymal transition (EMT) is a complex cellular programme involved in the progression of epithelial cancers to a metastatic stage. Along this process, epithelial traits are repressed in favour of a motile mesenchymal phenotype. In this context characteristic epithelial marker proteins such as E-Cadherin, zona occludens proteins, occludin, cortical actin, membrane associated β-catenin and mesenchymal markers including vimentin, N-Cadherin and EMT-transriptionfactors (snail, slug, twist etc.) are described.

We have developed a set of nano- and chromobodies against EMT relevant marker proteins (ZO-1, beta-Catenin, Snail, Occludin and Vimentin). With an intracellularly functional chromobody against vimentin we were able for the first time to visualize the spatiotemporal organization of endogenous vimentin during the EMT process. Based on this chromobody we generated stable chromobody lung cancer models (A549 cells). We further expanded this model for high-content analysis to identify and quantify the effects of EMT-modulating compounds.

Currently we are optimizing this assay to perform screens of EMT relevant compound libraries. To increase the physiological relevance we are adapting the current cell model to a 3D cell cultures system.

β-catenin-chromobodies

β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a many cellular processes. We have selected nanobodies against the N-terminal, core or C-terminal domain of beta-catenin and apply them as selective affinity reagents to capture endogenous beta-catenin-containing multi-protein complexes in sandwich immunoassays and co-immunoprecipitations.

To monitor subcellular localization and abundance of beta-catenin in living cells we engineer anti-beta-catenin chromobodies and corresponding stable cell lines. Using high-content imaging we monitor drug action on endogenous beta-catenin in real-time.