Observing proteins precisely within cells is extremely important for many branches of research but has been a significant technical challenge -- especially in living cells, as the required fluorescent labelling had to be individually attached to each protein. The research group led by Stefan Kubicek at CeMM has now overcome this hurdle: With a method called "vpCells," it is possible to label many proteins simultaneously, using five different fluorescent colours. This automated high-throughput approach, aided by AI-assisted image recognition, opens up entirely new applications in various disciplines, from fundamental cell biology to drug discovery.

 

The new method presented by Stefan Kubicek's group, called "visual proteomics Cells" (abbreviated vpCells), allows proteins to be fluorescently labelled in a way that preserves their endogenous regulatory mechanisms. Instead of labelling one protein at a time, vpCells can fuse many proteins simultaneously with a fluorescent tag in a so-called multiplex approach. A precursor of this method was already described by Kubicek's team in 2020 for studying metabolic enzymes (Reicher et al. Genome Res. 2020, DOI: 10.1101/gr.261503.120). Now it has been expanded and improved in three ways: Firstly, vpCells can label all theoretically possible proteins using the CRISPR/Cas9 gene-editing tool to genetically attach fluorescent proteins to the proteins under investigation. Secondly, vpCells use not only one fluorescent colour but a total of five complementary colours. In each cell, two different proteins to be tracked are marked. Thirdly, this colour scheme enables not only to generate visually appealing images, but also to optically recognize and discriminate the different proteins.

 

The method has already demonstrated its utility in two applications: On the one hand, more than 4,500 cell lines were generated as reporters for more than 1,100 proteins. On the other hand, the living reporter cells were used for a specific research question: Kubicek's team examined the effect of more than 1000 small-molecule substances on 61 proteins relevant to cancer cells. "These results provide a first glimpse into the versatility of the vpCells method," says Kubicek. "We expect many more future applications , from fundamental cell biology to applied drug discovery."