A common technique to visualise DNA or RNA molecules in cells is to incorporate into the target molecule multiple copies of a sequence that a fluorescently tagged protein can bind to. The intensity of the signal can then be turned up by adding more copies of the sequence, which allows for more fluorescent proteins to bind.
Despite the success of multimerising nucleic-acid-based motifs within RNA and DNA, no comparable, generic system existed for proteins. And so a team led by the postdoc Marvin Tanenbaum from Ron Vale’s lab at the University of California, San Francisco developed such a protein labelling system and called it SunTag (link is external).
Before SunTag the main way to visualise a protein in cells was to recode its sequence to contain a fluorescent domain, for example GFP. But, proteins with a single GFP domain attached are often too dim for fluorescence microscopy and adding more GFP domains often doesn’t work as the protein becomes too large and unwieldy within the cell.
With the SunTag system proteins of interest are recoded to contain multiple copies (up to 24) of a tiny peptide epitope, GNC4. The protein is then coexpressed with GFP-tagged single-chain antibodies (scFv) that recognize the epitope and bind. The antibodies are conjugated to either a fluorescent protein or a transcriptional regulator for imaging or expression studies respectively.
The fluorescent signal emitted from the SunTag system shows nearly stoichiometric signal amplification, where four copies of the peptide epitope resulted in a four-fold increase in the fluorescent signal and 24 copies of the peptide epitope showed a further six-fold increase.
Using the 24-epitope SunTag and GFP-conjugated antibody the team at UCSF have tracked and imaged single molecules in the cell membrane, nucleus, mitochondria and cytoskeleton, and used this to measure the dynamics of individual microtubules and the average run length of single cytoskeletal motors over time.
To assess its functionality in expression studies the team coupled the tag to a catalytically inactive Cas9 protein (dCas9) and coexpressed it with the antibody-transcriptional activator fusion, VP64. When delivered along with a small guide RNA that directed dCas9 to specific genomic addresses, the SunTag was associated with a change in expression of the reporter genes of over 40-fold. This application of the system has been already been exploited for genome-scale screens of gene function by a group of researchers from Stanford University.
SunTag’s high signal intensity may mean that cells can be imaged under dimmer light, reducing phototoxicity and enabling longer-term studies for live cells. The system also allows for lower expression levels than would otherwise be required for the visualisation of intracellular molecules, which could reduce potential experimental pitfalls from over-expressed proteins, and can be used to effectively visualise single molecules within the cell. Given the system’s design it can be easily adapted to new functions- conjugate a new effector molecule to the antibody and it could act as a transcriptional repressor, for instance.
However, as with all antibody labelling systems SunTag is big. When bound by 24 antibody-GFP complexes it measures 1.4MDa. The authors of the study show that this size does impact on protein mobility and may sometimes alter protein function or localisation. Additionally the poor expression of antibodies within the cellular cytoplasm restrict this system. They state that “The suitability of SunTag will need to be determined on a protein-by-protein basis”.
Nonetheless, where it does function this protein-tagging system could allow a host of new insights into the life inside the cell and demonstrates an exciting new approach to immunocytochemistry.
The researchers note that to overcome the inherent difficulties with poor antibody expression and the large size of the SunTag that a second generation of the system may have to be based upon alternative affinity proteins, like Affimer reagents, which are smaller in size and show greater cytoplasmic expression and stability yet retain the important characteristics of highly specific, high affinity binding to their target protein.
Further developments to the SunTag system that the authors have recognised include a second, orthogonal system allowing two-colour single-molecule imagine and dual transcriptional regulation, raising the possibility of activation and silencing different genes in the same cell, and an inducible recruitment system, which could enable temporal gene regulation.